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Sample records for early developing zebrafish

  1. Changes in Neurotransmitter Profiles during Early Zebrafish (Danio rerio) Development and after Pesticide Exposure.

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    Tufi, Sara; Leonards, Pim; Lamoree, Marja; de Boer, Jacob; Legler, Juliette; Legradi, Jessica

    2016-03-15

    During early development, neurotransmitters are important stimulants for the development of the central nervous system. Although the development of different neuronal cell types during early zebrafish (Danio rerio) development is well-studied, little is known of the levels of neurotransmitters, their precursors and metabolites during development, and how these levels are affected by exposure to environmental contaminants. A method based on hydrophilic interaction liquid chromatography coupled to tandem mass spectrometry has been applied for the first time to zebrafish embryos and larvae to study five neurotransmitter systems in parallel, including the dopaminergic-andrenergic, glutaminergic-GABAnergic, serotoninergic, histaminergic, and cholinergic systems. Our method enables the quantification of neurotransmitters and their precursors and metabolites in whole zebrafish from the period of zygote to free-swimming larvae 6 days postfertilization (dpf). We observed a developmental stage-dependent pattern with clear differences between the first 2 days of development and the following days. Whereas the neurotransmitter levels steadily increased, the precursors showed a peak at 3 dpf. After exposure to several pesticides, significant differences in concentrations of neurotransmitters and precursors were observed. Our study revealed new insights about neurotransmitter systems during early zebrafish development and showed the usefulness of our approach for environmental neurotoxicity studies.

  2. Early gonad development in zebrafish ( Danio rerio ) | Okuthe ...

    African Journals Online (AJOL)

    Gonadogenesis in zebrafish goes through an initial ovarian phase then subsequently into either ovarian or testicular phases. How germ cells choose to commit to an oogenic fate and enter meiosis or alternatively not enter meiosis and commit to a spermatogenetic fate remains a key question. This study investigated events ...

  3. Fibroblast growth factor signaling is required for early somatic gonad development in zebrafish.

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    Leerberg, Dena M; Sano, Kaori; Draper, Bruce W

    2017-09-01

    The vertebrate ovary and testis develop from a sexually indifferent gonad. During early development of the organism, primordial germ cells (the gamete lineage) and somatic gonad cells coalesce and begin to undergo growth and morphogenesis to form this bipotential gonad. Although this aspect of development is requisite for a fertile adult, little is known about the genetic regulation of early gonadogenesis in any vertebrate. Here, we provide evidence that fibroblast growth factor (Fgf) signaling is required for the early growth phase of a vertebrate bipotential gonad. Based on mutational analysis in zebrafish, we show that the Fgf ligand 24 (Fgf24) is required for proliferation, differentiation, and morphogenesis of the early somatic gonad, and as a result, most fgf24 mutants are sterile as adults. Additionally, we describe the ultrastructural elements of the early zebrafish gonad and show that distinct somatic cell populations can be identified soon after the gonad forms. Specifically, we show that fgf24 is expressed in an epithelial population of early somatic gonad cells that surrounds an inner population of mesenchymal somatic gonad cells that are in direct contact with the germ cells, and that fgf24 is required for stratification of the somatic tissue. Furthermore, based on gene expression analysis, we find that differentiation of the inner mesenchymal somatic gonad cells into functional cell types in the larval and early juvenile-stage gonad is dependent on Fgf24 signaling. Finally, we argue that the role of Fgf24 in zebrafish is functionally analogous to the role of tetrapod FGF9 in early gonad development.

  4. Toxicity and cardiac effects of carbaryl in early developing zebrafish (Danio rerio) embryos

    International Nuclear Information System (INIS)

    Lin, C.C.; Hui, Michelle N.Y.; Cheng, S.H.

    2007-01-01

    Carbaryl, an acetylcholinesterase inhibitor, is known to be moderately toxic to adult zebrafish and has been reported to cause heart malformations and irregular heartbeat in medaka. We performed experiments to study the toxicity of carbaryl, specifically its effects on the heart, in early developing zebrafish embryos. LC50 and EC50 values for carbaryl at 28 h post-fertilization were 44.66 μg/ml and 7.52 μg/ml, respectively, and 10 μg/ml carbaryl was used in subsequent experiments. After confirming acetylcholinesterase inhibition by carbaryl using an enzymatic method, we observed red blood cell accumulation, delayed hatching and pericardial edema, but not heart malformation as described in some previous reports. Our chronic exposure data also demonstrated carbaryl-induced bradycardia, which is a common effect of acetylcholinesterase inhibitors due to the accumulation of acetylcholine, in embryos from 1 day post-fertilization (dpf) to 5 dpf. The distance between the sinus venosus, the point where blood enters the atrium, and the bulbus arteriosus, the point where blood leaves the ventricle, indicated normal looping of the heart tube. Immunostaining of myosin heavy chains with the ventricle-specific antibody MF20 and the atrium-specific antibody S46 showed normal development of heart chambers. At the same time, acute exposure resulted in carbaryl-induced bradycardia. Heart rate dropped significantly after a 10-min exposure to 100 μg/ml carbaryl but recovered when carbaryl was removed. The novel observation of carbaryl-induced bradycardia in 1- and 2-dpf embryos suggested that carbaryl affected cardiac function possibly through an alternative mechanism other than acetylcholinesterase inhibition such as inhibition of calcium ion channels, since acetylcholine receptors in zebrafish are not functional until 3 dpf. However, the exact nature of this mechanism is currently unknown, and thus further studies are required

  5. The behavior of larval zebrafish reveals stressor-mediated anorexia during early vertebrate development

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    De Marco, Rodrigo J.; Groneberg, Antonia H.; Yeh, Chen-Min; Treviño, Mario; Ryu, Soojin

    2014-01-01

    The relationship between stress and food consumption has been well documented in adults but less so in developing vertebrates. Here we demonstrate that an encounter with a stressor can suppress food consumption in larval zebrafish. Furthermore, we provide indication that food intake suppression cannot be accounted for by changes in locomotion, oxygen consumption and visual responses, as they remain unaffected after exposure to a potent stressor. We also show that feeding reoccurs when basal levels of cortisol (stress hormone in humans and teleosts) are re-established. The results present evidence that the onset of stress can switch off the drive for feeding very early in vertebrate development, and add a novel endpoint for analyses of metabolic and behavioral disorders in an organism suitable for high-throughput genetics and non-invasive brain imaging. PMID:25368561

  6. Deiodinase knockdown during early zebrafish development affects growth, development, energy metabolism, motility and phototransduction.

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    Enise Bagci

    Full Text Available Thyroid hormone (TH balance is essential for vertebrate development. Deiodinase type 1 (D1 and type 2 (D2 increase and deiodinase type 3 (D3 decreases local intracellular levels of T3, the most important active TH. The role of deiodinase-mediated TH effects in early vertebrate development is only partially understood. Therefore, we investigated the role of deiodinases during early development of zebrafish until 96 hours post fertilization at the level of the transcriptome (microarray, biochemistry, morphology and physiology using morpholino (MO knockdown. Knockdown of D1+D2 (D1D2MO and knockdown of D3 (D3MO both resulted in transcriptional regulation of energy metabolism and (muscle development in abdomen and tail, together with reduced growth, impaired swim bladder inflation, reduced protein content and reduced motility. The reduced growth and impaired swim bladder inflation in D1D2MO could be due to lower levels of T3 which is known to drive growth and development. The pronounced upregulation of a large number of transcripts coding for key proteins in ATP-producing pathways in D1D2MO could reflect a compensatory response to a decreased metabolic rate, also typically linked to hypothyroidism. Compared to D1D2MO, the effects were more pronounced or more frequent in D3MO, in which hyperthyroidism is expected. More specifically, increased heart rate, delayed hatching and increased carbohydrate content were observed only in D3MO. An increase of the metabolic rate, a decrease of the metabolic efficiency and a stimulation of gluconeogenesis using amino acids as substrates may have been involved in the observed reduced protein content, growth and motility in D3MO larvae. Furthermore, expression of transcripts involved in purine metabolism coupled to vision was decreased in both knockdown conditions, suggesting that both may impair vision. This study provides new insights, not only into the role of deiodinases, but also into the importance of a correct

  7. Swimming Effects on Developing Zebrafish

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    Kranenbarg, S.; Pelster, B.

    2013-01-01

    Zebrafish represent an important vertebrate model species in developmental biology. This chapter reviews the effects of exercise on the development of the musculoskeletal system, the cardiovascular system, metabolic capacities of developing zebrafish, and regulation of these processes on the gene

  8. Cloning of zebrafish Mustn1 orthologs and their expression during early development.

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    Camarata, Troy; Vasilyev, Aleksandr; Hadjiargyrou, Michael

    2016-11-15

    Mustn1 is a small nuclear protein that is involved in the development and regeneration of the musculoskeletal system. Previous work established a role for Mustn1 in myogenic and chondrogenic differentiation. In addition, recent evidence suggests a potential role for Mustn1 in cilia function in zebrafish. A detailed study of Mustn1 expression has yet to be conducted in zebrafish. As such, we report herein the cloning of the zebrafish Mustn1 orthologs, mustn1a and mustn1b, and their expression during zebrafish embryonic and larval development. Results indicate a 44% nucleotide identity between the two paralogs. Phylogenetic analysis further confirmed that the Mustn1a and 1b predicted proteins were highly related to other vertebrate members of the Mustn1 protein family. Whole mount in situ hybridization revealed expression of both mustn1a and 1b at the 7-somite stage through 72hpf in structures such as Kupffer's vesicle, segmental mesoderm, head structures, and otic vesicle. Additionally, in 5day old larva, mustn1a and 1b expression is detected in the neurocranium, otic capsule, and the gut. Although both were expressed in the neurocranium, mustn1a was localized in the hypophyseal fenestra whereas mustn1b was found near the posterior basicapsular commissure. mustn1b also displayed expression in the ceratohyal and ceratobranchial elements of the pharyngeal skeleton. These expression patterns were verified temporally by q-PCR analysis. Taken together, we conclude that Mustn1 expression is conserved in vertebrates and that the variations in expression of the two zebrafish paralogs suggest different modes of molecular regulation. Copyright © 2016 Elsevier B.V. All rights reserved.

  9. The effects of waterborne uranium on the hatching success, development, and survival of early life stages of zebrafish (Danio rerio)

    International Nuclear Information System (INIS)

    Bourrachot, Stephanie; Simon, Olivier; Gilbin, Rodolphe

    2008-01-01

    In this study, we investigated the effects of the radioactive metal uranium (U) on the embryonic development, hatching success, growth rate, and survival of juvenile zebrafish (Danio rerio). We studied the effects of depleted uranium (20-500 μg L -1 of DU), inducing mainly chemical toxicity due to its low specific activity, and the combined effects of chemical and radiological toxicity by using a higher specific activity uranium isotope (20 and 100 μg L -1 of 233 U). Results showed that early life stages are significantly affected by uranium exposure through both chemical and combined (chemical and radiological) toxicity. Experiments showed significant effects of U on hatching success starting at the concentration of 250 μg L -1 of DU, causing a 42% delay in median hatching times relative to control. Furthermore, a reduction of growth (decrease in body length and weight) was observed followed by a high mortality of pro-larvae stage (up to 100% at DU concentrations of 250 μg L -1 upon a 15 day exposure). Bioaccumulation measurements highlighted that U was mainly localised in the chorion but penetrated in the embryo inside eggs at a higher concentration. The effects differed depending on the isotopic composition of the uranium: sublethal defects in the tail detachment process were more pronounced for 233 U than DU exposure, while the presence of 233 U specifically affected embryo development and led to higher mortality rates of the prolarvae. The results from this study showed that the early life stages of zebrafish seems to be more sensitive to uranium contamination than more mature stages, and underline the importance of including pro-larval stages into toxicity tests in order to improve the relevancy for environmental risk assessments

  10. The effect of excess expression of GFP in a novel heart-specific green fluorescence zebrafish regulated by nppa enhancer at early embryonic development.

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    Huang, Wen; Deng, Yun; Dong, Wei; Yuan, Wuzhou; Wan, Yongqi; Mo, Xiaoyan; Li, Yongqing; Wang, Zequn; Wang, Yuequn; Ocorr, Karen; Zhang, Bo; Lin, Shuo; Wu, Xiushan

    2011-02-01

    In order to study the impalpable effect of GFP in homozygous heart-specific GFP-positive zebrafish during the early stage, the researchers analyzed the heart function of morphology and physiology at the first 3 days after fertilization. This zebrafish line was produced by a large-scale Tol2 transposon mediated enhancer trap screen that generated a transgenic zebrafish with a heart-specific expression of green fluorescent protein (GFP)-tagged under control of the nppa enhancer. In situ hybridization experiments showed that the nppa:GFP line faithfully recapitulated both the spatial and temporal expressions of the endogenous nppa. Green fluorescence was intensively and specifically expressed in the myocardial cells located both in the heart chambers and in the atrioventricular canal. The embryonic heart of nppa:GFP line developed normally compared with those in the wild type. There was no difference between the nappa:GFP and wild type lines with respect to heart rate, overall size, ejection volume, and fractional shortening. Thus the excess expression of GFP in this transgenic line seemed to exert no detrimental effects on zebrafish hearts during the early stages.

  11. Expression pattern of cdkl5 during zebrafish early development: implications for use as model for atypical Rett syndrome.

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    Vitorino, Marta; Cunha, Nídia; Conceição, Natércia; Cancela, M Leonor

    2018-05-11

    Atypical Rett syndrome is a child neurodevelopmental disorder induced by mutations in CDKL5 gene and characterized by a progressive regression in development with loss of purposeful use of the hands, slowed brain and head growth, problems with walking, seizures, and intellectual disability. At the moment, there is no cure for this pathology and little information is available concerning animal models capable of mimicking its phenotypes, thus the development of additional animal models should be of interest to gain more knowledge about the disease. Zebrafish has been used successfully as model organism for many human genetic diseases; however, no information is available concerning the spatial and temporal expression of cdkl5 orthologous in this organism. In the present study, we identified the developmental expression patterns of cdkl5 in zebrafish by quantitative PCR and whole-mount in situ hybridization. cdkl5 is expressed maternally at low levels during the first 24 h of development. After that the expression of the gene increases significantly and it starts to be expressed mainly in the nervous system and in several brain structures, such as telencephalon, mesencephalon and diencephalon. The expression patterns of cdkl5 in zebrafish is in accordance with the tissues known to be affected in humans and associated to symptoms and deficits observed in Rett syndrome patients thus providing the first evidence that zebrafish could be an alternative model to study the molecular pathways of this disease as well as to test possible therapeutic approaches capable of rescuing the phenotype.

  12. BISPHENOL A EXPOSURE DURING EARLY DEVELOPMENT INDUCES SEX-SPECIFIC CHANGES IN ADULT ZEBRAFISH SOCIAL INTERACTIONS

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    Weber, Daniel N.; Hoffmann, Raymond G.; Hoke, Elizabeth S.; Tanguay, Robert L.

    2014-01-01

    Developmental bisphenol A (BPA) exposure is associated with adverse behavioral effects, although underlying modes of action remain unclear. Because BPA is a suspected xenoestrogen, the objective was to identify sex-based changes in adult zebrafish social behavior developmentally exposed to BPA (0.0, 0.1 or 1 μM) or one of two control compounds (0.1μM 17β-estradiol [E2], and 0.1 μM GSK4716, a synthetic estrogen-related receptor γ ligand). A test chamber was divided lengthwise so each arena held one fish unable to detect the presence of the other fish. A mirror was inserted at one end of each arena; baseline activity levels were determined without mirror. Arenas were divided into 3, computer-generated zones to represent different distances from mirror image. Circadian rhythm patterns were evaluated at 1–3 (= AM) and 5–8 (= PM) hr postprandial. Adult zebrafish were placed into arenas and monitored by digital camera for 5 min. Total distance traveled, % time spent at mirror image, and number of attacks on mirror image were quantified. E2, GSK4716, and all BPA treatments dampened male activity and altered male circadian activity patterns; there was no marked effect on female activity. BPA induced non-monotonic effects (response curve changes direction within range of concentrations examined) on male % time at mirror only in AM. All treatments produced increased % time at the mirror during PM. Male attacks on the mirror were reduced by BPA exposure only during AM. There were sex-specific effects of developmental BPA on social interactions and time-of-day of observation affected results. PMID:25424546

  13. The utility of zebrafish to study the mechanisms by which ethanol affects social behavior and anxiety during early brain development.

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    Parker, Matthew O; Annan, Leonette V; Kanellopoulos, Alexandros H; Brock, Alistair J; Combe, Fraser J; Baiamonte, Matteo; Teh, Muy-Teck; Brennan, Caroline H

    2014-12-03

    Exposure to moderate levels of ethanol during brain development has a number of effects on social behavior but the molecular mechanisms that mediate this are not well understood. Gaining a better understanding of these factors may help to develop therapeutic interventions in the future. Zebrafish offer a potentially useful model in this regard. Here, we introduce a zebrafish model of moderate prenatal ethanol exposure. Embryos were exposed to 20mM ethanol for seven days (48hpf-9dpf) and tested as adults for individual social behavior and shoaling. We also tested their basal anxiety with the novel tank diving test. We found that the ethanol-exposed fish displayed reductions in social approach and shoaling, and an increase in anxiety in the novel tank test. These behavioral differences corresponded to differences in hrt1aa, slc6a4 and oxtr expression. Namely, acute ethanol caused a spike in oxtr and ht1aa mRNA expression, which was followed by down-regulation at 7dpf, and an up-regulation in slc6a4 at 72hpf. This study confirms the utility of zebrafish as a model system for studying the molecular basis of developmental ethanol exposure. Furthermore, it proposes a putative developmental mechanism characterized by ethanol-induced OT inhibition leading to suppression of 5-HT and up-regulation of 5-HT1A, which leads, in turn, to possible homeostatic up-regulation of 5-HTT at 72hpf and subsequent imbalance of the 5-HT system. Copyright © 2014 Elsevier Inc. All rights reserved.

  14. In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development.

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    Verbueken, Evy; Alsop, Derek; Saad, Moayad A; Pype, Casper; Van Peer, Els M; Casteleyn, Christophe R; Van Ginneken, Chris J; Wilson, Joanna; Van Cruchten, Steven J

    2017-01-22

    At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP)-a group of drug-metabolizing enzymes-in microsomes from whole zebrafish embryos (ZEM) of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf) by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR). The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM) to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA) as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

  15. In Vitro Biotransformation of Two Human CYP3A Probe Substrates and Their Inhibition during Early Zebrafish Development

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    Evy Verbueken

    2017-01-01

    Full Text Available At present, the zebrafish embryo is increasingly used as an alternative animal model to screen for developmental toxicity after exposure to xenobiotics. Since zebrafish embryos depend on their own drug-metabolizing capacity, knowledge of their intrinsic biotransformation is pivotal in order to correctly interpret the outcome of teratogenicity assays. Therefore, the aim of this in vitro study was to assess the activity of cytochrome P450 (CYP—a group of drug-metabolizing enzymes—in microsomes from whole zebrafish embryos (ZEM of 5, 24, 48, 72, 96 and 120 h post-fertilization (hpf by means of a mammalian CYP substrate, i.e., benzyloxy-methyl-resorufin (BOMR. The same CYP activity assays were performed in adult zebrafish liver microsomes (ZLM to serve as a reference for the embryos. In addition, activity assays with the human CYP3A4-specific Luciferin isopropyl acetal (Luciferin-IPA as well as inhibition studies with ketoconazole and CYP3cide were carried out to identify CYP activity in ZLM. In the present study, biotransformation of BOMR was detected at 72 and 96 hpf; however, metabolite formation was low compared with ZLM. Furthermore, Luciferin-IPA was not metabolized by the zebrafish. In conclusion, the capacity of intrinsic biotransformation in zebrafish embryos appears to be lacking during a major part of organogenesis.

  16. The transcriptomics of glucocorticoid receptor signaling in developing zebrafish.

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    Dinushan Nesan

    Full Text Available Cortisol is the primary corticosteroid in teleosts that is released in response to stressor activation of the hypothalamus-pituitary-interrenal axis. The target tissue action of this hormone is primarily mediated by the intracellular glucocorticoid receptor (GR, a ligand-bound transcription factor. In developing zebrafish (Danio rerio embryos, GR transcripts and cortisol are maternally deposited into the oocyte prior to fertilization and influence early embryogenesis. To better understand of the molecular mechanisms involved, we investigated changes in the developmental transcriptome prior to hatch, in response to morpholino oligonucleotide knockdown of GR using the Agilent zebrafish microarray platform. A total of 1313 and 836 mRNA transcripts were significantly changed at 24 and 36 hours post fertilization (hpf, respectively. Functional analysis revealed numerous developmental processes under GR regulation, including neurogenesis, eye development, skeletal and cardiac muscle formation. Together, this study underscores a critical role for glucocorticoid signaling in programming molecular events essential for zebrafish development.

  17. Molecular genetics of pituitary development in zebrafish.

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    Pogoda, Hans-Martin; Hammerschmidt, Matthias

    2007-08-01

    The pituitary gland of vertebrates consists of two major parts, the neurohypophysis (NH) and the adenohypophysis (AH). As a central part of the hypothalamo-hypophyseal system (HHS), it constitutes a functional link between the nervous and the endocrine system to regulate basic body functions, such as growth, metabolism and reproduction. The development of the AH has been intensively studied in mouse, serving as a model for organogenesis and differential cell specification. However, given that the AH is a relatively recent evolutionary advance of the chordate phylum, it is also interesting to understand its development in lower chordate systems. In recent years, the zebrafish has emerged as a powerful lower vertebrate system for developmental studies, being amenable for large-scale genetic approaches, embryological manipulations, and in vivo imaging. Here, we present an overview of current knowledge of the mechanisms and genetic control of pituitary formation during zebrafish development. First, we describe the components of the zebrafish HHS, and the different pituitary cell types and hormones, followed by a description of the different steps of normal pituitary development. The central part of the review deals with the genes found to be essential for zebrafish AH development, accompanied by a description of the corresponding mutant phenotypes. Finally, we discuss future directions, with particular focus on evolutionary aspects, and some novel functional aspects with growing medical and social relevance.

  18. The toxicity of chlorpyrifos on the early life stage of zebrafish: a survey on the endpoints at development, locomotor behavior, oxidative stress and immunotoxicity.

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    Jin, Yuanxiang; Liu, Zhenzhen; Peng, Tao; Fu, Zhengwei

    2015-04-01

    Chlorpyrifos (CPF) is one of the most toxic pesticides in aquatic ecosystem, but its toxicity mechanisms to fish are still not fully understood. This study examined the toxicity targets of CPF in early life stage of zebrafish on the endpoints at developmental toxicity, neurotoxicity, oxidative stress and immunotoxicity. Firstly, CPF exposure decreased the body length, inhibited the hatchability and heart rate, and resulted in a number of morphological abnormalities, primarily spinal deformities (SD) and pericardial edema (PE), in larval zebrafish. Secondly, the free swimming activities and the swimming behaviors of the larvae in response to the stimulation of light-to-dark photoperiod transition were significantly influenced by the exposure to 100 and 300 μg/L CPF. In addition, the activity of acetylcholinesterase (AChE) and the transcription of some genes related to neurotoxicity were also influenced by CPF exposure. Thirdly, CPF exposure induced oxidative stress in the larval zebrafish. The malondialdehyde (MDA) levels increased and the glutathione (GSH) contents decreased significantly in a concentration-dependent manner after the exposure to CPF for 96 hours post fertilization (hpf). CPF affected not only the activities of superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPX) and glutathione S-transferase (GST), but also the transcriptional levels of their respective genes. Finally, the mRNA levels of the main cytokines including tumor necrosis factor α (Tnfα), interferon (Ifn), interleukin-1 beta (Il-1β), interleukin 6 (Il6), complement factor 4 (C4) in the larvae increased significantly after the exposure to 100 or 300 μg/L CPF for 96 hpf, suggesting that the innate immune system disturbed by CPF in larvae. Taken together, our results suggested that CPF had the potential to cause developmental toxicity, behavior alterations, oxidative stress and immunotoxicity in the larval zebrafish. Copyright © 2015 Elsevier Ltd. All rights

  19. Assessment of cardiotoxicity and effects of malathion on the early development of zebrafish (Danio rerio) using computer vision for heart rate quantification.

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    Simoneschi, Daniele; Simoneschi, Francesco; Todd, Nancy E

    2014-06-01

    Malathion, a common organophosphate insecticide, is a proven acetylcholinesterase inhibitor and is the most applied organophosphate insecticide in the United States. The use of zebrafish as a model to study the effects of pesticides on development is an innovative approach yielding relevant implications for determining the potential toxic effects of these pesticides on humans. In this study, a simple noninvasive technique was developed to investigate the cardiotoxicity of malathion on Danio rerio embryos, and to detect and quantify its effect on heart rate. Videos were recorded under a stereomicroscope and examined with our custom-made software (FishBeat) to determine the heart rate of the embryos. The pixel average intensity frequency (PI) of the videos was computed at its maximum probability to indicate the average number of heartbeats per second. Experimental observations successfully demonstrated that this method was able to detect the heart rate of zebrafish embryos as compared with manual stopwatch counting, with no significant difference. Embryos were treated acutely with increasing malathion concentrations (33.3 and 50 μg/mL malathion) at 52, 76, and 96 hpf. Embryos treated with 33.3 μg/mL malathion had significant bradycardia at 52 and 76 hpf, whereas embryos treated with 50 μg/mL malathion presented bradycardia at all hpf. These novel observations confirmed that malathion, acting as an acetylcholinesterase inhibitor, induced heartbeat irregularity in zebrafish embryos.

  20. Analysis of Lethality and Malformations During Zebrafish (Danio rerio) Development.

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    Raghunath, Azhwar; Perumal, Ekambaram

    2018-01-01

    The versatility offered by zebrafish (Danio rerio) makes it a powerful and an attractive vertebrate model in developmental toxicity and teratogenicity assays. Apart from the newly introduced chemicals as drugs, xenobiotics also induce abnormal developmental abnormalities and congenital malformations in living organisms. Over the recent decades, zebrafish embryo/larva has emerged as a potential tool to test teratogenicity potential of these chemicals. Zebrafish responds to compounds as mammals do as they share similarities in their development, metabolism, physiology, and signaling pathways with that of mammals. The methodology used by the different scientists varies enormously in the zebrafish embryotoxicity test. In this chapter, we present methods to assess lethality and malformations during zebrafish development. We propose two major malformations scoring systems: binomial and relative morphological scoring systems to assess the malformations in zebrafish embryos/larvae. Based on the scoring of the malformations, the test compound can be classified as a teratogen or a nonteratogen and its teratogenic potential is evaluated.

  1. Identification of estrogen target genes during zebrafish embryonic development through transcriptomic analysis.

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    Ruixin Hao

    Full Text Available Estrogen signaling is important for vertebrate embryonic development. Here we have used zebrafish (Danio rerio as a vertebrate model to analyze estrogen signaling during development. Zebrafish embryos were exposed to 1 µM 17β-estradiol (E2 or vehicle from 3 hours to 4 days post fertilization (dpf, harvested at 1, 2, 3 and 4 dpf, and subjected to RNA extraction for transcriptome analysis using microarrays. Differentially expressed genes by E2-treatment were analyzed with hierarchical clustering followed by biological process and tissue enrichment analysis. Markedly distinct sets of genes were up and down-regulated by E2 at the four different time points. Among these genes, only the well-known estrogenic marker vtg1 was co-regulated at all time points. Despite this, the biological functional categories targeted by E2 were relatively similar throughout zebrafish development. According to knowledge-based tissue enrichment, estrogen responsive genes were clustered mainly in the liver, pancreas and brain. This was in line with the developmental dynamics of estrogen-target tissues that were visualized using transgenic zebrafish containing estrogen responsive elements driving the expression of GFP (Tg(5xERE:GFP. Finally, the identified embryonic estrogen-responsive genes were compared to already published estrogen-responsive genes identified in male adult zebrafish (Gene Expression Omnibus database. The expressions of a few genes were co-regulated by E2 in both embryonic and adult zebrafish. These could potentially be used as estrogenic biomarkers for exposure to estrogens or estrogenic endocrine disruptors in zebrafish. In conclusion, our data suggests that estrogen effects on early embryonic zebrafish development are stage- and tissue- specific.

  2. Metabolomics and transcriptomics reveal the toxicity of difenoconazole to the early life stages of zebrafish (Danio rerio).

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    Teng, Miaomiao; Zhu, Wentao; Wang, Dezhen; Qi, Suzhen; Wang, Yao; Yan, Jin; Dong, Kai; Zheng, Mingqi; Wang, Chengju

    2018-01-01

    Difenoconazole is widely used to inhibit the growth of fungi, but its residue in the water environment may threaten ecosystem and human health. Here, 1 H nuclear magnetic resonance (NMR) and LC-MS/MS based metabolomics and transcriptomics approaches were used to assess the response of zebrafish to difenoconazole exposure. Early life stages of zebrafish were exposed to difenoconazole at environmentally relevant concentrations for 168h. Their comparison with the control group suggested an adverse development and disturbance of steroid hormones and VTG. KEGG pathway analysis identified five biological processes on the basis of differentially expressed genes (DEGs), as well as altered metabolites and amino acids in zebrafish following difenoconazole exposure. These affected processes included energy metabolism, amino acids metabolism, lipid metabolism, nucleotide metabolism, and an immune-related pathway. Collectively, these results bring us closer to an incremental understanding of the toxic effects of difenoconazole on zebrafish in its early development, and lend support to the continued use of the early life stages of zebrafish as a classical model to evaluate underlying environmental risks of xenobiotics in aquatic organisms. Copyright © 2017 Elsevier B.V. All rights reserved.

  3. Expression of voltage-activated calcium channels in the early zebrafish embryo.

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    Sanhueza, Dayán; Montoya, Andro; Sierralta, Jimena; Kukuljan, Manuel

    2009-05-01

    Increases in cytosolic calcium concentrations regulate many cellular processes, including aspects of early development. Calcium release from intracellular stores and calcium entry through non-voltage-gated channels account for signalling in non-excitable cells, whereas voltage-gated calcium channels (CaV) are important in excitable cells. We report the expression of multiple transcripts of CaV, identified by its homology to other species, in the early embryo of the zebrafish, Danio rerio, at stages prior to the differentiation of excitable cells. CaV mRNAs and proteins were detected as early as the 2-cell stages, which indicate that they arise from both maternal and zygotic transcription. Exposure of embryos to pharmacological blockers of CaV does not perturb early development significantly, although late effects are appreciable. These results suggest that CaV may have a role in calcium homeostasis and control of cellular process during early embryonic development.

  4. Transcriptome analysis of severe hypoxic stress during development in zebrafish

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    I.G. Woods

    2015-12-01

    Full Text Available Hypoxia causes critical cellular injury both in early human development and in adulthood, leading to cerebral palsy, stroke, and myocardial infarction. Interestingly, a remarkable phenomenon known as hypoxic preconditioning arises when a brief hypoxia exposure protects target organs against subsequent, severe hypoxia. Although hypoxic preconditioning has been demonstrated in several model organisms and tissues including the heart and brain, its molecular mechanisms remain poorly understood. Accordingly, we used embryonic and larval zebrafish to develop a novel vertebrate model for hypoxic preconditioning, and used this model to identify conserved hypoxia-regulated transcripts for further functional study as published in Manchenkov et al. (2015 in G3: Genes|Genomes|Genetics. In this Brief article, we provide extensive annotation for the most strongly hypoxia-regulated genes in zebrafish, including their human orthologs, and describe in detail the methods used to identify, filter, and annotate hypoxia-regulated transcripts for downstream functional and bioinformatic assays using the source data provided in Gene Expression Omnibus Accession GSE68473.

  5. Cadmium affects retinogenesis during zebrafish embryonic development

    International Nuclear Information System (INIS)

    Hen Chow, Elly Suk; Yu Hui, Michelle Nga; Cheng, Chi Wa; Cheng, Shuk Han

    2009-01-01

    Ocular malformations are commonly observed in embryos of aquatic species after exposure to toxicants. Using zebrafish embryos as the model organism, we showed that cadmium exposure from sphere stage (4 hpf) to end of segmentation stage (24 hpf) induced microphthalmia in cadmium-treated embryos. Embryos with eye defects were then assessed for visual abilities. Cadmium-exposed embryos were behaviorally blind, showing hyperpigmentation and loss of camouflage response to light. We investigated the cellular basis of the formation of the small eyes phenotype and the induction of blindness by studying retina development and retinotectal projections. Retinal progenitors were found in cadmium-treated embryos albeit in smaller numbers. The number of retinal ganglion cells (RGC), the first class of retinal cells to differentiate during retinogenesis, was reduced, while photoreceptor cells, the last batch of retinal neurons to differentiate, were absent. Cadmium also affected the propagation of neurons in neurogenic waves. The neurons remained in the ventronasal area and failed to spread across the retina. Drastically reduced RGC axons and disrupted optic stalk showed that the optic nerves did not extend from the retina beyond the chiasm into the tectum. Our data suggested that impairment in neuronal differentiation of the retina, disruption in RGC axon formation and absence of cone photoreceptors were the causes of microphthalmia and visual impairment in cadmium-treated embryos

  6. Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

    Science.gov (United States)

    Lei, Yong; Xiao, Qi; Huang, Shan; Xu, Wansu; Zhang, Zhe; He, Zhike; Liu, Yi; Deng, Fengjiao

    2011-12-01

    Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

  7. Impact of CdSe/ZnS quantum dots on the development of zebrafish embryos

    International Nuclear Information System (INIS)

    Lei Yong; Xiao Qi; Huang Shan; Xu Wansu; Zhang Zhe; He Zhike; Liu Yi; Den, Fengjiao

    2011-01-01

    Due to their unique fluorescent characteristics, quantum dots (QDs) have been successfully applied in the fields of biotechnology and medicine, but there is very limited information regarding their biodistribution and chronic toxicity in vivo. In this article, the biological behavior and toxic effects of mercaptoacetic acid-CdSe/ZnS QDs (MAA-QDs) in developing zebrafish embryos were investigated by in vivo tests. The MAA-QDs were introduced into zebrafish through microinjection at early stage. The results showed that the MAA-QDs at certain concentrations influenced the survival of zebrafish embryos, but treated embryos without developmental defects were also observed. MAA-QDs injected into the cytoplasm at the one-cell stage were allocated to progeny blastoderm cells during proliferation and almost never entered the yolk. The formation of notochord and primordial germ cells with normal morphologies was detected in the treated embryos by whole-mount in situ hybridization. Furthermore, traces of the element cadmium were mainly discovered in the tissue of liver and kidney of 3-month-old-treated zebrafish by quantitative assessment with inductively coupled plasma mass spectrometry. Thus, we hypothesized that low concentration MAA-QDs have chronic toxicities when they were delivered into zebrafish organs.

  8. Chondroitin / dermatan sulfate modification enzymes in zebrafish development.

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    Judith Habicher

    Full Text Available Chondroitin/dermatan sulfate (CS/DS proteoglycans consist of unbranched sulfated polysaccharide chains of repeating GalNAc-GlcA/IdoA disaccharide units, attached to serine residues on specific proteins. The CS/DS proteoglycans are abundant in the extracellular matrix where they have essential functions in tissue development and homeostasis. In this report a phylogenetic analysis of vertebrate genes coding for the enzymes that modify CS/DS is presented. We identify single orthologous genes in the zebrafish genome for the sulfotransferases chst7, chst11, chst13, chst14, chst15 and ust and the epimerase dse. In contrast, two copies were found for mammalian sulfotransferases CHST3 and CHST12 and the epimerase DSEL, named chst3a and chst3b, chst12a and chst12b, dsela and dselb, respectively. Expression of CS/DS modification enzymes is spatially and temporally regulated with a large variation between different genes. We found that CS/DS 4-O-sulfotransferases and 6-O-sulfotransferases as well as CS/DS epimerases show a strong and partly overlapping expression, whereas the expression is restricted for enzymes with ability to synthesize di-sulfated disaccharides. A structural analysis further showed that CS/DS sulfation increases during embryonic development mainly due to synthesis of 4-O-sulfated GalNAc while the proportion of 6-O-sulfated GalNAc increases in later developmental stages. Di-sulfated GalNAc synthesized by Chst15 and 2-O-sulfated GlcA/IdoA synthesized by Ust are rare, in accordance with the restricted expression of these enzymes. We also compared CS/DS composition with that of heparan sulfate (HS. Notably, CS/DS biosynthesis in early zebrafish development is more dynamic than HS biosynthesis. Furthermore, HS contains disaccharides with more than one sulfate group, which are virtually absent in CS/DS.

  9. Zebrafish foxo3b negatively regulates canonical Wnt signaling to affect early embryogenesis.

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    Xun-wei Xie

    Full Text Available FOXO genes are involved in many aspects of development and vascular homeostasis by regulating cell apoptosis, proliferation, and the control of oxidative stress. In addition, FOXO genes have been showed to inhibit Wnt/β-catenin signaling by competing with T cell factor to bind to β-catenin. However, how important of this inhibition in vivo, particularly in embryogenesis is still unknown. To demonstrate the roles of FOXO genes in embryogenesis will help us to further understand their relevant physiological functions. Zebrafish foxo3b gene, an orthologue of mammalian FOXO3, was expressed maternally and distributed ubiquitously during early embryogenesis and later restricted to brain. After morpholino-mediated knockdown of foxo3b, the zebrafish embryos exhibited defects in axis and neuroectoderm formation, suggesting its critical role in early embryogenesis. The embryo-developmental marker gene staining at different stages, phenotype analysis and rescue assays revealed that foxo3b acted its role through negatively regulating both maternal and zygotic Wnt/β-catenin signaling. Moreover, we found that foxo3b could interact with zebrafish β-catenin1 and β-catenin2 to suppress their transactivation in vitro and in vivo, further confirming its role relevant to the inhibition of Wnt/β-catenin signaling. Taken together, we revealed that foxo3b played a very important role in embryogenesis and negatively regulated maternal and zygotic Wnt/β-catenin signaling by directly interacting with both β-catenin1 and β-catenin2. Our studies provide an in vivo model for illustrating function of FOXO transcription factors in embryogenesis.

  10. Cadmium inhibits neurogenesis in zebrafish embryonic brain development

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    Chow, Elly Suk Hen [Division of Biology, California Institute of Technology, 1200 California Boulevard, Pasadena, CA 91125 (United States); Hui, Michelle Nga Yu; Lin Chunchi [Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong (China); Cheng Shukhan [Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong (China)], E-mail: bhcheng@cityu.edu.hk

    2008-05-01

    Cadmium is a non-essential heavy metal found abundantly in the environment. Children of women exposed to cadmium during pregnancy display lower motor and perceptual abilities. High cadmium body burden in children is also related to impaired intelligence and lowered school achievement. However, little is known about the molecular and cellular basis of developmental neurotoxicity in the sensitive early life stages of animals. In this study, we explore neurological deficits caused by cadmium during early embryonic stages in zebrafish by examining regionalization of the neural tube, pattern formation and cell fate determination, commitment of proneural genes and induction of neurogenesis. We show that cadmium-treated embryos developed a smaller head with unclear boundaries between the brain subdivisions, particularly in the mid-hindbrain region. Embryos display normal anterior to posterior regionalization; however, the commitment of neural progenitor cells was affected by cadmium. We observe prominent reductions in the expression of several proneuronal genes including ngn1 in cell clusters, zash1a in the developing optic tectum, and zash1b in the telencephalon and tectum. Cadmium-treated embryos also have fewer differentiated neurons and glia in the facial sensory ganglia as indicated by decreased zn-12 expression. Also, a lower transcription level of neurogenic genes, ngn1 and neuroD, is observed in neurons. Our data suggest that cadmium-induced neurotoxicity can be caused by impaired neurogenesis, resulting in markedly reduced neuronal differentiation and axonogenesis.

  11. Cadmium inhibits neurogenesis in zebrafish embryonic brain development

    International Nuclear Information System (INIS)

    Chow, Elly Suk Hen; Hui, Michelle Nga Yu; Lin Chunchi; Cheng Shukhan

    2008-01-01

    Cadmium is a non-essential heavy metal found abundantly in the environment. Children of women exposed to cadmium during pregnancy display lower motor and perceptual abilities. High cadmium body burden in children is also related to impaired intelligence and lowered school achievement. However, little is known about the molecular and cellular basis of developmental neurotoxicity in the sensitive early life stages of animals. In this study, we explore neurological deficits caused by cadmium during early embryonic stages in zebrafish by examining regionalization of the neural tube, pattern formation and cell fate determination, commitment of proneural genes and induction of neurogenesis. We show that cadmium-treated embryos developed a smaller head with unclear boundaries between the brain subdivisions, particularly in the mid-hindbrain region. Embryos display normal anterior to posterior regionalization; however, the commitment of neural progenitor cells was affected by cadmium. We observe prominent reductions in the expression of several proneuronal genes including ngn1 in cell clusters, zash1a in the developing optic tectum, and zash1b in the telencephalon and tectum. Cadmium-treated embryos also have fewer differentiated neurons and glia in the facial sensory ganglia as indicated by decreased zn-12 expression. Also, a lower transcription level of neurogenic genes, ngn1 and neuroD, is observed in neurons. Our data suggest that cadmium-induced neurotoxicity can be caused by impaired neurogenesis, resulting in markedly reduced neuronal differentiation and axonogenesis

  12. Inhibition of signaling between human CXCR4 and zebrafish ligands by the small molecule IT1t impairs the formation of triple-negative breast cancer early metastases in a zebrafish xenograft model

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    Claudia Tulotta

    2016-02-01

    Full Text Available Triple-negative breast cancer (TNBC is a highly aggressive and recurrent type of breast carcinoma that is associated with poor patient prognosis. Because of the limited efficacy of current treatments, new therapeutic strategies need to be developed. The CXCR4-CXCL12 chemokine signaling axis guides cell migration in physiological and pathological processes, including breast cancer metastasis. Although targeted therapies to inhibit the CXCR4-CXCL12 axis are under clinical experimentation, still no effective therapeutic approaches have been established to block CXCR4 in TNBC. To unravel the role of the CXCR4-CXCL12 axis in the formation of TNBC early metastases, we used the zebrafish xenograft model. Importantly, we demonstrate that cross-communication between the zebrafish and human ligands and receptors takes place and human tumor cells expressing CXCR4 initiate early metastatic events by sensing zebrafish cognate ligands at the metastatic site. Taking advantage of the conserved intercommunication between human tumor cells and the zebrafish host, we blocked TNBC early metastatic events by chemical and genetic inhibition of CXCR4 signaling. We used IT1t, a potent CXCR4 antagonist, and show for the first time its promising anti-tumor effects. In conclusion, we confirm the validity of the zebrafish as a xenotransplantation model and propose a pharmacological approach to target CXCR4 in TNBC.

  13. Kita driven expression of oncogenic HRAS leads to early onset and highly penetrant melanoma in zebrafish.

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    Cristina Santoriello

    2010-12-01

    Full Text Available Melanoma is the most aggressive and lethal form of skin cancer. Because of the increasing incidence and high lethality of melanoma, animal models for continuously observing melanoma formation and progression as well as for testing pharmacological agents are needed.Using the combinatorial Gal4-UAS system, we have developed a zebrafish transgenic line that expresses oncogenic HRAS under the kita promoter. Already at 3 days transgenic kita-GFP-RAS larvae show a hyper-pigmentation phenotype as earliest evidence of abnormal melanocyte growth. By 2-4 weeks, masses of transformed melanocytes form in the tail stalk of the majority of kita-GFP-RAS transgenic fish. The adult tumors evident between 1-3 months of age faithfully reproduce the immunological, histological and molecular phenotypes of human melanoma, but on a condensed time-line. Furthermore, they show transplantability, dependence on mitfa expression and do not require additional mutations in tumor suppressors. In contrast to kita expressing melanocyte progenitors that efficiently develop melanoma, mitfa expressing progenitors in a second Gal4-driver line were 4 times less efficient in developing melanoma during the three months observation period.This indicates that zebrafish kita promoter is a powerful tool for driving oncogene expression in the right cells and at the right level to induce early onset melanoma in the presence of tumor suppressors. Thus our zebrafish model provides a link between kita expressing melanocyte progenitors and melanoma and offers the advantage of a larval phenotype suitable for large scale drug and genetic modifier screens.

  14. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    LENUS (Irish Health Repository)

    Sapetto-Rebow, Beata

    2011-11-23

    Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A) are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm), a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization). Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT) and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

  15. Maternal topoisomerase II alpha, not topoisomerase II beta, enables embryonic development of zebrafish top2a-/- mutants

    Directory of Open Access Journals (Sweden)

    Sapetto-Rebow Beata

    2011-11-01

    Full Text Available Abstract Background Genetic alterations in human topoisomerase II alpha (TOP2A are linked to cancer susceptibility. TOP2A decatenates chromosomes and thus is necessary for multiple aspects of cell division including DNA replication, chromosome condensation and segregation. Topoisomerase II alpha is also required for embryonic development in mammals, as mouse Top2a knockouts result in embryonic lethality as early as the 4-8 cell stage. The purpose of this study was to determine whether the extended developmental capability of zebrafish top2a mutants arises from maternal expression of top2a or compensation from its top2b paralogue. Results Here, we describe bloody minded (blm, a novel mutant of zebrafish top2a. In contrast to mouse Top2a nulls, zebrafish top2a mutants survive to larval stages (4-5 day post fertilization. Developmental analyses demonstrate abundant expression of maternal top2a but not top2b. Inhibition or poisoning of maternal topoisomerase II delays embryonic development by extending the cell cycle M-phase. Zygotic top2a and top2b are co-expressed in the zebrafish CNS, but endogenous or ectopic top2b RNA appear unable to prevent the blm phenotype. Conclusions We conclude that maternal top2a enables zebrafish development before the mid-zygotic transition (MZT and that zebrafish top2a and top2b are not functionally redundant during development after activation of the zygotic genome.

  16. Egfl6 is involved in zebrafish notochord development.

    Science.gov (United States)

    Wang, Xueqian; Wang, Xin; Yuan, Wei; Chai, Renjie; Liu, Dong

    2015-08-01

    The epidermal growth factor (EGF) repeat motif defines a superfamily of diverse protein involved in regulating a variety of cellular and physiological processes, such as cell cycle, cell adhesion, proliferation, migration, and neural development. Egfl6, an EGF protein, also named MAGE was first cloned in human tissue. Up to date, the study of zebrafish Egfl6 expression pattern and functional analysis of Egfl6 involved in embryonic development of vertebrate in vivo is thus far lacking. Here we reported that Egfl6 was involved in zebrafish notochord development. It was shown that Egfl6 mRNA was expressed in zebrafish, developing somites, fin epidermis, pharyngeal arches, and hindbrain region. Particularly the secreted Egfl6 protein was significantly accumulated in notochord. Loss of Egfl6 function in zebrafish embryos resulted in curved body with distorted notochord in the posterior trunk. It was observed that expression of all Notch ligand and receptors in notochord of 28 hpf Egfl6 morphants was not affected, except notch2, which was up-regulated. We found that inhibition of Notch signaling by DAPT efficiently rescued notochord developmental defect of Egfl6 deficiency embryos.

  17. The dlx5a/dlx6a genes play essential roles in the early development of zebrafish median fin and pectoral structures.

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    Eglantine Heude

    Full Text Available The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution.

  18. The dlx5a/dlx6a Genes Play Essential Roles in the Early Development of Zebrafish Median Fin and Pectoral Structures

    Science.gov (United States)

    Heude, Églantine; Shaikho, Sarah; Ekker, Marc

    2014-01-01

    The Dlx5 and Dlx6 genes encode homeodomain transcription factors essential for the proper development of limbs in mammalian species. However, the role of their teleost counterparts in fin development has received little attention. Here, we show that dlx5a is an early marker of apical ectodermal cells of the pectoral fin buds and of the median fin fold, but also of cleithrum precursor cells during pectoral girdle development. We propose that early median fin fold establishment results from the medial convergence of dlx5a-expressing cells at the lateral edges of the neural keel. Expression analysis also shows involvement of dlx5a during appendage skeletogenesis. Using morpholino-mediated knock down, we demonstrate that disrupted dlx5a/6a function results in pectoral fin agenesis associated with misexpression of bmp4, fgf8a, and1 and msx genes. In contrast, the median fin fold presents defects in mesenchymal cell migration and actinotrichia formation, whereas the initial specification seems to occur normally. Our results demonstrate that the dlx5a/6a genes are essential for the induction of pectoral fin outgrowth, but are not required during median fin fold specification. The dlx5a/6a knock down also causes a failure of cleithrum formation associated with a drastic loss of runx2b and col10a1 expression. The data indicate distinct requirements for dlx5a/6a during median and pectoral fin development suggesting that initiation of unpaired and paired fin formation are not directed through the same molecular mechanisms. Our results refocus arguments on the mechanistic basis of paired appendage genesis during vertebrate evolution. PMID:24858471

  19. Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

    Science.gov (United States)

    Beck, Aaron P; Watt, Roland M; Bonner, Jennifer

    2014-02-28

    The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

  20. Control over the morphology and segregation of Zebrafish germ cell granules during embryonic development

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    Nakkrasae La-Iad

    2008-05-01

    Full Text Available Abstract Background Zebrafish germ cells contain granular-like structures, organized around the cell nucleus. These structures share common features with polar granules in Drosophila, germinal granules in Xenopus and chromatoid bodies in mice germ cells, such as the localization of the zebrafish Vasa, Piwi and Nanos proteins, among others. Little is known about the structure of these granules as well as their segregation in mitosis during early germ-cell development. Results Using transgenic fish expressing a fluorescently labeled novel component of Zebrafish germ cell granules termed Granulito, we followed the morphology and distribution of the granules. We show that whereas these granules initially exhibit a wide size variation, by the end of the first day of development they become a homogeneous population of medium size granules. We investigated this resizing event and demonstrated the role of microtubules and the minus-end microtubule dependent motor protein Dynein in the process. Last, we show that the function of the germ cell granule resident protein the Tudor domain containing protein-7 (Tdrd7 is required for determination of granule morphology and number. Conclusion Our results suggest that Zebrafish germ cell granules undergo a transformation process, which involves germ cell specific proteins as well as the microtubular network.

  1. The microcephaly gene aspm is involved in brain development in zebrafish

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    Kim, Hyun-Taek; Lee, Mi-Sun; Choi, Jung-Hwa [Department of Biology and GRAST, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Jung, Ju-Yeon [Department of Biotechnology, Konkuk University, Chungju 380-701 (Korea, Republic of); Ahn, Dae-Gwon [Department of Biology and GRAST, Chungnam National University, Daejeon 305-764 (Korea, Republic of); Yeo, Sang-Yeob [Department of Biotechnology, Division of Applied Chemistry and Biotechnology, Hanbat National University, Daejeon 305-719 (Korea, Republic of); Choi, Dong-Kug, E-mail: choidk@kku.ac.kr [Department of Biotechnology, Konkuk University, Chungju 380-701 (Korea, Republic of); Kim, Cheol-Hee, E-mail: zebrakim@cnu.ac.kr [Department of Biology and GRAST, Chungnam National University, Daejeon 305-764 (Korea, Republic of)

    2011-06-17

    Highlights: {yields} We identified a zebrafish aspm/mcph5 gene that is expressed in proliferating cells in the CNS during early development. {yields} Embryos injected with the aspm MO consistently showed a reduced head and eye size but were otherwise grossly normal, closely mimicking the known phenotypes of human microcephaly patients. {yields} Knock-down of aspm causes cell cycle arrest and apoptotic cell death during early development. -- Abstract: MCPH is a neurodevelopmental disorder characterized by a global reduction in cerebral cortical volume. Homozygous mutation of the MCPH5 gene, also known as ASPM, is the most common cause of the MCPH phenotype. To elucidate the roles of ASPM during embryonic development, the zebrafish aspm was identified, which is specifically expressed in proliferating cells in the CNS. Morpholino-mediated knock-down of aspm resulted in a significant reduction in head size. Furthermore, aspm-deficient embryos exhibited a mitotic arrest during early development. These findings suggest that the reduction in brain size in MCPH might be caused by lack of aspm function in the mitotic cell cycle and demonstrate that the zebrafish can provide a model system for congenital diseases of the human nervous system.

  2. The microcephaly gene aspm is involved in brain development in zebrafish

    International Nuclear Information System (INIS)

    Kim, Hyun-Taek; Lee, Mi-Sun; Choi, Jung-Hwa; Jung, Ju-Yeon; Ahn, Dae-Gwon; Yeo, Sang-Yeob; Choi, Dong-Kug; Kim, Cheol-Hee

    2011-01-01

    Highlights: → We identified a zebrafish aspm/mcph5 gene that is expressed in proliferating cells in the CNS during early development. → Embryos injected with the aspm MO consistently showed a reduced head and eye size but were otherwise grossly normal, closely mimicking the known phenotypes of human microcephaly patients. → Knock-down of aspm causes cell cycle arrest and apoptotic cell death during early development. -- Abstract: MCPH is a neurodevelopmental disorder characterized by a global reduction in cerebral cortical volume. Homozygous mutation of the MCPH5 gene, also known as ASPM, is the most common cause of the MCPH phenotype. To elucidate the roles of ASPM during embryonic development, the zebrafish aspm was identified, which is specifically expressed in proliferating cells in the CNS. Morpholino-mediated knock-down of aspm resulted in a significant reduction in head size. Furthermore, aspm-deficient embryos exhibited a mitotic arrest during early development. These findings suggest that the reduction in brain size in MCPH might be caused by lack of aspm function in the mitotic cell cycle and demonstrate that the zebrafish can provide a model system for congenital diseases of the human nervous system.

  3. Redundant roles of PRDM family members in zebrafish craniofacial development.

    Science.gov (United States)

    Ding, Hai-Lei; Clouthier, David E; Artinger, Kristin B

    2013-01-01

    PRDM proteins are evolutionary conserved Zn-Finger transcription factors that share a characteristic protein domain organization. Previous studies have shown that prdm1a is required for the specification and differentiation of neural crest cells in the zebrafish. Here we examine other members of this family, specifically prdm3, 5, and 16, in the differentiation of the zebrafish craniofacial skeleton. prdm3 and prdm16 are strongly expressed in the pharyngeal arches, while prdm5 is expressed specifically in the area of the forming neurocranium. Knockdown of prdm3 and prdm16 results in a reduction in the neural crest markers dlx2a and barx1 and defects in both the viscerocranium and the neurocranium. The knockdown of prdm3 and prdm16 in combination is additive in the neurocranium, but not in the viscerocranium. Injection of sub-optimal doses of prdm1a with prdm3 or prdm16 Morpholinos together leads to more severe phenotypes in the viscerocranium and neurocranium. prdm5 mutants have defects in the neurocranium and prdm1a and prdm5 double mutants also show more severe phenotypes. Overall, our data reveal that prdm3, 5, and 16 are involved in the zebrafish craniofacial development and that prdm1a may interact with prdm3, 5, and 16 in the formation of the craniofacial skeleton in zebrafish. Copyright © 2012 Wiley Periodicals, Inc.

  4. Intrinsic Xenobiotic Metabolizing Enzyme Activities in Early Life Stages of Zebrafish (Danio rerio).

    Science.gov (United States)

    Otte, Jens C; Schultz, Bernadette; Fruth, Daniela; Fabian, Eric; van Ravenzwaay, Bennard; Hidding, Björn; Salinas, Edward R

    2017-09-01

    Early life stages of zebrafish (Danio rerio, zf) are gaining attention as an alternative invivo test system for drug discovery, early developmental toxicity screenings and chemical testing in ecotoxicological and toxicological testing strategies. Previous studies have demonstrated transcriptional evidence for xenobiotic metabolizing enzymes (XME) during early zf development. However, elaborate experiments on XME activities during development are incomplete. In this work, the intrinsic activities of representative phase I and II XME were monitored by transformation of putative zf model substrates analyzed using photometry and high pressure liquid chromatography techniques. Six different defined stages of zf development (between 2.5 h postfertilization (hpf) to 120 hpf) were investigated by preparing a subcellular fraction from whole organism homogenates. We demonstrated that zf embryos as early as 2.5 hpf possess intrinsic metabolic activities for esterase, Aldh, Gst, and Cyp1a above the methodological detection limit. The activities of the enzymes Cyp3a and Nat were measurable during later stages in development. Activities represent dynamic patterns during development. The role of XME activities revealed in this work is relevant for the assessing toxicity in this test system and therefore contributes to a valuable characterization of zf embryos as an alternative testing organism in toxicology. © The Author 2017. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  5. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in-vitro culture in cryopreservation studies.

    Science.gov (United States)

    Anil, Siji; Rawson, David; Zhang, Tiantian

    2018-05-29

    Development of in vitro culture protocol for early stage ovarian follicles of zebrafish is important since cryopreserved early stage ovarian follicles would need to be matured in vitro following cryopreservation before they can be fertilised. Development of molecular markers for zebrafish (Danio rerio) ovarian follicle growth assessment following in vitro culture of early stage zebrafish ovarian follicles in ovarian tissue fragments is reported here for the first time although some work has been reported for in vitro culture of isolated early stage zebrafish ovarian follicles. The main aim of the present study was to develop molecular markers in an optimised in vitro culture protocol for stage I and stage II zebrafish ovarian follicles in ovarian tissue fragments. The effect of concentration of the hormones human chorionic gonadotropin and follicle stimulating hormones, and additives such as Foetal Bovine Serum and Bovine Serum Albumin were studied. The results showed that early stage zebrafish ovarian fragments containing stage I and stage II follicles which are cultured in vitro for 24 h in 20% FBS and 100mIU/ml FSH in 90% L-15 medium at 28 °C can grow to the size of stage II and stage III ovarian follicles respectively. More importantly the follicle growth from stage I to stage II and from stage II to stage III were confirmed using molecular markers such as cyp19a1a (also known as P450aromA) and vtg1 genes respectively. However, no follicle growth was observed following cryopreservation and in vitro culture. Copyright © 2018 Elsevier Inc. All rights reserved.

  6. Impacts of triclosan exposure on zebrafish early-life stage: Toxicity and acclimation mechanisms.

    Science.gov (United States)

    Falisse, Elodie; Voisin, Anne-Sophie; Silvestre, Frédéric

    2017-08-01

    Triclosan (TCS) is a broad spectrum antibacterial agent widely used in personal care products and present in most aquatic ecosystems. This study investigated the occurrence of triclosan acclimation and the biological mechanisms underlying the stress response triggered in early-life stage of zebrafish. Zebrafish eggs were first exposed to four different sublethal concentrations of TCS (2, 20, 50 and 100μg/L) for 7days following fertilization and subsequently exposed to a lethal concentration of TCS (1000μg/L). During the time-to-death exposure (TTD), mortality was continuously recorded to evaluate if increased resistance occurred. Overall, larvae exposed to 50μg/L of TCS demonstrated higher sensitivity, with delayed hatching and increased mortality during the sub-lethal exposure and significant lower mean time-to-death (TTD) value compared to the other groups. Interestingly, fish exposed to the highest concentration of TCS (100μg/L) presented a similar mean TTD value as controls and a significantly better survival in comparison with embryos exposed to 50μg/L, suggesting that acclimation process has been triggered at this concentration. Proteomic and enzymatic analyses were conducted on 7days post fertilization (dpf) larvae exposed to 50μg/L and 100μg/L of TCS giving insights into the functional changes triggered at those specific concentrations. TCS seemed to affect proteins involved in cytoskeleton, stress response, eyes and neuronal development. This was endorsed by the enzymatic results, which suggest impairment in glutathione metabolism and acute neurotoxicity. A significant 2.5-fold and 3-fold increase of AChE activity was observed following TCS exposure. Moreover, GPx activity was significantly increased whereas a significant inhibition of GR activity was observed, suggesting that de novo synthesis of reduced GSH might occur in order to maintain the ratio between reduced and oxidized GSH. Proteomic results revealed possible candidate protein involved in

  7. Vitamin D receptor deficiency impairs inner ear development in zebrafish

    International Nuclear Information System (INIS)

    Kwon, Hye-Joo

    2016-01-01

    The biological actions of vitamin D are largely mediated through binding to the vitamin D receptor (VDR), a member of the nuclear hormone receptor family, which regulates gene expression in a wide variety of tissues and cells. Mutations in VDR gene have been implicated in ear disorders (hearing loss and balance disorder) but the mechanisms are not well established. In this study, to investigate the role of VDR in inner ear development, morpholino-mediated gene knockdown approaches were used in zebrafish model system. Two paralogs for VDR, vdra and vdrb, have been identified in zebrafish. Knockdown of vdra had no effect on ear development, whereas knockdown of vdrb displayed morphological ear defects including smaller otic vesicles with malformed semicircular canals and abnormal otoliths. Loss-of-vdrb resulted in down-regulation of pre-otic markers, pax8 and pax2a, indicating impairment of otic induction. Furthermore, zebrafish embryos lacking vdrb produced fewer sensory hair cells in the ears and showed disruption of balance and motor coordination. These data reveal that VDR signaling plays an important role in ear development. - Highlights: • VDR signaling is involved in ear development. • Knockdown of vdrb causes inner ear malformations during embryogenesis. • Knockdown of vdrb affects otic placode induction. • Knockdown of vdrb reduces the number of sensory hair cells in the inner ear. • Knockdown of vdrb disrupts balance and motor coordination.

  8. Functional and Genetic Analysis of Choroid Plexus Development in Zebrafish

    Directory of Open Access Journals (Sweden)

    Hannah Elizabeth Henson

    2014-11-01

    Full Text Available The choroid plexus, an epithelial-based structure localized in the brain ventricle, is the major component of the blood-cerebrospinal fluid barrier. The choroid plexus produces the cerebrospinal fluid and regulates the components of the cerebrospinal fluid. Abnormal choroid plexus function is associated with neurodegenerative diseases, tumor formation in the choroid plexus epithelium, and hydrocephaly. In this study, we used zebrafish (Danio rerio as a model system to understand the genetic components of choroid plexus development. We generated an enhancer trap line, Et(cp:EGFPsj2, that expresses enhanced green fluorescent protein (EGFP in the choroid plexus epithelium. Using immunohistochemistry and fluorescent tracers, we demonstrated that the zebrafish choroid plexus possesses brain barrier properties such as tight junctions and transporter activity. Thus, we have established zebrafish as a functionally relevant model to study choroid plexus development. Using an unbiased approach, we performed a forward genetic dissection of the choroid plexus to identify genes essential for its formation and function. Using Et(cp:EGFPsj2, we isolated 10 recessive mutant lines with choroid plexus abnormalities, which were grouped into five classes based on GFP intensity, epithelial localization, and overall choroid plexus morphology. We also mapped the mutation for two mutant lines to chromosomes 4 and 21, respectively. The mutants generated in this study can be used to elucidate specific genes and signaling pathways essential for choroid plexus development, function, and/or maintenance and will provide important insights into how these genetic mutations contribute to disease.

  9. Znrg, a novel gene expressed mainly in the developing notochord of zebrafish.

    Science.gov (United States)

    Zhou, Yaping; Xu, Yan; Li, Jianzhen; Liu, Yao; Zhang, Zhe; Deng, Fengjiao

    2010-06-01

    The notochord, a defining characteristic of the chordate embryo is a critical midline structure required for axial skeletal formation in vertebrates, and acts as a signaling center throughout embryonic development. We utilized the digital differential display program of the National Center for Biotechnology Information, and identified a contig of expressed sequence tags (no. Dr. 83747) from the zebrafish ovary library in Genbank. Full-length cDNA of the identified gene was cloned by 5'- and 3'- RACE, and the resulting sequence was confirmed by polymerase chain reaction and sequencing. The cDNA clone contains 2,505 base pairs and encodes a novel protein of 707 amino acids that shares no significant homology with any known proteins. This gene was expressed in mature oocytes and at the one-cell stage, and persisted until the 5th day of development, as determined by RT-PCR. Transcripts were detected by whole-mount RNA in situ hybridization from the two-cell stage to 72 h of embryonic development. This gene was uniformly distributed from the cleavage stage up to the blastula stage. During early gastrulation, it was present in the dorsal region, and became restricted to the notochord and pectoral fin at 48 and 72 h of embryonic development. Based on its abundance in the notochord, we hypothesized that the novel gene may play an important role in notochord development in zebrafish; we named this gene, zebrafish notochord-related gene, or znrg.

  10. Deletion of Pr130 Interrupts Cardiac Development in Zebrafish

    Directory of Open Access Journals (Sweden)

    Jie Yang

    2016-11-01

    Full Text Available Protein phosphatase 2 regulatory subunit B, alpha (PPP2R3A, a regulatory subunit of protein phosphatase 2A (PP2A, is a major serine/threonine phosphatase that regulates crucial function in development and growth. Previous research has implied that PPP2R3A was involved in heart failure, and PR130, the largest transcription of PPP2R3A, functioning in the calcium release of sarcoplasmic reticulum (SR, plays an important role in the excitation-contraction (EC coupling. To obtain a better understanding of PR130 functions in myocardium and cardiac development, two pr130-deletion zebrafish lines were generated using clustered regularly interspaced short palindromic repeats (CRISPR/CRISPR-associated proteins (Cas system. Pr130-knockout zebrafish exhibited cardiac looping defects and decreased cardiac function (decreased fractional area and fractional shortening. Hematoxylin and eosin (H&E staining demonstrated reduced cardiomyocytes. Subsequent transmission electron microscopy revealed that the bright and dark bands were narrowed and blurred, the Z- and M-lines were fogged, and the gaps between longitudinal myocardial fibers were increased. Additionally, increased apoptosis was observed in cardiomyocyte in pr130-knockout zebrafish compared to wild-type (WT. Taken together, our results suggest that pr130 is required for normal myocardium formation and efficient cardiac contractile function.

  11. Time-lapse imaging of neural development: zebrafish lead the way into the fourth dimension.

    Science.gov (United States)

    Rieger, Sandra; Wang, Fang; Sagasti, Alvaro

    2011-07-01

    Time-lapse imaging is often the only way to appreciate fully the many dynamic cell movements critical to neural development. Zebrafish possess many advantages that make them the best vertebrate model organism for live imaging of dynamic development events. This review will discuss technical considerations of time-lapse imaging experiments in zebrafish, describe selected examples of imaging studies in zebrafish that revealed new features or principles of neural development, and consider the promise and challenges of future time-lapse studies of neural development in zebrafish embryos and adults. Copyright © 2011 Wiley-Liss, Inc.

  12. Morphofunctional transformations of the yolk syncytial layer during zebrafish development.

    Science.gov (United States)

    Kondakova, Ekaterina Alexandrovna A; Efremov, Vladimir Ivanovich I

    2014-02-01

    The yolk syncytial layer (YSL) is a provisory extraembryonic structure of teleost fishes and representatives of some other taxa with meroblastic cleavage. The YSL of teleosts is a symplast with polymorphous polyploid nuclei. It is known to perform nutritional, morphogenetic, immune, and, probably, other functions. Data about the YSL organization, functioning and regulation is fragmentary. Although gene expression patterns and other aspects of YSL functioning have been studied in Danio rerio, the morphology of its YSL has not been described in detail. The study of zebrafish YSL structure on sequential developmental stages is necessary to recognize specific features of this important polyfunctional system in this model organism and to extend our knowledge about provisory systems. The thickness of the YSL and the distribution of its nuclei are not uniform on each stage and change during development. During oblong and sphere stages the internal YSL (I-YSL) is filled with yolk inclusions; interphase yolk syncytial nuclei (YSN) and mitotic asters can be seen. During doming and epiboly the external YSL (E-YSL) is thicker than I-YSL. On the subsequent stages the YSL is thickened caudally. The dorsal YSL part is thickened during early segmentation stages and becomes the thinnest YSL region later. The anterior part of the YSL is thin, but enlarges during larval period. The YSN of different size and diverse forms, from regular to lobed, are present and form clusters. The number of irregular-shaped nuclei increases during development. The YSL thickens in the end of endotrophic and in the course of endo-exotrophic period, and its cytoplasm contains numerous yolk inclusions. After yolk exhaustion the YSL is flat. As the YSL degrades, the YSN become pycnotic, and the YSL remnant probably is cleared by phagocytes. Copyright © 2013 Wiley Periodicals, Inc.

  13. Maternal Cortisol Mediates Hypothalamus-Pituitary-Interrenal Axis Development in Zebrafish

    Science.gov (United States)

    Nesan, Dinushan; Vijayan, Mathilakath M.

    2016-01-01

    In zebrafish (Danio rerio), de novo synthesis of cortisol in response to stressor exposure commences only after hatch. Maternally deposited cortisol is present during embryogenesis, but a role for this steroid in early development is unclear. We tested the hypothesis that maternal cortisol is essential for the proper development of hypothalamus-pituitary-interrenal (HPI) axis activity and the onset of the stressor-induced cortisol response in larval zebrafish. In this study, zygotic cortisol content was manipulated by microinjecting antibody to sequester this steroid, thereby making it unavailable during embryogenesis. This was compared with embryos containing excess cortisol by microinjection of exogenous steroid. The resulting larval phenotypes revealed distinct treatment effects, including deformed mesoderm structures when maternal cortisol was unavailable and cardiac edema after excess cortisol. Maternal cortisol unavailability heightened the cortisol stress response in post-hatch larvae, whereas excess cortisol abolished the stressor-mediated cortisol elevation. This contrasting hormonal response corresponded with altered expression of key HPI axis genes, including crf, 11B hydroxylase, pomca, and star, which were upregulated in response to reduced cortisol availability and downregulated when embryos had excess cortisol. These findings for the first time underscore a critical role for maternally deposited cortisol in programming HPI axis development and function in zebrafish. PMID:26940285

  14. Apoptosis-related genes induced in response to ketamine during early life stages of zebrafish.

    Science.gov (United States)

    Félix, Luís M; Serafim, Cindy; Valentim, Ana M; Antunes, Luís M; Matos, Manuela; Coimbra, Ana M

    2017-09-05

    Increasing evidence supports that ketamine, a widely used anaesthetic, potentiates apoptosis during development through the mitochondrial pathway of apoptosis. Defects in the apoptotic machinery can cause or contribute to the developmental abnormalities previously described in ketamine-exposed zebrafish. The involvement of the apoptotic machinery in ketamine-induced teratogenicity was addressed by assessing the apoptotic signals at 8 and 24 hpf following 20min exposure to ketamine at three stages of early zebrafish embryo development (256 cell, 50% epiboly and 1-4 somites stages). Exposure at the 256-cell stage to ketamine induced an up-regulation of casp8 and pcna at 8 hpf while changes in pcna at the mRNA level were observed at 24 hpf. After the 50% epiboly stage exposure, the mRNA levels of casp9 were increased at 8 and 24 hpf while aifm1 was affected at 24 hpf. Both tp53 and pcna expressions were increased at 8 hpf. After exposure during the 1-4 somites stage, no meaningful changes on transcript levels were observed. The distribution of apoptotic cells and the caspase-like enzymatic activities of caspase-3 and -9 were not affected by ketamine exposure. It is proposed that ketamine exposure at the 256-cell stage induced a cooperative mechanism between proliferation and cellular death while following exposure at the 50% epiboly, a p53-dependent and -independent caspase activation may occur. Finally, at the 1-4 somites stage, the defence mechanisms are already fully in place to protect against ketamine-insult. Thus, ketamine teratogenicity seems to be dependent on the functional mechanisms present in each developmental stage. Copyright © 2017 Elsevier B.V. All rights reserved.

  15. Exposure to low dose benzo[a]pyrene during early life stages causes symptoms similar to cardiac hypertrophy in adult zebrafish.

    Science.gov (United States)

    Huang, Lixing; Gao, Dongxu; Zhang, Youyu; Wang, Chonggang; Zuo, Zhenghong

    2014-07-15

    Growing evidence indicates that polycyclic aromatic hydrocarbons (PAHs) can lead to cardiac hypertrophy and recent research indicates that exposure to low dose crude oil during early embryonic development may lead to impacts on heart health at later life stages. The aim of this study was to evaluate whether exposure during early life stages to low dose benzo[a]pyrene (BaP), as a high-ring PAH, would lead to cardiac hypertrophy at later life stages. Zebrafish were exposed to low dose BaP until 96 hpf, then transferred to clean water and maintained for a year before histological and molecular biological analysis. Our results showed that exposure to low level BaP during early life stages increased heart weight to body weight ratios and deposited collagen in the heart of adult zebrafish. ANP, BNP and c-Myc were also induced in the heart of adult zebrafish by BaP. These results proved that low level BaP exposure during early life stages caused symptoms similar to cardiac hypertrophy in adult zebrafish. Our results displayed an elevated expression of CdC42, RhoA, p-ERK1, 2 and Rac1. Therefore, the mechanism of the cardiac hypertrophy caused by BaP exposure during early life stages may be through inducing the expression of CdC42, RhoA and Rac1, together with activating ERK1, 2. Copyright © 2014 Elsevier B.V. All rights reserved.

  16. Mutagenesis and phenotyping resources in zebrafish for studying development and human disease

    Science.gov (United States)

    Varshney, Gaurav Kumar

    2014-01-01

    The zebrafish (Danio rerio) is an important model organism for studying development and human disease. The zebrafish has an excellent reference genome and the functions of hundreds of genes have been tested using both forward and reverse genetic approaches. Recent years have seen an increasing number of large-scale mutagenesis projects and the number of mutants or gene knockouts in zebrafish has increased rapidly, including for the first time conditional knockout technologies. In addition, targeted mutagenesis techniques such as zinc finger nucleases, transcription activator-like effector nucleases and clustered regularly interspaced short sequences (CRISPR) or CRISPR-associated (Cas), have all been shown to effectively target zebrafish genes as well as the first reported germline homologous recombination, further expanding the utility and power of zebrafish genetics. Given this explosion of mutagenesis resources, it is now possible to perform systematic, high-throughput phenotype analysis of all zebrafish gene knockouts. PMID:24162064

  17. Zebrafish: an exciting model for investigating the spatio-temporal pattern of enteric nervous system development.

    LENUS (Irish Health Repository)

    Doodnath, Reshma

    2012-02-01

    AIM: Recently, the zebrafish (Danio rerio) has been shown to be an excellent model for human paediatric research. Advantages over other models include its small size, externally visually accessible development and ease of experimental manipulation. The enteric nervous system (ENS) consists of neurons and enteric glia. Glial cells permit cell bodies and processes of neurons to be arranged and maintained in a proper spatial arrangement, and are essential in the maintenance of basic physiological functions of neurons. Glial fibrillary acidic protein (GFAP) is expressed in astrocytes, but also expressed outside of the central nervous system. The aim of this study was to investigate the spatio-temporal pattern of GFAP expression in developing zebrafish ENS from 24 h post-fertilization (hpf), using transgenic fish that express green fluorescent protein (GFP). METHODS: Zebrafish embryos were collected from transgenic GFP Tg(GFAP:GFP)(mi2001) adult zebrafish from 24 to 120 hpf, fixed and processed for whole mount immunohistochemistry. Antibodies to Phox2b were used to identify enteric neurons. Specimens were mounted on slides and imaging was performed using a fluorescent laser confocal microscope. RESULTS: GFAP:GFP labelling outside the spinal cord was identified in embryos from 48 hpf. The patterning was intracellular and consisted of elongated profiles that appeared to migrate away from the spinal cord into the periphery. At 72 and 96 hpf, GFAP:GFP was expressed dorsally and ventrally to the intestinal tract. At 120 hpf, GFAP:GFP was expressed throughout the intestinal wall, and clusters of enteric neurons were identified using Phox2b immunofluorescence along the pathway of GFAP:GFP positive processes, indicative of a migratory pathway of ENS precursors from the spinal cord into the intestine. CONCLUSION: The pattern of migration of GFAP:GFP expressing cells outside the spinal cord suggests an organized, early developing migratory pathway to the ENS. This shows for the

  18. Alternative splicing of sept9a and sept9b in zebrafish produces multiple mRNA transcripts expressed throughout development.

    Directory of Open Access Journals (Sweden)

    Megan L Landsverk

    2010-05-01

    Full Text Available Septins are involved in a number of cellular processes including cytokinesis and organization of the cytoskeleton. Alterations in human septin-9 (SEPT9 levels have been linked to multiple cancers, whereas mutations in SEPT9 cause the episodic neuropathy, hereditary neuralgic amyotrophy (HNA. Despite its important function in human health, the in vivo role of SEPT9 is unknown.Here we utilize zebrafish to study the role of SEPT9 in early development. We show that zebrafish possess two genes, sept9a and sept9b that, like humans, express multiple transcripts. Knockdown or overexpression of sept9a transcripts results in specific developmental alterations including circulation defects and aberrant epidermal development.Our work demonstrates that sept9 plays an important role in zebrafish development, and establishes zebrafish as a valuable model organism for the study of SEPT9.

  19. Early gonad development in zebrafish (Danio rerio)

    African Journals Online (AJOL)

    SAM

    2014-08-13

    Aug 13, 2014 ... as a relatively larger proportion of proliferating "gonial" germ cells in the latter gonads was considered ... solution in 100 ml tank water) as described by Westerfield (1993). They were ... 90°C hot plate with toluidine blue and thereafter examined and ... morphological sex differentiation, sex differences could.

  20. Effects of probiotic administration on zebrafish development and reproduction.

    Science.gov (United States)

    Carnevali, O; Avella, M A; Gioacchini, G

    2013-07-01

    As the consumption of probiotics increases worldwide, scientists focus on identifying bacterial strains able to improve human life quality and evidence the biological pathways affected by probiotic treatment. In this review, some recent observations on the effects of changes of microbiota on zebrafish metabolism were discussed. In addition, the effects of Lactobacillus rhamnosus - a component of the human gut microflora - as a diet supplement on Danio rerio were presented. When administered chronically, L. rhamnosus may affect larval development and the physiology of reproductive system in the zebrafish model. It was hypothesized exogenous L. rhamnosus accelerates larval growth and backbone development by acting on insulin-like growth factors-I (igfI) and -II (igfII), peroxisome proliferator activated receptors-α and -β, (pparα,β) vitamin D receptor-α (vdrα) and retinoic acid receptor-γ (rarγ). Gonadal differentiation was anticipated at 6weeks together with a higher expression of gnrh3 at the larval stage when L. rhamnosus was administered throughout development. Moreover, brood stock alimented with a L. rhamnosus-supplemented diet showed better reproductive performances as per follicles development, ovulated oocytes quantification and embryos quality. A plausible involvement of factors such as leptin, and kiss1 and 2 in the improvements was concluded. The observations made on the physiology of female reproduction were correlated with the gene expression of a gigantic number of factors as the aromatase cytochrome p 19 (cyp19a), the vitellogenin (vtg) and the α isoform of the E2 receptor (erα), luteinizing hormone receptor (lhr), 20-β hydroxysteroid dehydrogenase (20β-hsd), membrane progesterone receptors α and β, cyclin B, activinβA1, smad2, transforming growth factor β1 (tgfβ1), growth differentiation factor9 (gdf9) and bone morphogenetic protein15 (bmp15.) A model in which the exogenous L. rhamnosus in the digestive tract of zebrafish from the

  1. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos

    OpenAIRE

    Sundvik, Maria; Nieminen, Heikki J.; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-01-01

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) a...

  2. Ftr82 Is Critical for Vascular Patterning during Zebrafish Development

    Directory of Open Access Journals (Sweden)

    Hsueh-Wei Chang

    2017-01-01

    Full Text Available Cellular components and signaling pathways are required for the proper growth of blood vessels. Here, we report for the first time that a teleost-specific gene ftr82 (finTRIM family, member 82 plays a critical role in vasculature during zebrafish development. To date, there has been no description of tripartite motif proteins (TRIM in vascular development, and the role of ftr82 is unknown. In this study, we found that ftr82 mRNA is expressed during the development of vessels, and loss of ftr82 by morpholino (MO knockdown impairs the growth of intersegmental vessels (ISV and caudal vein plexus (CVP, suggesting that ftr82 plays a critical role in promoting ISV and CVP growth. We showed the specificity of ftr82 MO by analyzing ftr82 expression products and expressing ftr82 mRNA to rescue ftr82 morphants. We further showed that the knockdown of ftr82 reduced ISV cell numbers, suggesting that the growth impairment of vessels is likely due to a decrease of cell proliferation and migration, but not cell death. In addition, loss of ftr82 affects the expression of vascular markers, which is consistent with the defect of vascular growth. Finally, we showed that ftr82 likely interacts with vascular endothelial growth factor (VEGF and Notch signaling. Together, we identify teleost-specific ftr82 as a vascular gene that plays an important role for vascular development in zebrafish.

  3. Identification and expression analysis of zebrafish glypicans during embryonic development.

    Directory of Open Access Journals (Sweden)

    Mansi Gupta

    Full Text Available Heparan sulfate Proteoglycans (HSPG are ubiquitous molecules with indispensable functions in various biological processes. Glypicans are a family of HSPG's, characterized by a Gpi-anchor which directs them to the cell surface and/or extracellular matrix where they regulate growth factor signaling during development and disease. We report the identification and expression pattern of glypican genes from zebrafish. The zebrafish genome contains 10 glypican homologs, as opposed to six in mammals, which are highly conserved and are phylogenetically related to the mammalian genes. Some of the fish glypicans like Gpc1a, Gpc3, Gpc4, Gpc6a and Gpc6b show conserved synteny with their mammalian cognate genes. Many glypicans are expressed during the gastrulation stage, but their expression becomes more tissue specific and defined during somitogenesis stages, particularly in the developing central nervous system. Existence of multiple glypican orthologs in fish with diverse expression pattern suggests highly specialized and/or redundant function of these genes during embryonic development.

  4. In vivo wall shear measurements within the developing zebrafish heart.

    Directory of Open Access Journals (Sweden)

    R Aidan Jamison

    Full Text Available Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.

  5. In vivo wall shear measurements within the developing zebrafish heart.

    Science.gov (United States)

    Jamison, R Aidan; Samarage, Chaminda R; Bryson-Richardson, Robert J; Fouras, Andreas

    2013-01-01

    Physical forces can influence the embryonic development of many tissues. Within the cardiovascular system shear forces resulting from blood flow are known to be one of the regulatory signals that shape the developing heart. A key challenge in investigating the role of shear forces in cardiac development is the ability to obtain shear force measurements in vivo. Utilising the zebrafish model system we have developed a methodology that allows the shear force within the developing embryonic heart to be determined. Accurate wall shear measurement requires two essential pieces of information; high-resolution velocity measurements near the heart wall and the location and orientation of the heart wall itself. We have applied high-speed brightfield imaging to capture time-lapse series of blood flow within the beating heart between 3 and 6 days post-fertilization. Cardiac-phase filtering is applied to these time-lapse images to remove the heart wall and other slow moving structures leaving only the red blood cell movement. Using particle image velocimetry to calculate the velocity of red blood cells in different regions within the heart, and using the signal-to-noise ratio of the cardiac-phase filtered images to determine the boundary of blood flow, and therefore the position of the heart wall, we have been able to generate the necessary information to measure wall shear in vivo. We describe the methodology required to measure shear in vivo and the application of this technique to the developing zebrafish heart. We identify a reduction in shear at the ventricular-bulbar valve between 3 and 6 days post-fertilization and demonstrate that the shear environment of the ventricle during systole is constantly developing towards a more uniform level.

  6. The essential role of endogenous ghrelin in growth hormone expression during zebrafish adenohypophysis development.

    Science.gov (United States)

    Li, Xi; He, Jiangyan; Hu, Wei; Yin, Zhan

    2009-06-01

    Ghrelin, a multifunctional hormone, including potent GH stimulation activity, has been suggested to be important during embryonic development. Expression of ghrelin has been confirmed in the zebrafish pancreas during embryonic stages. Interfering with ghrelin function using two specific antisense morpholino oligonucleotides causes defects during zebrafish embryonic development. In ghrelin morphants the expression of GH was abolished in zebrafish somatotropes, whereas the expression patterns of the other key molecules involved in hypothalamic-pituitary development and distinct pituitary hormones genes remain largely intact at the appropriate time during zebrafish adenohypophysis development. Effective rescue of the ghrelin morphants with exogenous ghrelin mRNA showed that the correct gene had been targeted. Moreover, by analyzing the efficiencies of the ghrelin morphants rescue experiments with various forms of exogenous mutant ghrelin mRNAs, we also demonstrated the essentiality of the form acyl-ghrelin on GH stimulation during zebrafish adenohypophysis development. Our in vivo experiments, for the first time, also provided evidence of the existence of functional obestatin in the C-terminal part of zebrafish proghrelin peptides. Our research here has demonstrated that zebrafish is a unique model for functional studies of endogenous ghrelin, especially during embryonic development.

  7. Biomarkers as a tool to assess effects of chromium (VI): comparison of responses in zebrafish early life stages and adults.

    Science.gov (United States)

    Domingues, Inês; Oliveira, Rhaul; Lourenço, Joana; Grisolia, Cesar Koppe; Mendo, Sónia; Soares, A M V M

    2010-09-01

    The present work aims to compare the sensitivity of embryos and adult zebrafish to chromium (VI) (as potassium dichromate) focusing on biomarkers (cholinesterase, glutathione S-transferase and lactate dehydrogenase) as endpoints. Zebrafish eggs showed less sensitivity to Cr (VI) (96 h-LC50=145.7 mg/L) than adults (96 h-LC50=39.4 mg/L) probably due to the protective action of the chorion. However, biomarkers were much more responsive in larvae than in adults and gave clear indications about Cr (VI) mode of action: it seems to be neurotoxic (inhibited cholinesterase), to inhibit glutathione S-transferase activity and to interfere with cellular metabolic activity (changes in lactate dehydrogenase activity) in larvae. In adults, only glutathione S-transferase was responsive, showing a clear inhibition. The responsiveness of the analyzed biomarkers in larvae reinforces the idea of the usefulness of early life stage assays in the assessment of chemicals effects. Moreover, early life stage assays also contributed with relevant information regarding anomalies in larvae development and behavior. Further research should focus on the use of biomarkers to assess long term effects which are ecologically more relevant. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  8. A dominant negative zebrafish Ahr2 partially protects developing zebrafish from dioxin toxicity.

    Directory of Open Access Journals (Sweden)

    Kevin A Lanham

    Full Text Available The toxicity by 2,3,7,8 tetrachlorodibenzo-p-dioxin (TCDD is thought to be caused by activation of the aryl hydrocarbon receptor (AHR. However, our understanding of how AHR activation by TCDD leads to toxic effects is poor. Ideally we would like to manipulate AHR activity in specific tissues and at specific times. One route to this is expressing dominant negative AHRs (dnAHRs. This work describes the construction and characterization of dominant negative forms of the zebrafish Ahr2 in which the C-terminal transactivation domain was either removed, or replaced with the inhibitory domain from the Drosophila engrailed repressor protein. One of these dnAhr2s was selected for expression from the ubiquitously active e2fα promoter in transgenic zebrafish. We found that these transgenic zebrafish expressing dnAhr2 had reduced TCDD induction of the Ahr2 target gene cyp1a, as measured by 7-ethoxyresorufin-O-deethylase activity. Furthermore, the cardiotoxicity produced by TCDD, pericardial edema, heart malformation, and reduced blood flow, were all mitigated in the zebrafish expressing the dnAhr2. These results provide in vivo proof-of-principle results demonstrating the effectiveness of dnAHRs in manipulating AHR activity in vivo, and demonstrating that this approach can be a means for blocking TCDD toxicity.

  9. A zebrafish larval model reveals early tissue-specific innate immune responses to Mucor circinelloides.

    Science.gov (United States)

    Voelz, Kerstin; Gratacap, Remi L; Wheeler, Robert T

    2015-11-01

    Mucormycosis is an emerging fungal infection that is clinically difficult to manage, with increasing incidence and extremely high mortality rates. Individuals with diabetes, suppressed immunity or traumatic injury are at increased risk of developing disease. These individuals often present with defects in phagocytic effector cell function. Research using mammalian models and phagocytic effector cell lines has attempted to decipher the importance of the innate immune system in host defence against mucormycosis. However, these model systems have not been satisfactory for direct analysis of the interaction between innate immune effector cells and infectious sporangiospores in vivo. Here, we report the first real-time in vivo analysis of the early innate immune response to mucormycete infection using a whole-animal zebrafish larval model system. We identified differential host susceptibility, dependent on the site of infection (hindbrain ventricle and swim bladder), as well as differential functions of the two major phagocyte effector cell types in response to viable and non-viable spores. Larval susceptibility to mucormycete spore infection was increased upon immunosuppressant treatment. We showed for the first time that macrophages and neutrophils were readily recruited in vivo to the site of infection in an intact host and that spore phagocytosis can be observed in real-time in vivo. While exploring innate immune effector recruitment dynamics, we discovered the formation of phagocyte clusters in response to fungal spores that potentially play a role in fungal spore dissemination. Spores failed to activate pro-inflammatory gene expression by 6 h post-infection in both infection models. After 24 h, induction of a pro-inflammatory response was observed only in hindbrain ventricle infections. Only a weak pro-inflammatory response was initiated after spore injection into the swim bladder during the same time frame. In the future, the zebrafish larva as a live whole

  10. Slc39a7/zip7 plays a critical role in development and zinc homeostasis in zebrafish.

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    Guang Yan

    Full Text Available BACKGROUND: Slc39a7/Zip7, also known as Ke4, is a member of solute carrier family 39 (Slc39a and plays a critical role in regulating cell growth and death. Because the function of Zip7 in vivo was unclear, the present study investigated the function of zip7 in vertebrate development and zinc metabolism using zebrafish as a model organism. PRINCIPAL FINDING: Using real-time PCR to determine the gene expression pattern of zip7 during zebrafish development, we found that zip7 mRNA is expressed throughout embryonic development and into maturity. Interestingly, whole mount in situ hybridization revealed that while zip7 mRNA is ubiquitously expressed until 12 hours post-fertilization (hpf; at 24 hpf and beyond, zip7 mRNA was specifically detected only in eyes. Morpholino-antisense (MO gene knockdown assay revealed that downregulation of zip7 expression resulted in several morphological defects in zebrafish including decreased head size, smaller eyes, shorter palates, and shorter and curved spinal cords. Analysis by synchrotron radiation X-ray fluorescence (SR-XRF showed reduced concentrations of zinc in brain, eyes, and gills of zip7-MO-injected embryos. Furthermore, incubation of the zip7 knockdown embryos in a zinc-supplemented solution was able to rescue the MO-induced morphological defects. SIGNIFICANCE: Our data suggest that zip7 is required for eye, brain, and skeleton formation during early embryonic development in zebrafish. Moreover, zinc supplementation can partially rescue defects resulting from zip7 gene knockdown. Taken together, our data provide critical insight into a novel function of zip7 in development and zinc homeostasis in vivo in zebrafish.

  11. Imaging retinal progenitor lineages in developing zebrafish embryos.

    Science.gov (United States)

    Jusuf, Patricia; Harris, William A; Poggi, Lucia

    2013-03-01

    In this protocol, we describe how to make and analyze four dimensional (4D) movies of retinal lineage in the zebrafish embryo in vivo. 4D consists of three spatial dimensions (3D) reconstructed from stacks of confocal planes plus one time dimension. Our imaging is performed on transgenic cells that express fluorescent proteins under the control of cell-specific promoters or on cells that transiently express such reporters in specific retinal cell progenitors. An important aspect of lineage tracing is the ability to follow individual cells as they undergo multiple cell divisions, final migration, and differentiation. This may mean many hours of 4D imaging, requiring that cells be kept healthy and maintained under conditions suitable for normal development. The longest movies we have made are ∼50 h. By analyzing these movies, we can see when a specific cell was born and who its sister was, allowing us to reconstruct its retinal lineages in vivo.

  12. Effects of acoustic levitation on the development of zebrafish, Danio rerio, embryos.

    Science.gov (United States)

    Sundvik, Maria; Nieminen, Heikki J; Salmi, Ari; Panula, Pertti; Hæggström, Edward

    2015-09-04

    Acoustic levitation provides potential to characterize and manipulate material such as solid particles and fluid in a wall-less environment. While attempts to levitate small animals have been made, the biological effects of such levitation have been scarcely documented. Here, our goal was to explore if zebrafish embryos can be levitated (peak pressures at the pressure node and anti-node: 135 dB and 144 dB, respectively) with no effects on early development. We levitated the embryos (n = 94) at 2-14 hours post fertilization (hpf) for 1000 (n = 47) or 2000 seconds (n = 47). We compared the size and number of trunk neuromasts and otoliths in sonicated samples to controls (n = 94), and found no statistically significant differences (p > 0.05). While mortality rate was lower in the control group (22.3%) compared to that in the 1000 s (34.0%) and 2000 s (42.6%) levitation groups, the differences were statistically insignificant (p > 0.05). The results suggest that acoustic levitation for less than 2000 sec does not interfere with the development of zebrafish embryos, but may affect mortality rate. Acoustic levitation could potentially be used as a non-contacting wall-less platform for characterizing and manipulating vertebrae embryos without causing major adverse effects to their development.

  13. Human amyloidogenic light chain proteins result in cardiac dysfunction, cell death, and early mortality in zebrafish.

    Science.gov (United States)

    Mishra, Shikha; Guan, Jian; Plovie, Eva; Seldin, David C; Connors, Lawreen H; Merlini, Giampaolo; Falk, Rodney H; MacRae, Calum A; Liao, Ronglih

    2013-07-01

    Systemic amyloid light-chain (AL) amyloidosis is associated with rapidly progressive and fatal cardiomyopathy resulting from the direct cardiotoxic effects of circulating AL light chain (AL-LC) proteins and the indirect effects of AL fibril tissue infiltration. Cardiac amyloidosis is resistant to standard heart failure therapies, and, to date, there are limited treatment options for these patients. The mechanisms underlying the development of cardiac amyloidosis and AL-LC cardiotoxicity are largely unknown, and their study has been limited by the lack of a suitable in vivo model system. Here, we establish an in vivo zebrafish model of human AL-LC-induced cardiotoxicity. AL-LC isolated from AL cardiomyopathy patients or control nonamyloidogenic LC protein isolated from multiple myeloma patients (Con-LC) was directly injected into the circulation of zebrafish at 48 h postfertilization. AL-LC injection resulted in impaired cardiac function, pericardial edema, and increased cell death relative to Con-LC, culminating in compromised survival with 100% mortality within 2 wk, independent of AL fibril deposition. Prior work has implicated noncanonical p38 MAPK activation in the pathogenesis of AL-LC-induced cardiotoxicity, and p38 MAPK inhibition via SB-203580 rescued AL-LC-induced cardiac dysfunction and cell death and attenuated mortality in zebrafish. This in vivo zebrafish model of AL-LC cardiotoxicity demonstrates that antagonism of p38 MAPK within the AL-LC cardiotoxic signaling response may serve to improve cardiac function and mortality in AL cardiomyopathy. Furthermore, this in vivo model system will allow for further study of the molecular underpinnings of AL cardiotoxicity and identification of novel therapeutic strategies.

  14. Zebrafish embryos exposed to alcohol undergo abnormal development of motor neurons and muscle fibers.

    Science.gov (United States)

    Sylvain, Nicole J; Brewster, Daniel L; Ali, Declan W

    2010-01-01

    Children exposed to alcohol in utero have significantly delayed gross and fine motor skills, as well as deficiencies in reflex development. The reasons that underlie the motor deficits caused by ethanol (EtOH) exposure remain to be fully elucidated. The present study was undertaken to investigate the effects of embryonic alcohol exposure (1.5%, 2% and 2.5% EtOH) on motor neuron and muscle fiber morphology in 3 days post fertilization (dpf) larval zebrafish. EtOH treated fish exhibited morphological deformities and fewer bouts of swimming in response to touch, compared with untreated fish. Immunolabelling with anti-acetylated tubulin indicated that fish exposed to 2.5% EtOH had significantly higher rates of motor neuron axon defects. Immunolabelling of primary and secondary motor neurons, using znp-1 and zn-8, revealed that fish exposed to 2% and 2.5% EtOH exhibited significantly higher rates of primary and secondary motor neuron axon defects compared to controls. Examination of red and white muscle fibers revealed that fish exposed to EtOH had significantly smaller fibers compared with controls. These findings indicate that motor neuron and muscle fiber morphology is affected by early alcohol exposure in zebrafish embryos, and that this may be related to deficits in locomotion. Copyright 2010 Elsevier Inc. All rights reserved.

  15. Alternative haplotypes of antigen processing genes in zebrafish diverged early in vertebrate evolution

    Science.gov (United States)

    McConnell, Sean C.; Hernandez, Kyle M.; Wcisel, Dustin J.; Kettleborough, Ross N.; Stemple, Derek L.; Andrade, Jorge; de Jong, Jill L. O.

    2016-01-01

    Antigen processing and presentation genes found within the MHC are among the most highly polymorphic genes of vertebrate genomes, providing populations with diverse immune responses to a wide array of pathogens. Here, we describe transcriptome, exome, and whole-genome sequencing of clonal zebrafish, uncovering the most extensive diversity within the antigen processing and presentation genes of any species yet examined. Our CG2 clonal zebrafish assembly provides genomic context within a remarkably divergent haplotype of the core MHC region on chromosome 19 for six expressed genes not found in the zebrafish reference genome: mhc1uga, proteasome-β 9b (psmb9b), psmb8f, and previously unknown genes psmb13b, tap2d, and tap2e. We identify ancient lineages for Psmb13 within a proteasome branch previously thought to be monomorphic and provide evidence of substantial lineage diversity within each of three major trifurcations of catalytic-type proteasome subunits in vertebrates: Psmb5/Psmb8/Psmb11, Psmb6/Psmb9/Psmb12, and Psmb7/Psmb10/Psmb13. Strikingly, nearby tap2 and MHC class I genes also retain ancient sequence lineages, indicating that alternative lineages may have been preserved throughout the entire MHC pathway since early diversification of the adaptive immune system ∼500 Mya. Furthermore, polymorphisms within the three MHC pathway steps (antigen cleavage, transport, and presentation) are each predicted to alter peptide specificity. Lastly, comparative analysis shows that antigen processing gene diversity is far more extensive than previously realized (with ancient coelacanth psmb8 lineages, shark psmb13, and tap2t and psmb10 outside the teleost MHC), implying distinct immune functions and conserved roles in shaping MHC pathway evolution throughout vertebrates. PMID:27493218

  16. Effects of rare earth elements La and Yb on the morphological and functional development of zebrafish embryos

    Institute of Scientific and Technical Information of China (English)

    Jun'an Cui; Zhiyong Zhang; Wei Bai; Ligang Zhang; Xiao He; Yuhui Ma; Yan Liu; Zhifang Chai

    2012-01-01

    In recent years,with the wide applications and mineral exploitation of rare earth elements,their potential environmental and health effects have caused increasing public concern.Effect of rare earth elements La and Yb on the morphological and functional development of zebrafish embryos were studied.The embryos were exposed to La3+ or Yb3+ at 0,0.01,0.1,0.3,0.5 and 1.0 mmol/L,respectively.Early life stage parameters such as egg and embryo mortality,gastrula development,tail detachment,eyes,somite formation,circulatory system,pigmentation,malformations,hatching rate,length of larvae and mortality were investigated.The results showed La3+ and Yb3+ delayed zebrafish embryo and larval development,decreased survival and hatching rates,and caused tail malformation in a concentration-dependent way.Moreover,heavy rare-earth ytterbium led to more severe acute toxicity of zebrafish embryo than light rare-earth lanthanum.

  17. Zebrafish scarb2a insertional mutant reveals a novel function for the Scarb2/Limp2 receptor in notochord development.

    Science.gov (United States)

    Diaz-Tellez, Abigail; Zampedri, Cecilia; Ramos-Balderas, Jose L; García-Hernández, Fernando; Maldonado, Ernesto

    2016-04-01

    Scarb2 or Limp2 belong to a subfamily of Scavenger receptors described as lysosomal transmembrane glycosylated receptors, that are mutated in the human syndrome AMRF (action myoclonus-renal failure). The zebrafish insertional mutant scarb2a(hi1463Tg) has notochord defects, the notochord is a defining feature of chordates running along the center of the longitudinal axis and it is essential for forming the spinal column in all vertebrates. There are three paralogous scarb2 genes in zebrafish; scarb2a, scarb2b, and scarb2c. Both Scarb2a and Scarb2b proteins lack the classical di-leucine motif. We found that scarb2a(hi1463Tg) homozygous zebrafish embryos have a null mutation impairing vacuole formation in the notochord and simultaneously disrupting proper formation of the basement membrane resulting in its thickening at the ventral side of the notochord, which may be the cause for the anomalous upward bending observed in the trunk. Through whole-mount in situ hybridization, we detected scarb2a mRNA expression in the notochord and in the brain early in development. However, it is puzzling that scarb2a notochord mRNA expression is short-lived in the presumptive notochord and precedes the complete differentiation of the notochord. This work describes a novel function for the Scarb2 receptor as an essential glycoprotein for notochord development. © 2016 Wiley Periodicals, Inc.

  18. Data Integration for Spatio-Temporal Patterns of Gene Expression of Zebrafish development: the GEMS database

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    Belmamoune Mounia

    2008-06-01

    Full Text Available The Gene Expression Management System (GEMS is a database system for patterns of gene expression. These patterns result from systematic whole-mount fluorescent in situ hybridization studies on zebrafish embryos. GEMS is an integrative platform that addresses one of the important challenges of developmental biology: how to integrate genetic data that underpin morphological changes during embryogenesis. Our motivation to build this system was by the need to be able to organize and compare multiple patterns of gene expression at tissue level. Integration with other developmental and biomolecular databases will further support our understanding of development. The GEMS operates in concert with a database containing a digital atlas of zebrafish embryo; this digital atlas of zebrafish development has been conceived prior to the expansion of the GEMS. The atlas contains 3D volume models of canonical stages of zebrafish development in which in each volume model element is annotated with an anatomical term. These terms are extracted from a formal anatomical ontology, i.e. the Developmental Anatomy Ontology of Zebrafish (DAOZ. In the GEMS, anatomical terms from this ontology together with terms from the Gene Ontology (GO are also used to annotate patterns of gene expression and in this manner providing mechanisms for integration and retrieval . The annotations are the glue for integration of patterns of gene expression in GEMS as well as in other biomolecular databases. At the one hand, zebrafish anatomy terminology allows gene expression data within GEMS to be integrated with phenotypical data in the 3D atlas of zebrafish development. At the other hand, GO terms extend GEMS expression patterns integration to a wide range of bioinformatics resources.

  19. Large-scale analysis of acute ethanol exposure in zebrafish development: a critical time window and resilience.

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    Shaukat Ali

    Full Text Available BACKGROUND: In humans, ethanol exposure during pregnancy causes a spectrum of developmental defects (fetal alcohol syndrome or FAS. Individuals vary in phenotypic expression. Zebrafish embryos develop FAS-like features after ethanol exposure. In this study, we ask whether stage-specific effects of ethanol can be identified in the zebrafish, and if so, whether they allow the pinpointing of sensitive developmental mechanisms. We have therefore conducted the first large-scale (>1500 embryos analysis of acute, stage-specific drug effects on zebrafish development, with a large panel of readouts. METHODOLOGY/PRINCIPAL FINDINGS: Zebrafish embryos were raised in 96-well plates. Range-finding indicated that 10% ethanol for 1 h was suitable for an acute exposure regime. High-resolution magic-angle spinning proton magnetic resonance spectroscopy showed that this produced a transient pulse of 0.86% concentration of ethanol in the embryo within the chorion. Survivors at 5 days postfertilisation were analysed. Phenotypes ranged from normal (resilient to severely malformed. Ethanol exposure at early stages caused high mortality (≥88%. At later stages of exposure, mortality declined and malformations developed. Pharyngeal arch hypoplasia and behavioral impairment were most common after prim-6 and prim-16 exposure. By contrast, microphthalmia and growth retardation were stage-independent. CONCLUSIONS: Our findings show that some ethanol effects are strongly stage-dependent. The phenotypes mimic key aspects of FAS including craniofacial abnormality, microphthalmia, growth retardation and behavioral impairment. We also identify a critical time window (prim-6 and prim-16 for ethanol sensitivity. Finally, our identification of a wide phenotypic spectrum is reminiscent of human FAS, and may provide a useful model for studying disease resilience.

  20. Cell fate determination in zebrafish embryonic and adult muscle development

    NARCIS (Netherlands)

    Tee, J.M.

    2010-01-01

    We are interested in how the genetic basis of muscle precursor cells determines the outcome of the muscle cell fate, and thus leading to disruption in muscle formation and maintenance. We utilized the zebrafish carrying mutations in both Axin1 and Apc1, resulting in overactivation of the

  1. Differential Toxicity of mDia Formin-Directed Functional Agonists and Antagonists in Developing Zebrafish

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    Hunter LeCorgne

    2018-04-01

    Full Text Available The mammalian Diaphanous-related (mDia formins are cytoskeletal regulators that assemble and, in some cases, bundle filamentous actin (F-actin, as well as stabilize microtubules. The development of small molecule antagonists and agonists that interrogate mDia formin function has allowed us to investigate the roles of formins in disease states. A small molecule inhibitor of FH2 domain (SMIFH2 inhibits mDia-dependent actin dynamics and abrogates tumor cell migration and cell division in vitro and ex vivo tissue explants. mDia formin activation with small molecule intramimics IMM01/02 and mDia2-DAD peptides inhibited glioblastoma motility and invasion in vitro and ex vivo rat brain slices. However, SMIFH2, IMMs, and mDia2 DAD efficacy in vivo remains largely unexplored and potential toxicity across a range of developmental phenotypes has not been thoroughly characterized. In this study, we performed an in vivo screen of early life-stage toxicity in Danio rerio zebrafish embryos 2 days post-fertilization (dpf in response to SMIFH2, IMM01/02, and mDia2 DAD. SMIFH2 at concentrations ≥5–10 μM induced significant defects in developing zebrafish, including shorter body lengths, tail curvature and defective tail cellularity, craniofacial malformations, pericardial edema, absent and/or compromised vasculature function and flow, depressed heart rates and increased mortality. Conversely, IMM and mDia2 DAD peptides were minimally toxic at concentrations up to 10–20 and 50 μM, respectively. SMIFH2's therapeutic potential may therefore be limited by its substantial in vivo toxicity at functional concentrations. mDia formin agonism with IMMs and mDia2 DADs may therefore be a more effective and less toxic anti-invasive therapeutic approach.

  2. A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development

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    Sugimoto Masahiko

    2010-07-01

    Full Text Available Abstract Background Development and maintenance of the blood-brain and blood-retinal barrier is critical for the homeostasis of brain and retinal tissue. Despite decades of research our knowledge of the formation and maintenance of the blood-brain (BBB and blood-retinal (BRB barrier is very limited. We have established an in vivo model to study the development and maintenance of these barriers by generating a transgenic zebrafish line that expresses a vitamin D-binding protein fused with enhanced green fluorescent protein (DBP-EGFP in blood plasma, as an endogenous tracer. Results The temporal establishment of the BBB and BRB was examined using this transgenic line and the results were compared with that obtained by injection of fluorescent dyes into the sinus venosus of embryos at various stages of development. We also examined the expression of claudin-5, a component of tight junctions during the first 4 days of development. We observed that the BBB of zebrafish starts to develop by 3 dpf, with expression of claudin-5 in the central arteries preceding it at 2 dpf. The hyaloid vasculature in the zebrafish retina develops a barrier function at 3 dpf, which endows the zebrafish with unique advantages for studying the BRB. Conclusion Zebrafish embryos develop BBB and BRB function simultaneously by 3 dpf, which is regulated by tight junction proteins. The Tg(l-fabp:DBP-EGFP zebrafish will have great advantages in studying development and maintenance of the blood-neural barrier, which is a new application for the widely used vertebrate model.

  3. Development of an opioid self-administration assay to study drug seeking in zebrafish.

    Science.gov (United States)

    Bossé, Gabriel D; Peterson, Randall T

    2017-09-29

    The zebrafish (Danio rerio) has become an excellent tool to study mental health disorders, due to its physiological and genetic similarity to humans, ease of genetic manipulation, and feasibility of small molecule screening. Zebrafish have been shown to exhibit characteristics of addiction to drugs of abuse in non-contingent assays, including conditioned place preference, but contingent assays have been limited to a single assay for alcohol consumption. Using inexpensive electronic, mechanical, and optical components, we developed an automated opioid self-administration assay for zebrafish, enabling us to measure drug seeking and gain insight into the underlying biological pathways. Zebrafish trained in the assay for five days exhibited robust self-administration, which was dependent on the function of the μ-opioid receptor. In addition, a progressive ratio protocol was used to test conditioned animals for motivation. Furthermore, conditioned fish continued to seek the drug despite an adverse consequence and showed signs of stress and anxiety upon withdrawal of the drug. Finally, we validated our assay by confirming that self-administration in zebrafish is dependent on several of the same molecular pathways as in other animal models. Given the ease and throughput of this assay, it will enable identification of important biological pathways regulating drug seeking and could lead to the development of new therapeutic molecules to treat addiction. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Development of Alginate Microspheres Containing Chuanxiong for Oral Administration to Adult Zebrafish

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    Li-Jen Lin

    2016-01-01

    Full Text Available Oral administration of Traditional Chinese Medicine (TCM by patients is the common way to treat health problems. Zebrafish emerges as an excellent animal model for the pharmacology investigation. However, the oral delivery system of TCM in zebrafish has not been established so far. This issue was addressed by development of alginate microparticles for oral delivery of chuanxiong, a TCM that displays antifibrotic and antiproliferative effects on hepatocytes. The delivery microparticles were prepared from gelification of alginate containing various levels of chuanxiong. The chuanxiong-encapsulated alginate microparticles were characterized for their solubility, structure, encapsulation efficiency, the cargo release profile, and digestion in gastrointestinal tract of zebrafish. Encapsulation of chuanxiong resulted in more compact structure and the smaller size of microparticles. The release rate of chuanxiong increased for alginate microparticles carrying more chuanxiong in simulated intestinal fluid. This remarkable feature ensures the controlled release of encapsulated cargos in the gastrointestinal tract of zebrafish. Moreover, chuanxiong-loaded alginate microparticles were moved to the end of gastrointestinal tract after oral administration for 6 hr and excreted from the body after 16 hr. Therefore, our developed method for oral administration of TCM in zebrafish is useful for easy and rapid evaluation of the drug effect on disease.

  5. Transient knockdown of tyrosine hydroxylase during development has persistent effects on behaviour in adult zebrafish (Danio rerio.

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    Isabel Formella

    Full Text Available Abnormal dopamine (DA signaling is often suggested as causative in schizophrenia. The other prominent hypothesis for this disorder, largely driven by epidemiological data, is that certain adverse events during the early stages of brain development increase an individual's risk of developing schizophrenia later in life. However, the clinical and preclinical literature consistently implicates behavioural, cognitive, and pharmacological abnormalities, implying that DA signaling is abnormal in the adult brain. How can we reconcile these two major hypotheses underlying much of the clinical and basic research into schizophrenia? In this study we have transiently knocked down tyrosine hydroxylase (TH, the rate limiting enzyme in DA synthesis gene expression in the early stages of brain development in zebrafish using morpholinos. We show that by adulthood, TH and DA levels have returned to normal and basic DA-mediated behaviours, such as locomotion, are also normal. However, when they were exposed to a novel environment the levels of freezing and immediate positioning in deeper zones were significantly reduced in these adult fish. The neurochemistry underlying these behaviours is complex, and the exact mechanisms for these abnormal behaviours remains unknown. This study demonstrates that early transient alterations in DA ontogeny can produce persistent alterations in adult brain function and suggests that the zebrafish may be a promising model animal for future studies directed at clarifying the basic neurodevelopmental mechanisms behind complex psychiatric disease.

  6. Lnx2 ubiquitin ligase is essential for exocrine cell differentiation in the early zebrafish pancreas.

    Science.gov (United States)

    Won, Minho; Ro, Hyunju; Dawid, Igor B

    2015-10-06

    The gene encoding the E3 ubiquitin ligase Ligand of Numb protein-X (Lnx)2a is expressed in the ventral-anterior pancreatic bud of zebrafish embryos in addition to its expression in the brain. Knockdown of Lnx2a by using an exon 2/intron 2 splice morpholino resulted in specific inhibition of the differentiation of ventral bud derived exocrine cell types, with little effect on endocrine cell types. A frame shifting null mutation in lnx2a did not mimic this phenotype, but a mutation that removed the exon 2 splice donor site did. We found that Lnx2b functions in a redundant manner with its paralog Lnx2a. Inhibition of lnx2a exon 2/3 splicing causes exon 2 skipping and leads to the production of an N-truncated protein that acts as an interfering molecule. Thus, the phenotype characterized by inhibition of exocrine cell differentiation requires inactivation of both Lnx2a and Lnx2b. Human LNX1 is known to destabilize Numb, and we show that inhibition of Numb expression rescues the Lnx2a/b-deficient phenotype. Further, Lnx2a/b inhibition leads to a reduction in the number of Notch active cells in the pancreas. We suggest that Lnx2a/b function to fine tune the regulation of Notch through Numb in the differentiation of cell types in the early zebrafish pancreas. Further, the complex relationships among genotype, phenotype, and morpholino effect in this case may be instructive in the ongoing consideration of morpholino use.

  7. Effects of decreased muscle activity on developing axial musculature in nic b107 mutant zebrafish (Danio rerio)

    NARCIS (Netherlands)

    Meulen, van der T.; Schipper, H.; Leeuwen, van J.L.; Kranenbarg, S.

    2005-01-01

    The present paper discusses the effects of decreased muscle activity (DMA) on embryonic development in the zebrafish. Wild-type zebrafish embryos become mobile around 18 h post-fertilisation, long before the axial musculature is fully differentiated. As a model for DMA, the nicb107 mutant was used.

  8. The Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease 2012

    Science.gov (United States)

    Zetterberg, Henrik

    2013-01-01

    Abstract The first Nordic Countries Meeting on the Zebrafish as a Model for Development and Disease took place at Karolinska Institutet in Stockholm, November 21–23, 2012. The meeting gathered 130 scientists, students, and company representatives from Iceland, Finland, Norway, Denmark, and Sweden, as well as invited guests and keynote speakers from England, Scotland, Germany, Poland, The Netherlands, Singapore, Japan, and the United States. Presentations covered a wide range of topics, including developmental biology, genetics, evolutionary biology, toxicology, behavioral studies, and disease mechanisms. The need for formal guidance and training in zebrafish housing, husbandry, and health monitoring was recognized, and the meeting expressed its support for the joint working group of the FELASA/COST action BM0804 EuFishBioMed. The decision was made to turn the Nordic meeting into an annual event and create a Nordic network of zebrafish researchers. PMID:23590403

  9. Label-free imaging of developing vasculature in zebrafish with phase variance optical coherence microscopy

    Science.gov (United States)

    Chen, Yu; Fingler, Jeff; Trinh, Le A.; Fraser, Scott E.

    2016-03-01

    A phase variance optical coherence microscope (pvOCM) has been created to visualize blood flow in the vasculature of zebrafish embryos, without using exogenous labels. The pvOCM imaging system has axial and lateral resolutions of 2 μm in tissue, and imaging depth of more than 100 μm. Imaging of 2-5 days post-fertilization zebrafish embryos identified the detailed structures of somites, spinal cord, gut and notochord based on intensity contrast. Visualization of the blood flow in the aorta, veins and intersegmental vessels was achieved with phase variance contrast. The pvOCM vasculature images were confirmed with corresponding fluorescence microscopy of a zebrafish transgene that labels the vasculature with green fluorescent protein. The pvOCM images also revealed functional information of the blood flow activities that is crucial for the study of vascular development.

  10. External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio

    International Nuclear Information System (INIS)

    Gagnaire, B.; Cavalié, I.; Pereira, S.; Floriani, M.; Dubourg, N.; Camilleri, V.; Adam-Guillermin, C.

    2015-01-01

    Highlights: • The present study aimed to evaluate the effects of gamma rays on zebrafish larvae. • Different techniques were used: gene expression, biochemistry, microscopy and macroscopical observations. • The results showed that gamma irradiation can alter embryo-larval development at several levels of organization. - Abstract: In the general context of validation of tools useful for the characterization of ecological risk linked to ionizing radiation, the effects of an external gamma irradiation were studied in zebrafish larvae irradiated for 96 h with two dose rates: 0.8 mGy/d, which is close to the level recommended to protect ecosystems from adverse effects of ionizing radiation (0.24 mGy/d) and a higher dose rate of 570 mGy/d. Several endpoints were investigated, such as mortality, hatching, and some parameters of embryo-larval development, immunotoxicity, apoptosis, genotoxicity, neurotoxicity and histological alterations. Results showed that an exposure to gamma rays induced an acceleration of hatching for both doses and a decrease of yolk bag diameter for the highest dose, which could indicate an increase of global metabolism. AChE activity decreased with the low dose rate of gamma irradiation and alterations were also shown in muscles of irradiated larvae. These results suggest that gamma irradiation can induce damages on larval neurotransmission, which could have repercussions on locomotion. DNA damages, basal ROS production and apoptosis were also induced by irradiation, while ROS stimulation index and EROD biotransformation activity were decreased and gene expression of acetylcholinesterase, choline acetyltransferase, cytochrome p450 and myeloperoxidase increased. These results showed that ionizing radiation induced an oxidative stress conducting to DNA damages. This study characterized further the modes of action of ionizing radiation in fish.

  11. External gamma irradiation-induced effects in early-life stages of zebrafish, Danio rerio

    Energy Technology Data Exchange (ETDEWEB)

    Gagnaire, B., E-mail: beatrice.gagnaire@irsn.fr [Institut de Radioprotection et de Sureté Nucléaire (IRSN), PRP-ENV/SERIS/LECO, Cadarache, Saint-Paul-lez-Durance 13115 (France); Cavalié, I. [Institut de Radioprotection et de Sureté Nucléaire (IRSN), PRP-ENV/SERIS/LECO, Cadarache, Saint-Paul-lez-Durance 13115 (France); Pereira, S. [Neolys Diagnostics, Lyon 69373 (France); Floriani, M.; Dubourg, N.; Camilleri, V.; Adam-Guillermin, C. [Institut de Radioprotection et de Sureté Nucléaire (IRSN), PRP-ENV/SERIS/LECO, Cadarache, Saint-Paul-lez-Durance 13115 (France)

    2015-12-15

    Highlights: • The present study aimed to evaluate the effects of gamma rays on zebrafish larvae. • Different techniques were used: gene expression, biochemistry, microscopy and macroscopical observations. • The results showed that gamma irradiation can alter embryo-larval development at several levels of organization. - Abstract: In the general context of validation of tools useful for the characterization of ecological risk linked to ionizing radiation, the effects of an external gamma irradiation were studied in zebrafish larvae irradiated for 96 h with two dose rates: 0.8 mGy/d, which is close to the level recommended to protect ecosystems from adverse effects of ionizing radiation (0.24 mGy/d) and a higher dose rate of 570 mGy/d. Several endpoints were investigated, such as mortality, hatching, and some parameters of embryo-larval development, immunotoxicity, apoptosis, genotoxicity, neurotoxicity and histological alterations. Results showed that an exposure to gamma rays induced an acceleration of hatching for both doses and a decrease of yolk bag diameter for the highest dose, which could indicate an increase of global metabolism. AChE activity decreased with the low dose rate of gamma irradiation and alterations were also shown in muscles of irradiated larvae. These results suggest that gamma irradiation can induce damages on larval neurotransmission, which could have repercussions on locomotion. DNA damages, basal ROS production and apoptosis were also induced by irradiation, while ROS stimulation index and EROD biotransformation activity were decreased and gene expression of acetylcholinesterase, choline acetyltransferase, cytochrome p450 and myeloperoxidase increased. These results showed that ionizing radiation induced an oxidative stress conducting to DNA damages. This study characterized further the modes of action of ionizing radiation in fish.

  12. Effect of X-ray Contrast Media, Chlorination, and Chloramination on Zebrafish Development

    Science.gov (United States)

    Effect of X-ray Contrast Media, Chlorination, and Chloramination on Zebrafish Development Little is known about the vertebrate developmental toxicity of chlorinated or chloraminated drinking water (DW), iodinated X-ray contrast media (ICM, a common contaminate of DW) or how the c...

  13. Endurance exercise differentially stimulates heart and axial muscle development in zebrafish (Danio rerio)

    NARCIS (Netherlands)

    Meulen, T. van der; Schipper, H.; Boogaart, J.G. van den; Huising, M.O.; Kranenbarg, S.; Leeuwen, J.L. van

    2006-01-01

    Mechanical load is an important factor in the differentiation of cells and tissues. To investigate the effects of increased mechanical load on development of muscle and bone, zebrafish were subjected to endurance swim training for 6 h/day for 10 wk starting at 14 days after fertilization. During the

  14. Identification and expression analysis of zebrafish polypeptide α-N-acetylgalactosaminyltransferase Y-subfamily genes during embryonic development.

    Science.gov (United States)

    Nakayama, Yoshiaki; Nakamura, Naosuke; Kawai, Tamiko; Kaneda, Eiichi; Takahashi, Yui; Miyake, Ayumi; Itoh, Nobuyuki; Kurosaka, Akira

    2014-09-01

    Mucin-type glycosylation is one of the most common posttranslational modifications of secretory and membrane proteins and has diverse physiological functions. The initial biosynthesis of mucin-type carbohydrates is catalyzed by UDP-GalNAc: polypeptide α-N-acetylgalactosaminyltransferases (GalNAc-Ts) encoded by GALNT genes. Among these, GalNAc-T8, -T9, -T17, and -T18 form a characteristic subfamily called "Y-subfamily" and have no or very low in vitro transferase activities when assayed with typical mucin peptides as acceptor substrates. Although the Y-subfamily isozymes have been reported to be possibly involved in various diseases, their in vivo functions have not been reported. Here, we isolated zebrafish Y-subfamily galnt genes, and determined their spatial and temporal expressions during the early development of zebrafish. Our study demonstrated that all the Y-subfamily isozymes were well conserved in zebrafish with GalNAc-T18 having two orthologs, galnt18a and galnt18b, and with the other three isozymes each having a corresponding ortholog, galnt8, galnt9, and galnt17. The galnt8 was expressed in the cephalic mesoderm and hatching gland during early developmental stages, and differently expressed in the head, somatic muscles, and liver in the later stages. The other three orthologs also exhibited the characteristic expression patterns, although their expressions were generally strong in the nervous systems. In addition to the expression in the brain, galnt17 and galnt18a were expressed in the somitic muscles, and galnt18a and galnt18b in the notochord. These expression patterns may contribute to the functional analysis of the Y-subfamily, whose physiological roles still remain to be elucidated. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Functional validation of GWAS gene candidates for abnormal liver function during zebrafish liver development

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    Leah Y. Liu

    2013-09-01

    Genome-wide association studies (GWAS have revealed numerous associations between many phenotypes and gene candidates. Frequently, however, further elucidation of gene function has not been achieved. A recent GWAS identified 69 candidate genes associated with elevated liver enzyme concentrations, which are clinical markers of liver disease. To investigate the role of these genes in liver homeostasis, we narrowed down this list to 12 genes based on zebrafish orthology, zebrafish liver expression and disease correlation. To assess the function of gene candidates during liver development, we assayed hepatic progenitors at 48 hours post fertilization (hpf and hepatocytes at 72 hpf using in situ hybridization following morpholino knockdown in zebrafish embryos. Knockdown of three genes (pnpla3, pklr and mapk10 decreased expression of hepatic progenitor cells, whereas knockdown of eight genes (pnpla3, cpn1, trib1, fads2, slc2a2, pklr, mapk10 and samm50 decreased cell-specific hepatocyte expression. We then induced liver injury in zebrafish embryos using acetaminophen exposure and observed changes in liver toxicity incidence in morphants. Prioritization of GWAS candidates and morpholino knockdown expedites the study of newly identified genes impacting liver development and represents a feasible method for initial assessment of candidate genes to instruct further mechanistic analyses. Our analysis can be extended to GWAS for additional disease-associated phenotypes.

  16. Differential induction of four msx homeobox genes during fin development and regeneration in zebrafish.

    Science.gov (United States)

    Akimenko, M A; Johnson, S L; Westerfield, M; Ekker, M

    1995-02-01

    To study the genetic regulation of growth control and pattern formation during fin development and regeneration, we have analysed the expression of four homeobox genes, msxA, msxB, msxC and msxD in zebrafish fins. The median fin fold, which gives rise to the unpaired fins, expresses these four msx genes during development. Transcripts of the genes are also present in cells of the presumptive pectoral fin buds. The most distal cells, the apical ectodermal ridge of the paired fins and the cleft and flanking cells of the median fin fold express all these msx genes with the exception of msxC. Mesenchymal cells underlying the most distal cells express all four genes. Expression of the msx genes in the fin fold and fin buds is transient and, by 3 days after fertilization, msx expression in the median fin fold falls below levels detectable by in situ hybridization. Although the fins of adult zebrafish normally have levels of msx transcripts undetectable by in situ hybridization, expression of all four genes is strongly reinduced during regeneration of both paired and unpaired fins. Induction of msx gene expression in regenerating caudal fins occurs as early as 30 hours postamputation. As the blastema forms, the levels of expression increase and reach a maximum between the third and fifth days. Then, msx expression progressively declines and disappears by day 12 when the caudal fin has grown back to its normal size. In the regenerating fin, the blastema cells that develop at the tip of each fin ray express msxB and msxC. Cells of the overlying epithelium express msxA and msxD, but do not express msxB or msxC. Amputations at various levels along the proximodistal axis of the fin suggest that msxB expression depends upon the position of the blastema, with cells of the rapidly proliferating proximal blastema expressing higher levels than the cells of the less rapidly proliferating distal blastema. Expression of msxC and msxD is independent of the position of the blastema cell

  17. Estrogenic effects of several BPA analogs in the developing zebrafish brain

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    Joel eCano-Nicolau

    2016-03-01

    Full Text Available Important set of studies have demonstrated the endocrine disrupting activity of Bisphenol A (BPA. The present work aimed at defining estrogenic-like activity of several BPA structural analogs, including BPS, BPF, BPAF, and BPAP, on 4-day or 7-day post-fertilization (dpf zebrafish larva as an in vivo model. We measured the induction level of the estrogen-sensitive marker cyp19a1b gene (Aromatase B, expressed in the brain, using three different in situ/in vivo strategies: 1 Quantification of cyp19a1b transcripts using RT-qPCR in wild type 7-dpf larva brains exposed to bisphenols ; 2 Detection and distribution of cyp19a1b transcripts using in situ hybridization on 7-dpf brain sections (hypothalamus; and 3 Quantification of the cyp19a1b promoter activity in live cyp19a1b-GFP transgenic zebrafish (EASZY assay at 4-dpf larval stage. These three different experimental approaches demonstrated that BPS, BPF or BPAF exposure, similarly to BPA, significantly activates the expression of the estrogenic marker in the brain of developing zebrafish. In vitro experiments using both reporter gene assay in a glial cell context and competitive ligand binding assays strongly suggested that up-regulation of cyp19a1b is largely mediated by the zebrafish estrogen nuclear receptor alpha (zfERα. Importantly, and in contrast to other tested bisphenol A analogs, the bisphenol AP (BPAP did not show estrogenic activity in our model.

  18. Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

    Science.gov (United States)

    Edeling, Melissa A; Sanker, Subramaniam; Shima, Takaki; Umasankar, P K; Höning, Stefan; Kim, Hye Y; Davidson, Lance A; Watkins, Simon C; Tsang, Michael; Owen, David J; Traub, Linton M

    2009-12-03

    PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

  19. Structural requirements for PACSIN/Syndapin operation during zebrafish embryonic notochord development.

    Directory of Open Access Journals (Sweden)

    Melissa A Edeling

    2009-12-01

    Full Text Available PACSIN/Syndapin proteins are membrane-active scaffolds that participate in endocytosis. The structure of the Drosophila Syndapin N-terminal EFC domain reveals a crescent shaped antiparallel dimer with a high affinity for phosphoinositides and a unique membrane-inserting prong upon the concave surface. Combined structural, biochemical and reverse genetic approaches in zebrafish define an important role for Syndapin orthologue, Pacsin3, in the early formation of the notochord during embryonic development. In pacsin3-morphant embryos, midline convergence of notochord precursors is defective as axial mesodermal cells fail to polarize, migrate and differentiate properly. The pacsin3 morphant phenotype of a stunted body axis and contorted trunk is rescued by ectopic expression of Drosophila Syndapin, and depends critically on both the prong that protrudes from the surface of the bowed Syndapin EFC domain and the ability of the antiparallel dimer to bind tightly to phosphoinositides. Our data confirm linkage between directional migration, endocytosis and cell specification during embryonic morphogenesis and highlight a key role for Pacsin3 in this coupling in the notochord.

  20. Development of a transient expression assay for detecting environmental oestrogens in zebrafish and medaka embryos

    Directory of Open Access Journals (Sweden)

    Lee Okhyun

    2012-06-01

    Full Text Available Abstract Background Oestrogenic contaminants are widespread in the aquatic environment and have been shown to induce adverse effects in both wildlife (most notably in fish and humans, raising international concern. Available detecting and testing systems are limited in their capacity to elucidate oestrogen signalling pathways and physiological impacts. Here we developed a transient expression assay to investigate the effects of oestrogenic chemicals in fish early life stages and to identify target organs for oestrogenic effects. To enhance the response sensitivity to oestrogen, we adopted the use of multiple tandem oestrogen responsive elements (EREc38 in a Tol2 transposon mediated Gal4ff-UAS system. The plasmid constructed (pTol2_ERE-TATA-Gal4ff, contains three copies of oestrogen response elements (3ERE that on exposure to oestrogen induces expression of Gal4ff which this in turn binds Gal4-responsive Upstream Activated Sequence (UAS elements, driving the expression of a second reporter gene, EGFP (Enhanced Green Fluorescent Protein. Results The response of our construct to oestrogen exposure in zebrafish embryos was examined using a transient expression assay. The two plasmids were injected into 1–2 cell staged zebrafish embryos, and the embryos were exposed to various oestrogens including the natural steroid oestrogen 17ß-oestradiol (E2, the synthetic oestrogen 17α- ethinyloestradiol (EE2, and the relatively weak environmental oestrogen nonylphenol (NP, and GFP expression was examined in the subsequent embryos using fluorescent microscopy. There was no GFP expression detected in unexposed embryos, but specific and mosaic expression of GFP was detected in the liver, heart, somite muscle and some other tissue cells for exposures to steroid oestrogen treatments (EE2; 10 ng/L, E2; 100 ng/L, after 72 h exposures. For the NP exposures, GFP expression was observed at 10 μg NP/L after 72 h (100 μg NP/L was toxic to the fish. We

  1. Myosin-1 inhibition by PClP affects membrane shape, cortical actin distribution and lipid droplet dynamics in early Zebrafish embryos.

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    Prabuddha Gupta

    Full Text Available Myosin-1 (Myo1 represents a mechanical link between the membrane and actin-cytoskeleton in animal cells. We have studied the effect of Myo1 inhibitor PClP in 1-8 cell Zebrafish embryos. Our results indicate a unique involvement of Myo1 in early development of Zebrafish embryos. Inhibition of Myo1 (by PClP and Myo2 (by Blebbistatin lead to arrest in cell division. While Myo1 isoforms appears to be important for both the formation and the maintenance of cleavage furrows, Myo2 is required only for the formation of furrows. We found that the blastodisc of the embryo, which contains a thick actin cortex (~13 μm, is loaded with cortical Myo1. Myo1 appears to be crucial for maintaining the blastodisc morphology and the actin cortex thickness. In addition to cell division and furrow formation, inhibition of Myo1 has a drastic effect on the dynamics and distribution of lipid droplets (LDs in the blastodisc near the cleavage furrow. All these results above are effects of Myo1 inhibition exclusively; Myo2 inhibition by blebbistatin does not show such phenotypes. Therefore, our results demonstrate a potential role for Myo1 in the maintenance and formation of furrow, blastodisc morphology, cell-division and LD organization within the blastodisc during early embryogenesis.

  2. Critical early roles for col27a1a and col27a1b in zebrafish notochord morphogenesis, vertebral mineralization and post-embryonic axial growth.

    Science.gov (United States)

    Christiansen, Helena E; Lang, Michael R; Pace, James M; Parichy, David M

    2009-12-29

    Fibrillar collagens are well known for their links to human diseases, with which all have been associated except for the two most recently identified fibrillar collagens, type XXIV collagen and type XXVII collagen. To assess functions and potential disease phenotypes of type XXVII collagen, we examined its roles in zebrafish embryonic and post-embryonic development. We identified two type XXVII collagen genes in zebrafish, col27a1a and col27a1b. Both col27a1a and col27a1b were expressed in notochord and cartilage in the embryo and early larva. To determine sites of type XXVII collagen function, col27a1a and col27a1b were knocked down using morpholino antisense oligonucleotides. Knockdown of col27a1a singly or in conjunction with col27a1b resulted in curvature of the notochord at early stages and formation of scoliotic curves as well as dysmorphic vertebrae at later stages. These defects were accompanied by abnormal distributions of cells and protein localization in the notochord, as visualized by transmission electron microscopy, as well as delayed vertebral mineralization as detected histologically. Together, our findings indicate a key role for type XXVII collagen in notochord morphogenesis and axial skeletogenesis and suggest a possible human disease phenotype.

  3. Kaempferol suppresses lipid accumulation by inhibiting early adipogenesis in 3T3-L1 cells and zebrafish.

    Science.gov (United States)

    Lee, Yeon-Joo; Choi, Hyeon-Son; Seo, Min-Jung; Jeon, Hui-Jeon; Kim, Kui-Jin; Lee, Boo-Yong

    2015-08-01

    Kaempferol is a flavonoid present in Kaempferia galanga and Opuntia ficus indica var. saboten. Recent studies have suggested that it has anti-oxidant, anti-inflammatory, anti-cancer, and anti-obesity effects. In this study, we focused on the anti-adipogenic effects of kaempferol during adipocyte differentiation. The results showed that kaempferol inhibits lipid accumulation in adipocytes and zebrafish. Oil Red O and Nile Red staining showed that the number of intracellular lipid droplets decreased in adipocytes and zebrafish treated with kaempferol. LPAATθ (lysophosphatidic acid acyltransferase), lipin1, and DGAT1 (triglyceride synthetic enzymes) and FASN and SREBP-1C (fatty acid synthetic proteins) showed decreased expression levels in the presence of kaempferol. In addition, treatment of kaempferol showed an inhibitory activity on cell cycle progression. Kaempferol delayed cell cycle progression from the S to G2/M phase through the regulation of cyclins in a dose-dependent manner. Kaempferol blocked the phosphorylation of AKT (protein kinase B) and mammalian target of rapamycin (mTOR) signaling pathway during the early stages of adipogenesis. In addition, kaempferol down-regulated pro-early adipogenic factors such as CCAAT-enhancer binding proteins β (C/EBPβ), and Krüppel-like factors (KLFs) 4 and 5, while anti-early adipogenic factors, such as KLF2 and pref-1(preadipocyte factor-1), were upregulated. These kaempferol-mediated regulations of early adipogenic factors resulted in the attenuation of late adipogenic factors such as C/EBPα and peroxisome proliferator-activated receptor γ (PPARγ). These results were supported in zebrafish based on the decrease in lipid accumulation and expression of adipogenic factors. Our results indicated that kaempferol might have an anti-obesity effect by regulating lipid metabolism.

  4. Vitamin D receptor signaling is required for heart development in zebrafish embryo

    Energy Technology Data Exchange (ETDEWEB)

    Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa [Biology Department, Texas A& M University, College Station, TX77843-3258 (United States); Biology Department, Princess Nourah University, Riyadh 11671 (Saudi Arabia)

    2016-02-12

    Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.

  5. Vitamin D receptor signaling is required for heart development in zebrafish embryo

    International Nuclear Information System (INIS)

    Kwon, Hye-Joo

    2016-01-01

    Vitamin D has been found to be associated with cardiovascular diseases. However, the role of vitamin D in heart development during embryonic period is largely unknown. Vitamin D induces its genomic effects through its nuclear receptor, the vitamin D receptor (VDR). The present study investigated the role of VDR on heart development by antisense-mediated knockdown approaches in zebrafish model system. In zebrafish embryos, two distinct VDR genes (vdra and vdrb) have been identified. Knockdown of vdra has little effect on heart development, whereas disrupting vdrb gene causes various cardiac phenotypes, characterized by pericardial edema, slower heart rate and laterality defects. Depletion of both vdra and vdrb (vdra/b) produce additive, but not synergistic effects. To determine whether atrioventricular (AV) cardiomyocytes are properly organized in these embryos, the expression of bmp4, which marks the developing AV boundary at 48 h post-fertilization, was examined. Notably, vdra/b-deficient embryos display ectopic expression of bmp4 towards the ventricle or throughout atrial and ventricular chambers. Taken together, these results suggest that VDR signaling plays an essential role in heart development. - Highlights: • VDR signaling is involved in embryonic heart development. • Knockdown of vdrb, but not vdra, causes decreased heart rate in zebrafish embryo. • Loss of vdr results in cardiac laterality defects. • Loss of vdra/b alters atrioventricular boundary formation. • Loss of vdra/b causes abnormal cardiac looping.

  6. A Versatile Mounting Method for Long Term Imaging of Zebrafish Development.

    Science.gov (United States)

    Hirsinger, Estelle; Steventon, Ben

    2017-01-26

    Zebrafish embryos offer an ideal experimental system to study complex morphogenetic processes due to their ease of accessibility and optical transparency. In particular, posterior body elongation is an essential process in embryonic development by which multiple tissue deformations act together to direct the formation of a large part of the body axis. In order to observe this process by long-term time-lapse imaging it is necessary to utilize a mounting technique that allows sufficient support to maintain samples in the correct orientation during transfer to the microscope and acquisition. In addition, the mounting must also provide sufficient freedom of movement for the outgrowth of the posterior body region without affecting its normal development. Finally, there must be a certain degree in versatility of the mounting method to allow imaging on diverse imaging set-ups. Here, we present a mounting technique for imaging the development of posterior body elongation in the zebrafish D. rerio. This technique involves mounting embryos such that the head and yolk sac regions are almost entirely included in agarose, while leaving out the posterior body region to elongate and develop normally. We will show how this can be adapted for upright, inverted and vertical light-sheet microscopy set-ups. While this protocol focuses on mounting embryos for imaging for the posterior body, it could easily be adapted for the live imaging of multiple aspects of zebrafish development.

  7. F-spondin/spon1b expression patterns in developing and adult zebrafish.

    Directory of Open Access Journals (Sweden)

    Veronica Akle

    Full Text Available F-spondin, an extracellular matrix protein, is an important player in embryonic morphogenesis and CNS development, but its presence and role later in life remains largely unknown. We generated a transgenic zebrafish in which GFP is expressed under the control of the F-spondin (spon1b promoter, and used it in combination with complementary techniques to undertake a detailed characterization of the expression patterns of F-spondin in developing and adult brain and periphery. We found that F-spondin is often associated with structures forming long neuronal tracts, including retinal ganglion cells, the olfactory bulb, the habenula, and the nucleus of the medial longitudinal fasciculus (nMLF. F-spondin expression coincides with zones of adult neurogenesis and is abundant in CSF-contacting secretory neurons, especially those in the hypothalamus. Use of this new transgenic model also revealed F-spondin expression patterns in the peripheral CNS, notably in enteric neurons, and in peripheral tissues involved in active patterning or proliferation in adults, including the endoskeleton of zebrafish fins and the continuously regenerating pharyngeal teeth. Moreover, patterning of the regenerating caudal fin following fin amputation in adult zebrafish was associated with F-spondin expression in the blastema, a proliferative region critical for tissue reconstitution. Together, these findings suggest major roles for F-spondin in the CNS and periphery of the developing and adult vertebrate.

  8. The effect of MRN complex and ATM kinase inhibitors on Zebrafish embryonic development

    Science.gov (United States)

    Kumaran, Malina; Fazry, Shazrul

    2018-04-01

    Zebrafish is an ideal animal model to study developmental biology due to its transparent embryos and rapid development stages of embryogenesis. Here we investigate the role of DNA damage proteins, specifically Mre11/Rad50/NBN (MRN) complex and ataxia-telangiectasia mutated (ATM) kinase during embryogenesis by inhibiting its function using specific MRN complex (Mirin) and ATM Kinase inhibitors (Ku60019 and Ku55933). Zebrafish embryos at midblastula transition (MBT) stage are treated with Mirin, Ku60019 and Ku55933. The embryonic development of the embryos was monitored at 24 hours-post fertilisation (hpf), 48 hpf and 72 hpf. We observed that at the lowest concentrations (3 µM of Mirin, 1.5 nM of Ku60019 and 3 nM of Ku55933), the inhibitors treated embryos have 100% survivability. However, with increasing inhibitor concentration, the survivability drops. Control or mock treatment of all embryos shows 100 % survivability rate. This study suggests that DNA damage repair proteins may be crucial for normal zebrafish embryo development and survival.

  9. Development of the zebrafish myoseptum with emphasis on the myotendinous junction.

    Science.gov (United States)

    Charvet, Benjamin; Malbouyres, Marilyne; Pagnon-Minot, Aurélie; Ruggiero, Florence; Le Guellec, Dominique

    2011-12-01

    Zebrafish myosepta connect two adjacent muscle cells and transmit muscular forces to axial structures during swimming via the myotendinous junction (MTJ). The MTJ establishes transmembrane linkages system consisting of extracellular matrix molecules (ECM) surrounding the basement membrane, cytoskeletal elements anchored to sarcolema, and all intermediate proteins that link ECM to actin filaments. Using a series of zebrafish specimens aged between 24 h post-fertilization and 2 years old, the present paper describes at the transmission electron microscope level the development of extracellular and intracellular elements of the MTJ. The transverse myoseptum development starts during the segmentation period by deposition of sparse and loosely organized collagen fibrils. During the hatching period, a link between actin filaments and sarcolemma is established. The basal lamina underlining sarcolemma is well differentiated. Later, collagen fibrils display an orthogonal orientation and fibroblast-like cells invade the myoseptal stroma. A dense network of collagen fibrils is progressively formed that both anchor myoseptal fibroblasts and sarcolemmal basement membrane. The differentiation of a functional MTJ is achieved when sarcolemma interacts with both cytoskeletal filaments and extracellular components. This solid structural link between contractile apparatus and ECM leads to sarcolemma deformations resulting in the formation of regular invaginations, and allows force transmission during muscle contraction. This paper presents the first ultrastructural atlas of the zebrafish MTJ development, which represents an useful tool to analyse the mechanisms of the myotendinous system formation and their disruption in muscle disorders.

  10. Assessment of Toxicological Perturbations and Variants of Pancreatic Islet Development in the Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Karilyn E. Sant

    2016-09-01

    Full Text Available The pancreatic islets, largely comprised of insulin-producing beta cells, play a critical role in endocrine signaling and glucose homeostasis. Because they have low levels of antioxidant defenses and a high perfusion rate, the endocrine islets may be a highly susceptible target tissue of chemical exposures. However, this endpoint, as well as the integrity of the surrounding exocrine pancreas, is often overlooked in studies of developmental toxicology. Disruption of development by toxicants can alter cell fate and migration, resulting in structural alterations that are difficult to detect in mammalian embryo systems, but that are easily observed in the zebrafish embryo model (Danio rerio. Using endogenously expressed fluorescent protein markers for developing zebrafish beta cells and exocrine pancreas tissue, we documented differences in islet area and incidence rates of islet morphological variants in zebrafish embryos between 48 and 96 h post fertilization (hpf, raised under control conditions commonly used in embryotoxicity assays. We identified critical windows for chemical exposures during which increased incidences of endocrine pancreas abnormalities were observed following exposure to cyclopamine (2–12 hpf, Mono-2-ethylhexyl phthalate (MEHP (3–48 hpf, and Perfluorooctanesulfonic acid (PFOS (3–48 hpf. Both islet area and length of the exocrine pancreas were sensitive to oxidative stress from exposure to the oxidant tert-butyl hydroperoxide during a highly proliferative critical window (72 hpf. Finally, pancreatic dysmorphogenesis following developmental exposures is discussed with respect to human disease.

  11. Dithiocarbamates are teratogenic to developing zebrafish through inhibition of lysyl oxidase activity

    International Nuclear Information System (INIS)

    Boxtel, Antonius L. van; Kamstra, Jorke H.; Fluitsma, Donna M.; Legler, Juliette

    2010-01-01

    Dithiocarbamates (DTCs) are a class of compounds that are extensively used in agriculture as pesticides. As such, humans and wildlife are undoubtedly exposed to these chemicals. Although DTCs are thought to be relatively safe due to their short half lives, it is well established that they are teratogenic to vertebrates, especially to fish. In zebrafish, these teratogenic effects are characterized by distorted notochord development and shortened anterior to posterior axis. DTCs are known copper (Cu) chelators but this does not fully explain the observed teratogenic effects. We show here that DTCs cause malformations in zebrafish that highly resemble teratogenic effects observed by direct inhibition of a group of cuproenzymes termed lysyl oxidases (LOX). Additionally, we demonstrate that partial knockdown of three LOX genes, lox, loxl1 and loxl5b, sensitizes the developing embryo to DTC exposure. Finally, we show that DTCs directly inhibit zebrafish LOX activity in an ex vivo amine oxidase assay. Taken together, these results provide the first evidence that DTC induced teratogenic effects are, at least in part, caused by direct inhibition of LOX activity.

  12. Isthmin 1 (ism1) is required for normal hematopoiesis in developing zebrafish.

    Science.gov (United States)

    Berrun, Arturo; Harris, Elena; Stachura, David L

    2018-01-01

    Hematopoiesis is an essential and highly regulated biological process that begins with hematopoietic stem cells (HSCs). In healthy organisms, HSCs are responsible for generating a multitude of mature blood cells every day, yet the molecular pathways that instruct HSCs to self-renew and differentiate into post-mitotic blood cells are not fully known. To understand these molecular pathways, we investigated novel genes expressed in hematopoietic-supportive cell lines from the zebrafish (Danio rerio), a model system increasingly utilized to uncover molecular pathways important in the development of other vertebrate species. We performed RNA sequencing of the transcriptome of three stromal cell lines derived from different stages of embryonic and adult zebrafish and identified hundreds of highly expressed transcripts. For our studies, we focused on isthmin 1 (ism1) due to its shared synteny with its human gene ortholog and because it is a secreted protein. To characterize ism1, we performed loss-of-function experiments to identify if mature blood cell production was disrupted. Myeloid and erythroid lineages were visualized and scored with transgenic zebrafish expressing lineage-specific markers. ism1 knockdown led to reduced numbers of neutrophils, macrophages, and erythrocytes. Analysis of clonal methylcellulose assays from ism1 morphants also showed a reduction in total hematopoietic stem and progenitor cells (HSPCs). Overall, we demonstrate that ism1 is required for normal generation of HSPCs and their downstream progeny during zebrafish hematopoiesis. Further investigation into ism1 and its importance in hematopoiesis may elucidate evolutionarily conserved processes in blood formation that can be further investigated for potential clinical utility.

  13. Isthmin 1 (ism1) is required for normal hematopoiesis in developing zebrafish

    Science.gov (United States)

    Berrun, Arturo; Harris, Elena

    2018-01-01

    Hematopoiesis is an essential and highly regulated biological process that begins with hematopoietic stem cells (HSCs). In healthy organisms, HSCs are responsible for generating a multitude of mature blood cells every day, yet the molecular pathways that instruct HSCs to self-renew and differentiate into post-mitotic blood cells are not fully known. To understand these molecular pathways, we investigated novel genes expressed in hematopoietic-supportive cell lines from the zebrafish (Danio rerio), a model system increasingly utilized to uncover molecular pathways important in the development of other vertebrate species. We performed RNA sequencing of the transcriptome of three stromal cell lines derived from different stages of embryonic and adult zebrafish and identified hundreds of highly expressed transcripts. For our studies, we focused on isthmin 1 (ism1) due to its shared synteny with its human gene ortholog and because it is a secreted protein. To characterize ism1, we performed loss-of-function experiments to identify if mature blood cell production was disrupted. Myeloid and erythroid lineages were visualized and scored with transgenic zebrafish expressing lineage-specific markers. ism1 knockdown led to reduced numbers of neutrophils, macrophages, and erythrocytes. Analysis of clonal methylcellulose assays from ism1 morphants also showed a reduction in total hematopoietic stem and progenitor cells (HSPCs). Overall, we demonstrate that ism1 is required for normal generation of HSPCs and their downstream progeny during zebrafish hematopoiesis. Further investigation into ism1 and its importance in hematopoiesis may elucidate evolutionarily conserved processes in blood formation that can be further investigated for potential clinical utility. PMID:29758043

  14. Patterns of free calcium in zebrafish embryos

    NARCIS (Netherlands)

    Creton, R; Speksnijder, JE; Jaffe, LF

    Direct knowledge of Ca2+ patterns in vertebrate development is largely restricted to early stages, in which they control fertilization, ooplasmic segregation and cleavage. To explore new roles of Ca2+ in vertebrate development, we injected the Ca2+ indicator aequorin into zebrafish eggs and imaged

  15. An Fgf-Shh signaling hierarchy regulates early specification of the zebrafish skull.

    Science.gov (United States)

    McCarthy, Neil; Sidik, Alfire; Bertrand, Julien Y; Eberhart, Johann K

    2016-07-15

    The neurocranium generates most of the craniofacial skeleton and consists of prechordal and postchordal regions. Although development of the prechordal is well studied, little is known of the postchordal region. Here we characterize a signaling hierarchy necessary for postchordal neurocranial development involving Fibroblast growth factor (Fgf) signaling for early specification of mesodermally-derived progenitor cells. The expression of hyaluron synthetase 2 (has2) in the cephalic mesoderm requires Fgf signaling and Has2 function, in turn, is required for postchordal neurocranial development. While Hedgehog (Hh)-deficient embryos also lack a postchordal neurocranium, this appears primarily due to a later defect in chondrocyte differentiation. Inhibitor studies demonstrate that postchordal neurocranial development requires early Fgf and later Hh signaling. Collectively, our results provide a mechanistic understanding of early postchordal neurocranial development and demonstrate a hierarchy of signaling between Fgf and Hh in the development of this structure. Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.

  16. A Student Team in a University of Michigan Biomedical Engineering Design Course Constructs a Microfluidic Bioreactor for Studies of Zebrafish Development

    Science.gov (United States)

    Shen, Yu-chi; Li, David; Al-Shoaibi, Ali; Bersano-Begey, Tom; Chen, Hao; Ali, Shahid; Flak, Betsy; Perrin, Catherine; Winslow, Max; Shah, Harsh; Ramamurthy, Poornapriya; Schmedlen, Rachael H.; Takayama, Shuichi

    2009-01-01

    Abstract The zebrafish is a valuable model for teaching developmental, molecular, and cell biology; aquatic sciences; comparative anatomy; physiology; and genetics. Here we demonstrate that zebrafish provide an excellent model system to teach engineering principles. A seven-member undergraduate team in a biomedical engineering class designed, built, and tested a zebrafish microfluidic bioreactor applying microfluidics, an emerging engineering technology, to study zebrafish development. During the semester, students learned engineering and biology experimental design, chip microfabrication, mathematical modeling, zebrafish husbandry, principles of developmental biology, fluid dynamics, microscopy, and basic molecular biology theory and techniques. The team worked to maximize each person's contribution and presented weekly written and oral reports. Two postdoctoral fellows, a graduate student, and three faculty instructors coordinated and directed the team in an optimal blending of engineering, molecular, and developmental biology skill sets. The students presented two posters, including one at the Zebrafish meetings in Madison, Wisconsin (June 2008). PMID:19292670

  17. Overexpression of miR-19b Impairs Cardiac Development in Zebrafish by Targeting ctnnb1

    Directory of Open Access Journals (Sweden)

    Mengmeng Li

    2014-07-01

    Full Text Available Background: MicroRNAs are broadly accepted as crucial regulators of cardiovascular development, and dysregulation of their expression has been linked to cardiac disease. MicroRNA cluster miR-17-92 has been implicated in cardiac development and function, yet its defined mechanisms of action in this context are uncertain. Here, we focused on miR-19b, a key component of the miR-17-92 cluster proven to induce cardiomyocyte proliferation in vitro. We aimed to identify the biological significance of miR-19b in cardiac development and its underlying molecular mechanism of action in vivo. Methods: We micro-injected zebrafish embryos with different concentrations (0, 2, 4 and 8 μm of miR-19b mimics or a negative control, and assessed the embryo malformation rate, mortality rate, hatching rate and heart abnormalities at 72 hours post-fertilization (72 hpf. Results: We found that overexpression of miR-19b impacted left-right symmetry and cardiac development of zebrafish embryos, characterized by pericardial edema, slower heart rate and cardiac looping defects in a dose-dependent manner. Moreover, several important signaling molecules in the Wnt signaling pathway were abnormally expressed, suggesting that overexpression of miR-19b induces the inhibition of the Wnt signaling pathway by directly targeting ctnnb1. Interestingly, the deformed cardiac phenotype was partially rescued by treatment with the GSK3β inhibitor lithium chloride. Conclusion: Our findings suggest that miR-19b regulates laterality development and heart looping in zebrafish embryos by targeting ctnnb1.

  18. Development and implementation of a three-choice serial reaction time task for zebrafish (Danio rerio).

    Science.gov (United States)

    Parker, Matthew O; Millington, Mollie E; Combe, Fraser J; Brennan, Caroline H

    2012-02-01

    Zebrafish are an established and widely utilized developmental genetic model system, but limitations in developed behavioral assays have meant that their potential as a model in behavioral neuroscience has yet to be fully realized. Here, we describe the development of a novel operant behavioral assay to examine a variety of aspects of stimulus control in zebrafish using a 3 choice serial reaction time task (3 CSRTT). Fish were briefly exposed to three spatially distinct, but perceptually identical stimuli, presented in a random order after a fixed-time inter-trial interval (ITI). Entries to the correct response aperture either during the stimulus presentation, or within a brief limited hold period following presentation, were reinforced with illumination of the magazine light and delivery of a small food reward. Following training, premature responding was probed with a long-ITI session three times; once at baseline, once following a saline injection and once following an injection of a low dose of amphetamine (AMPH; 0.025 mg/kg). We predicted that if premature responding was related to impulsivity (as in rodents) it would be reduced following the AMPH injection. Results confirmed that zebrafish could learn to perform a complex operant task similar to tasks developed for rodents which are used to probe sustained attention and impulsivity, but the results from the AMPH trials were inconclusive. This study provides the foundations for development and further validation of this species as a model for some aspects of human attentional and impulse control disorders, such as substance abuse disorder. Copyright © 2011 Elsevier B.V. All rights reserved.

  19. Exposure to a PBDE/OH-BDE mixture alters juvenile zebrafish (Danio rerio) development.

    Science.gov (United States)

    Macaulay, Laura J; Chernick, Melissa; Chen, Albert; Hinton, David E; Bailey, Jordan M; Kullman, Seth W; Levin, Edward D; Stapleton, Heather M

    2017-01-01

    Polybrominated diphenyl ethers (PBDEs) and their metabolites (e.g., hydroxylated BDEs [OH-BDEs]) are contaminants frequently detected together in human tissues and are structurally similar to thyroid hormones. Thyroid hormones partially mediate metamorphic transitions between life stages in zebrafish, making this a critical developmental window that may be vulnerable to chemicals disrupting thyroid signaling. In the present study, zebrafish were exposed to 6-OH-BDE-47 (30 nM; 15 μg/L) alone, or to a low-dose (30 μg/L) or high-dose (600 μg/L) mixture of PentaBDEs, 6-OH-BDE-47 (0.5-6 μg/L), and 2,4,6-tribromophenol (5-100 μg/L) during juvenile development (9-23 d postfertilization) and evaluated for developmental endpoints mediated by thyroid hormone signaling. Fish were sampled at 3 time points and examined for developmental and skeletal morphology, apical thyroid and skeletal gene markers, and modifications in swimming behavior (as adults). Exposure to the high-dose mixture resulted in >85% mortality within 1 wk of exposure, despite being below reported acute toxicity thresholds for individual congeners. The low-dose mixture and 6-OH-BDE-47 groups exhibited reductions in body length and delayed maturation, specifically relating to swim bladder, fin, and pigmentation development. Reduced skeletal ossification was also observed in 6-OH-BDE-47-treated fish. Assessment of thyroid and osteochondral gene regulatory networks demonstrated significantly increased expression of genes that regulate skeletal development and thyroid hormones. Overall, these results indicate that exposures to PBDE/OH-BDE mixtures adversely impact zebrafish maturation during metamorphosis. Environ Toxicol Chem 2017;36:36-48. © 2016 SETAC. © 2016 SETAC.

  20. Effects of copper oxide nanoparticles on developing zebrafish embryos and larvae

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    Sun Y

    2016-03-01

    Full Text Available Yan Sun, Gong Zhang, Zizi He, Yajie Wang, Jianlin Cui, Yuhao Li Department of Pathology, Key Laboratory of Tumor Microenvironment and Neurovascular Regulation, Nankai University School of Medicine, Tianjin, People’s Republic of China Abstract: Copper oxide nanoparticles (CuO NPs are used for a variety of purposes in a wide range of commercially available products. Some CuO NPs probably end up in the aquatic systems, thus raising concerns about aqueous exposure toxicity, and the impact of CuO NPs on liver development and neuronal differentiation remains unclear. In this study, particles were characterized using Fourier transform infrared spectra, scanning electron microscopy, and transmission electron microscopy. Zebrafish embryos were continuously exposed to CuO NPs from 4 hours postfertilization at concentrations of 50, 25, 12.5, 6.25, or 1 mg/L. The expression of gstp1 and cyp1a was examined by quantitative reverse transcription polymerase chain reaction. The expression of tumor necrosis factor alpha and superoxide dismutase 1 was examined by quantitative reverse transcription polymerase chain reaction and Western blotting. Liver development and retinal neurodifferentiation were analyzed by whole-mount in situ hybridization, hematoxylin–eosin staining, and immunohistochemistry, and a behavioral test was performed to track the movement of larvae. We show that exposure of CuO NPs at low doses has little effect on embryonic development. However, exposure to CuO NPs at concentrations of 12.5 mg/L or higher leads to abnormal phenotypes and induces an inflammatory response in a dose-dependent pattern. Moreover, exposure to CuO NPs at high doses results in an underdeveloped liver and a delay in retinal neurodifferentiation accompanied by reduced locomotor ability. Our data demonstrate that short-term exposure to CuO NPs at high doses shows hepatotoxicity and neurotoxicity in zebrafish embryos and larvae. Keywords: copper oxide nanoparticles

  1. Bio-electrosprayed multicellular zebrafish embryos are viable and develop normally

    International Nuclear Information System (INIS)

    Clarke, Jonathan D W; Jayasinghe, Suwan N

    2008-01-01

    Bio-electrosprays are rapidly emerging as a viable protocol for directly engineering living cells. This communication reports the bio-electrospraying of multicellular organisms, namely zebrafish embryos. The results demonstrate that the bio-electrospray protocol fails to induce any embryological perturbations. In addition to analysing overall embryo morphology, we use transgenic embryos that express green fluorescent protein in specific brain neurons to determine that neuronal numbers and organization are completely normal. These results demonstrate that the bio-electrospraying protocol does not interfere with the complex gene regulation and cell movements required for the development of a multicellular organism. (communication)

  2. Hedgehog-PKA signaling and gnrh3 regulate the development of zebrafish gnrh3 neurons.

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    Ming-Wei Kuo

    Full Text Available GnRH neurons secrete GnRH that controls the development of the reproduction system. Despite many studies, the signals controlling the development of GnRH neurons from its progenitors have not been fully established. To understand the development of GnRH neurons, we examined the development of gnrh3-expressing cells using a transgenic zebrafish line that expresses green fluorescent protein (GFP and LacZ driven by the gnrh3 promoter. GFP and LacZ expression recapitulated that of gnrh3 in the olfactory region, olfactory bulb and telencephalon. Depletion of gnrh3 by morpholinos led to a reduction of GFP- and gnrh3-expressing cells, while over-expression of gnrh3 mRNA increased the number of these cells. This result indicates a positive feed-forward regulation of gnrh3 cells by gnrh3. The gnrh3 cells were absent in embryos that lack Hedgehog signaling, but their numbers were increased in embryos overexpressing shhb. We manipulated the amounts of kinase that antagonizes the Hedgehog signaling pathway, protein kinase A (PKA, by treating embryos with PKA activator forskolin or by injecting mRNAs encoding its constitutively active catalytic subunit (PKA* and dominant negative regulatory subunit (PKI into zebrafish embryos. PKA* misexpression or forskolin treatment decreased GFP cell numbers, while PKI misexpression led to ectopic production of GFP cells. Our data indicate that the Hedgehog-PKA pathway participates in the development of gnrh3-expressing neurons during embryogenesis.

  3. A novel role for MAPKAPK2 in morphogenesis during zebrafish development.

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    Beth A Holloway

    2009-03-01

    Full Text Available One of the earliest morphogenetic processes in the development of many animals is epiboly. In the zebrafish, epiboly ensues when the animally localized blastoderm cells spread, thin over, and enclose the vegetally localized yolk. Only a few factors are known to function in this fundamental process. We identified a maternal-effect mutant, betty boop (bbp, which displays a novel defect in epiboly, wherein the blastoderm margin constricts dramatically, precisely when half of the yolk cell is covered by the blastoderm, causing the yolk cell to burst. Whole-blastoderm transplants and mRNA microinjection rescue demonstrate that Bbp functions in the yolk cell to regulate epiboly. We positionally cloned the maternal-effect bbp mutant gene and identified it as the zebrafish homolog of the serine-threonine kinase Mitogen Activated Protein Kinase Activated Protein Kinase 2, or MAPKAPK2, which was not previously known to function in embryonic development. We show that the regulation of MAPKAPK2 is conserved and p38 MAP kinase functions upstream of MAPKAPK2 in regulating epiboly in the zebrafish embryo. Dramatic alterations in calcium dynamics, together with the massive marginal constrictive force observed in bbp mutants, indicate precocious constriction of an F-actin network within the yolk cell, which first forms at 50% epiboly and regulates epiboly progression. We show that MAPKAPK2 activity and its regulator p38 MAPK function in the yolk cell to regulate the process of epiboly, identifying a new pathway regulating this cell movement process. We postulate that a p38 MAPKAPK2 kinase cascade modulates the activity of F-actin at the yolk cell margin circumference allowing the gradual closure of the blastopore as epiboly progresses.

  4. Inhibition of calcium uptake during hypoxia in developing zebrafish is mediated by hypoxia-inducible factor.

    Science.gov (United States)

    Kwong, Raymond W M; Kumai, Yusuke; Tzaneva, Velislava; Azzi, Estelle; Hochhold, Nina; Robertson, Cayleih; Pelster, Bernd; Perry, Steve F

    2016-12-15

    The present study investigated the potential role of hypoxia-inducible factor (HIF) in calcium homeostasis in developing zebrafish (Danio rerio). It was demonstrated that zebrafish raised in hypoxic water (30 mmHg; control, 155 mmHg P O 2 ) until 4 days post-fertilization exhibited a substantial reduction in whole-body Ca 2+ levels and Ca 2+ uptake. Ca 2+ uptake in hypoxia-treated fish did not return to pre-hypoxia (control) levels within 2 h of transfer back to normoxic water. Results from real-time PCR showed that hypoxia decreased the whole-body mRNA expression levels of the epithelial Ca 2+ channel (ecac), but not plasma membrane Ca 2+ -ATPase (pmca2) or Na + /Ca 2+ -exchanger (ncx1b). Whole-mount in situ hybridization revealed that the number of ecac-expressing ionocytes was reduced in fish raised in hypoxic water. These findings suggested that hypoxic treatment suppressed the expression of ecac, thereby reducing Ca 2+ influx. To further evaluate the potential mechanisms for the effects of hypoxia on Ca 2+ regulation, a functional gene knockdown approach was employed to prevent the expression of HIF-1αb during hypoxic treatment. Consistent with a role for HIF-1αb in regulating Ca 2+ balance during hypoxia, the results demonstrated that the reduction of Ca 2+ uptake associated with hypoxic exposure was not observed in fish experiencing HIF-1αb knockdown. Additionally, the effects of hypoxia on reducing the number of ecac-expressing ionocytes was less pronounced in HIF-1αb-deficient fish. Overall, the current study revealed that hypoxic exposure inhibited Ca 2+ uptake in developing zebrafish, probably owing to HIF-1αb-mediated suppression of ecac expression. © 2016. Published by The Company of Biologists Ltd.

  5. Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain.

    Science.gov (United States)

    Roussigne, Myriam; Blader, Patrick; Wilson, Stephen W

    2012-03-01

    How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.

  6. GLUT2-mediated glucose uptake and availability are required for embryonic brain development in zebrafish.

    Science.gov (United States)

    Marín-Juez, Rubén; Rovira, Mireia; Crespo, Diego; van der Vaart, Michiel; Spaink, Herman P; Planas, Josep V

    2015-01-01

    Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

  7. Smyd3 is required for the development of cardiac and skeletal muscle in zebrafish.

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    Tomoaki Fujii

    Full Text Available Modifications of histone tails are involved in the regulation of a wide range of biological processes including cell cycle, cell survival, cell division, and cell differentiation. Among the modifications, histone methylation plays a critical role in cardiac and skeletal muscle differentiation. In our earlier studies, we found that SMYD3 has methyltransferase activity to histone H3 lysine 4, and that its up-regulation is involved in the tumorigenesis of human colon, liver, and breast. To clarify the role of Smyd3 in development, we have studied its expression patterns in zebrafish embryos and the effect of its suppression on development using Smyd3-specific antisense morpholino-oligonucleotides. We here show that transcripts of smyd3 were expressed in zebrafish embryos at all developmental stages examined and that knockdown of smyd3 in embryos resulted in pericardial edema and defects in the trunk structure. In addition, these phenotypes were associated with abnormal expression of three heart-chamber markers including cmlc2, amhc and vmhc, and abnormal expression of myogenic regulatory factors including myod and myog. These data suggest that Smyd3 plays an important role in the development of heart and skeletal muscle.

  8. cables1 Is Required for Embryonic Neural Development: Molecular, Cellular, and Behavioral Evidence From the Zebrafish

    Science.gov (United States)

    GROENEWEG, JOLIJN W.; WHITE, YVONNE A.R.; KOKEL, DAVID; PETERSON, RANDALL T.; ZUKERBERG, LAWRENCE R.; BERIN, INNA; RUEDA, BO R.; WOOD, ANTONY W.

    2014-01-01

    SUMMARY In vitro studies have suggested that the Cables1 gene regulates epithelial cell proliferation, whereas other studies suggest a role in promoting neural differentiation. In efforts to clarify the functions of Cables1 in vivo, we conducted gain- and loss-of-function studies targeting its ortholog (cables1) in the zebrafish embryo. Similar to rodents, zebrafish cables1 mRNA expression is detected most robustly in embryonic neural tissues. Antisense knockdown of cables1 leads to increased numbers of apoptotic cells, particularly in brain tissue, in addition to a distinct behavioral phenotype, characterized by hyperactivity in response to stimulation. Apoptosis and the behavioral abnormality could be rescued by co-expression of a morpholino-resistant cables1 construct. Suppression of p53 expression in cables1 morphants partially rescued both apoptosis and the behavioral phenotype, suggesting that the phenotype of cables1 morphants is due in part to p53-dependent apoptosis. Alterations in the expression patterns of several neural transcription factors were observed in cables1 morphants during early neurulation, suggesting that cables1 is required for early neural differentiation. Ectopic overexpression of cables1 strongly disrupted embryonic morphogenesis, while overexpression of a cables1 mutant lacking the C-terminal cyclin box had little effect, suggesting functional importance of the cyclin box. Lastly, marked reductions in p35, but not Cdk5, were observed in cables1 morphants. Collectively, these data suggest that cables1 is important for neural differentiation during embryogenesis, in a mechanism that likely involves interactions with the Cdk5/p35 kinase pathway. PMID:21268180

  9. Intraspinal serotonergic neurons consist of two, temporally distinct populations in developing zebrafish.

    Science.gov (United States)

    Montgomery, Jacob E; Wiggin, Timothy D; Rivera-Perez, Luis M; Lillesaar, Christina; Masino, Mark A

    2016-06-01

    Zebrafish intraspinal serotonergic neuron (ISN) morphology and distribution have been examined in detail at different ages; however, some aspects of the development of these cells remain unclear. Although antibodies to serotonin (5-HT) have detected ISNs in the ventral spinal cord of embryos, larvae, and adults, the only tryptophan hydroxylase (tph) transcript that has been described in the spinal cord is tph1a. Paradoxically, spinal tph1a is only expressed transiently in embryos, which brings the source of 5-HT in the ISNs of larvae and adults into question. Because the pet1 and tph2 promoters drive transgene expression in the spinal cord, we hypothesized that tph2 is expressed in spinal cords of zebrafish larvae. We confirmed this hypothesis through in situ hybridization. Next, we used 5-HT antibody labeling and transgenic markers of tph2-expressing neurons to identify a transient population of ISNs in embryos that was distinct from ISNs that appeared later in development. The existence of separate ISN populations may not have been recognized previously due to their shared location in the ventral spinal cord. Finally, we used transgenic markers and immunohistochemical labeling to identify the transient ISN population as GABAergic Kolmer-Agduhr double-prime (KA″) neurons. Altogether, this study revealed a novel developmental paradigm in which KA″ neurons are transiently serotonergic before the appearance of a stable population of tph2-expressing ISNs. © 2015 Wiley Periodicals, Inc.

  10. Identification of Spt5 target genes in zebrafish development reveals its dual activity in vivo.

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    Keerthi Krishnan

    Full Text Available Spt5 is a conserved essential protein that represses or stimulates transcription elongation in vitro. Immunolocalization studies on Drosophila polytene chromosomes suggest that Spt5 is associated with many loci throughout the genome. However, little is known about the prevalence and identity of Spt5 target genes in vivo during development. Here, we identify direct target genes of Spt5 using fog(sk8 zebrafish mutant, which disrupts the foggy/spt5 gene. We identified that fog(sk8 and their wildtype siblings differentially express less than 5% of genes examined. These genes participate in diverse biological processes from stress response to cell fate specification. Up-regulated genes exhibit shorter overall gene length compared to all genes examined. Through chromatin immunoprecipitation in zebrafish embryos, we identified a subset of developmentally critical genes that are bound by both Spt5 and RNA polymerase II. The protein occupancy patterns on these genes are characteristic of both repressive and stimulatory elongation regulation. Together our findings establish Spt5 as a dual regulator of transcription elongation in vivo and identify a small but diverse set of target genes critically dependent on Spt5 during development.

  11. pitx2 Deficiency results in abnormal ocular and craniofacial development in zebrafish.

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    Yi Liu

    Full Text Available Human PITX2 mutations are associated with Axenfeld-Rieger syndrome, an autosomal-dominant developmental disorder that involves ocular anterior segment defects, dental hypoplasia, craniofacial dysmorphism and umbilical abnormalities. Characterization of the PITX2 pathway and identification of the mechanisms underlying the anomalies associated with PITX2 deficiency is important for better understanding of normal development and disease; studies of pitx2 function in animal models can facilitate these analyses. A knockdown of pitx2 in zebrafish was generated using a morpholino that targeted all known alternative transcripts of the pitx2 gene; morphant embryos generated with the pitx2(ex4/5 splicing-blocking oligomer produced abnormal transcripts predicted to encode truncated pitx2 proteins lacking the third (recognition helix of the DNA-binding homeodomain. The morphological phenotype of pitx2(ex4/5 morphants included small head and eyes, jaw abnormalities and pericardial edema; lethality was observed at ∼6-8-dpf. Cartilage staining revealed a reduction in size and an abnormal shape/position of the elements of the mandibular and hyoid pharyngeal arches; the ceratobranchial arches were also decreased in size. Histological and marker analyses of the misshapen eyes of the pitx2(ex4/5 morphants identified anterior segment dysgenesis and disordered hyaloid vasculature. In summary, we demonstrate that pitx2 is essential for proper eye and craniofacial development in zebrafish and, therefore, that PITX2/pitx2 function is conserved in vertebrates.

  12. Genetic Regulation of Vascular Development: Building the Zebrafish Vascular Tree

    NARCIS (Netherlands)

    R.L.J.M. Herpers (Robert)

    2010-01-01

    textabstractThe extensive networks of blood and lymphatic vessels within the vertebrate body are essential for the transport and delivery of fluids, gases, macromolecules and cells, and play important roles in facilitating immune responses. The development of the vascular tree requires a highly

  13. Dioxin inhibition of swim bladder development in zebrafish: is it secondary to heart failure?

    Science.gov (United States)

    Yue, Monica S; Peterson, Richard E; Heideman, Warren

    2015-05-01

    The swim bladder is a gas-filled organ that is used for regulating buoyancy and is essential for survival in most teleost species. In zebrafish, swim bladder development begins during embryogenesis and inflation occurs within 5 days post fertilization (dpf). Embryos exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) before 96 h post fertilization (hpf) developed swim bladders normally until the growth/elongation phase, at which point growth was arrested. It is known that TCDD exposure causes heart malformations that lead to heart failure in zebrafish larvae, and that blood circulation is a key factor in normal development of the swim bladder. The adverse effects of TCDD exposure on the heart occur during the same period of time that swim bladder development and growth occurs. Based on this coincident timing, and the dependence of swim bladder development on proper circulatory development, we hypothesized that the adverse effects of TCDD on swim bladder development were secondary to heart failure. We compared swim bladder development in TCDD-exposed embryos to: (1) silent heart morphants, which lack cardiac contractility, and (2) transiently transgenic cmlc2:caAHR-2AtRFP embryos, which mimic TCDD-induced heart failure via heart-specific, constitutive activation of AHR signaling. Both of these treatment groups, which were not exposed to TCDD, developed hypoplastic swim bladders of comparable size and morphology to those found in TCDD-exposed embryos. Furthermore, in all treatment groups swim bladder development was arrested during the growth/elongation phase. Together, these findings support a potential role for heart failure in the inhibition of swim bladder development caused by TCDD. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Effects of oxytetracycline and amoxicillin on development and biomarkers activities of zebrafish (Danio rerio).

    Science.gov (United States)

    Oliveira, Rhaul; McDonough, Sakchai; Ladewig, Jessica C L; Soares, Amadeu M V M; Nogueira, António J A; Domingues, Inês

    2013-11-01

    Antibiotics have been widely used in human and veterinary medicine to treat or prevent diseases. Residues of antibiotics have been found in aquatic environments, but their effects on fish have been not properly investigated. This work aimed to assess the sub-lethal effects of oxytetracycline and amoxicillin on zebrafish development and biomarkers. Embryos and adults were exposed during 96 h to amoxicillin and oxytetracycline following OECD guidelines. Tissues of adults and pools of embryos were used for catalase, glutathione-S-transferases and lactate dehydrogenase determinations. Amoxicillin caused premature hatching (48 h-EC50=132.4 mg/l) whereas oxytetracycline cause delayed hatching of embryos (72 h-EC50=127.6 mg/l). Moreover, both antibiotics inhibited catalase and induced glutathione-S-transferases in zebrafish adults. However, only oxytetracycline induced lactate dehydrogenase. Short-term effects of antibiotics were observed at high doses (mg/l) indicating that physiological impairment in fish populations is unlike to occur. However, effects of chronic exposures to low doses of ABs must be investigated. Copyright © 2013 Elsevier B.V. All rights reserved.

  15. Transcriptomic analysis in the developing zebrafish embryo after compound exposure: Individual gene expression and pathway regulation

    Energy Technology Data Exchange (ETDEWEB)

    Hermsen, Sanne A.B., E-mail: Sanne.Hermsen@rivm.nl [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.178, 3508 TD, Utrecht (Netherlands); Pronk, Tessa E. [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Department of Toxicogenomics, Maastricht University, P.O. Box 616, 6200 MD, Maastricht (Netherlands); Brandhof, Evert-Jan van den [Centre for Environmental Quality, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Ven, Leo T.M. van der [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Piersma, Aldert H. [Centre for Health Protection, National Institute for Public Health and the Environment (RIVM), P.O. Box 1, 3720 BA Bilthoven (Netherlands); Institute for Risk Assessment Sciences (IRAS), Utrecht University, P.O. Box 80.178, 3508 TD, Utrecht (Netherlands)

    2013-10-01

    The zebrafish embryotoxicity test is a promising alternative assay for developmental toxicity. Classically, morphological assessment of the embryos is applied to evaluate the effects of compound exposure. However, by applying differential gene expression analysis the sensitivity and predictability of the test may be increased. For defining gene expression signatures of developmental toxicity, we explored the possibility of using gene expression signatures of compound exposures based on commonly expressed individual genes as well as based on regulated gene pathways. Four developmental toxic compounds were tested in concentration-response design, caffeine, carbamazepine, retinoic acid and valproic acid, and two non-embryotoxic compounds, D-mannitol and saccharin, were included. With transcriptomic analyses we were able to identify commonly expressed genes, which were mostly development related, after exposure to the embryotoxicants. We also identified gene pathways regulated by the embryotoxicants, suggestive of their modes of action. Furthermore, whereas pathways may be regulated by all compounds, individual gene expression within these pathways can differ for each compound. Overall, the present study suggests that the use of individual gene expression signatures as well as pathway regulation may be useful starting points for defining gene biomarkers for predicting embryotoxicity. - Highlights: • The zebrafish embryotoxicity test in combination with transcriptomics was used. • We explored two approaches of defining gene biomarkers for developmental toxicity. • Four compounds in concentration-response design were tested. • We identified commonly expressed individual genes as well as regulated gene pathways. • Both approaches seem suitable starting points for defining gene biomarkers.

  16. Impacts of chemical modification on the toxicity of diverse nanocellulose materials to developing zebrafish.

    Science.gov (United States)

    Harper, Bryan J; Clendaniel, Alicea; Sinche, Federico; Way, Daniel; Hughes, Michael; Schardt, Jenna; Simonsen, John; Stefaniak, Aleksandr B; Harper, Stacey L

    2016-06-01

    Cellulose is an abundant and renewable resource currently being investigated for utility in nanomaterial form for various promising applications ranging from medical and pharmaceutical uses to mechanical reinforcement and biofuels. The utility of nanocellulose and wide implementation ensures increasing exposure to humans and the environment as nanocellulose-based technologies advance. Here, we investigate how differences in aspect ratio and changes to surface chemistry, as well as synthesis methods, influence the biocompatibility of nanocellulose materials using the embryonic zebrafish. Investigations into the toxicity of neutral, cationic and anionic surface functionalities revealed that surface chemistry had a minimal influence on the overall toxicity of nanocellulose materials. Higher aspect ratio cellulose nanofibers produced by mechanical homogenization were, in some cases, more toxic than other cellulose-based nanofibers or nanocrystals produced by chemical synthesis methods. Using fluorescently labeled nanocellulose we were able to show that nanocellulose uptake did occur in embryonic zebrafish during development. We conclude that the benign nature of nanocellulose materials makes them an ideal platform to systematically investigate the inherent surface features driving nanomaterial toxicity in order to create safer design principles for engineered nanoparticles.

  17. Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

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    Maide Ö. Raeker

    2011-01-01

    Full Text Available During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues.

  18. Obscurin Depletion Impairs Organization of Skeletal Muscle in Developing Zebrafish Embryos

    Science.gov (United States)

    Raeker, Maide Ö.; Russell, Mark W.

    2011-01-01

    During development, skeletal myoblasts differentiate into myocytes and skeletal myotubes with mature contractile structures that are precisely oriented with respect to surrounding cells and tissues. Establishment of this highly ordered structure requires reciprocal interactions between the differentiating myocytes and the surrounding extracellular matrix to form correctly positioned and well-organized attachments from the skeletal muscle to the bony skeleton. Using the developing zebrafish embryo as a model, we examined the relationship between new myofibril assembly and the organization of the membrane domains involved in cell-extracellular matrix interactions. We determined that depletion of obscurin, a giant muscle protein, resulted in irregular cell morphology and disturbed extracellular matrix organization during skeletal muscle development. The resulting impairment of myocyte organization was associated with disturbance of the internal architecture of the myocyte suggesting that obscurin participates in organizing the internal structure of the myocyte and translating those structural cues to surrounding cells and tissues. PMID:22190853

  19. Combinatorial effects of zinc deficiency and arsenic exposure on zebrafish (Danio rerio development.

    Directory of Open Access Journals (Sweden)

    Laura M Beaver

    Full Text Available Zinc deficiency and chronic low level exposures to inorganic arsenic in drinking water are both significant public health concerns that affect millions of people including pregnant women. These two conditions can co-exist in the human population but little is known about their interaction, and in particular, whether zinc deficiency sensitizes individuals to arsenic exposure and toxicity, especially during critical windows of development. To address this, we utilized the Danio rerio (zebrafish model to test the hypothesis that parental zinc deficiency sensitizes the developing embryo to low-concentration arsenic toxicity, leading to altered developmental outcomes. Adult zebrafish were fed defined zinc deficient and zinc adequate diets and were spawned resulting in zinc adequate and zinc deficient embryos. The embryos were treated with environmentally relevant concentrations of 0, 50, and 500 ppb arsenic. Arsenic exposure significantly reduced the amount of zinc in the developing embryo by ~7%. The combination of zinc deficiency and low-level arsenic exposures did not sensitize the developing embryo to increased developmental malformations or mortality. The combination did cause a 40% decline in physical activity of the embryos, and this decline was significantly greater than what was observed with zinc deficiency or arsenic exposure alone. Significant changes in RNA expression of genes that regulate zinc homeostasis, response to oxidative stress and insulin production (including zip1, znt7, nrf2, ogg1, pax4, and insa were found in zinc deficient, or zinc deficiency and arsenic exposed embryos. Overall, the data suggests that the combination of zinc deficiency and arsenic exposure has harmful effects on the developing embryo and may increase the risk for developing chronic diseases like diabetes.

  20. Role of Active Contraction and Tropomodulins in Regulating Actin Filament Length and Sarcomere Structure in Developing Zebrafish Skeletal Muscle.

    Science.gov (United States)

    Mazelet, Lise; Parker, Matthew O; Li, Mei; Arner, Anders; Ashworth, Rachel

    2016-01-01

    Whilst it is recognized that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1 (ts25) ) which lacks functional voltage-gated calcium channels (dihydropyridine receptors) in the muscle and pharmacological immobilization of embryos with a reversible anesthetic (Tricaine), allowed the study of paralysis (in mutants and anesthetized fish) and recovery of movement (reversal of anesthetic treatment). The effect of paralysis in early embryos (aged between 17 and 24 hours post-fertilization, hpf) on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localization of the actin capping proteins Tropomodulin 1 & 4 (Tmod) in fish aged from 17 hpf until 42 hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post-fertilization (dpf). Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf) resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralyzed fish by 42 hpf. In conclusion, myofibril organization is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localization of Tmod1 to its sarcomeric

  1. Role of active contraction and tropomodulins in regulating actin filament length and sarcomere structure in developing zebrafish skeletal muscle

    Directory of Open Access Journals (Sweden)

    Lise eMazelet

    2016-03-01

    Full Text Available Whilst it is recognised that contraction plays an important part in maintaining the structure and function of mature skeletal muscle, its role during development remains undefined. In this study the role of movement in skeletal muscle maturation was investigated in intact zebrafish embryos using a combination of genetic and pharmacological approaches. An immotile mutant line (cacnb1ts25 which lacks functional voltage-gated calcium channels (dihydropyridine receptors in the muscle and pharmacological immobilisation of embryos with a reversible anaesthetic (Tricaine, allowed the study of paralysis (in mutants and anaesthetised fish and recovery of movement (reversal of anaesthetic treatment. The effect of paralysis in early embryos (aged between 17-24 hours post fertilisation, hpf on skeletal muscle structure at both myofibrillar and myofilament level was determined using both immunostaining with confocal microscopy and small angle X-ray diffraction. The consequences of paralysis and subsequent recovery on the localisation of the actin capping proteins Tropomodulin 1 &4 (Tmod in fish aged from 17hpf until 42hpf was also assessed. The functional consequences of early paralysis were investigated by examining the mechanical properties of the larval muscle. The length-force relationship, active and passive tension, was measured in immotile, recovered and control skeletal muscle at 5 and 7 day post fertilisation (dpf. Recovery of muscle function was also assessed by examining swimming patterns in recovered and control fish. Inhibition of the initial embryonic movements (up to 24 hpf resulted in an increase in myofibril length and a decrease in width followed by almost complete recovery in both moving and paralysed fish by 42hpf. In conclusion, myofibril organisation is regulated by a dual mechanism involving movement-dependent and movement-independent processes. The initial contractile event itself drives the localisation of Tmod1 to its sarcomeric

  2. ESX-5-deficient Mycobacterium marinum is hypervirulent in adult zebrafish

    KAUST Repository

    Weerdenburg, Eveline M.

    2012-02-15

    ESX-5 is a mycobacterial type VII protein secretion system responsible for transport of numerous PE and PPE proteins. It is involved in the induction of host cell death and modulation of the cytokine response in vitro. In this work, we studied the effects of ESX-5 in embryonic and adult zebrafish using Mycobacterium marinum. We found that ESX-5-deficient M.marinum was slightly attenuated in zebrafish embryos. Surprisingly, the same mutant showed highly increased virulence in adult zebrafish, characterized by increased bacterial loads and early onset of granuloma formation with rapid development of necrotic centres. This early onset of granuloma formation was accompanied by an increased expression of pro-inflammatory cytokines and tissue remodelling genes in zebrafish infected with the ESX-5 mutant. Experiments using RAG-1-deficient zebrafish showed that the increased virulence of the ESX-5 mutant was not dependent on the adaptive immune system. Mixed infection experiments with wild-type and ESX-5 mutant bacteria showed that the latter had a specific advantage in adult zebrafish and outcompeted wild-type bacteria. Together our experiments indicate that ESX-5-mediated protein secretion is used by M.marinum to establish a moderate and persistent infection. © 2012 Blackwell Publishing Ltd.

  3. Proprotein convertase subtilisin/kexin type 7 (PCSK7) is essential for the zebrafish development and bioavailability of transforming growth factor β1a (TGFβ1a).

    Science.gov (United States)

    Turpeinen, Hannu; Oksanen, Anna; Kivinen, Virpi; Kukkurainen, Sampo; Uusimäki, Annemari; Rämet, Mika; Parikka, Mataleena; Hytönen, Vesa P; Nykter, Matti; Pesu, Marko

    2013-12-20

    Proprotein convertase subtilisin/kexin (PCSK) enzymes convert proproteins into bioactive end products. Although other PCSK enzymes are known to be essential for biological processes ranging from cholesterol metabolism to host defense, the in vivo importance of the evolutionarily ancient PCSK7 has remained enigmatic. Here, we quantified the expressions of all pcsk genes during the 1st week of fish development and in several tissues. pcsk7 expression was ubiquitous and evident already during the early development. To compare mammalian and zebrafish PCSK7, we prepared homology models, which demonstrated remarkable structural conservation. When the PCSK7 function in developing larvae was inhibited, we found that PCSK7-deficient fish have defects in various organs, including the brain, eye, and otic vesicle, and these result in mortality within 7 days postfertilization. A genome-wide analysis of PCSK7-dependent gene expression showed that, in addition to developmental processes, several immune system-related pathways are also regulated by PCSK7. Specifically, the PCSK7 contributed to the mRNA expression and proteolytic cleavage of the cytokine TGFβ1a. Consequently, tgfβ1a morphant fish displayed phenotypical similarities with pcsk7 morphants, underscoring the importance of this cytokine in the zebrafish development. Targeting PCSK activity has emerged as a strategy for treating human diseases. Our results suggest that inhibiting PCSK7 might interfere with normal vertebrate development.

  4. Analyses of pancreas development by generation of gfp transgenic zebrafish using an exocrine pancreas-specific elastaseA gene promoter

    International Nuclear Information System (INIS)

    Wan Haiyan; Korzh, Svitlana; Li Zhen; Mudumana, Sudha Puttur; Korzh, Vladimir; Jiang Yunjin; Lin Shuo; Gong Zhiyuan

    2006-01-01

    In contrast to what we know on development of endocrine pancreas, the formation of exocrine pancreas remains poorly understood. To create an animal model that allows observation of exocrine cell differentiation, proliferation, and morphogenesis in living animals, we used the zebrafish elastaseA (elaA) regulatory sequence to develop transgenic zebrafish that display highly specific exocrine pancreas expression of GFP in both larvae and adult. By following GFP expression, we found that the pancreas in early development was a relatively compact organ and later extended posterior along the intestine. By transferring the elaA:gfp transgene into slow muscle omitted mutant that is deficient in receiving Hedgehog signals, we further showed that Hedgehog signaling is required for exocrine morphogenesis but not for cell differentiation. We also applied the morpholino knockdown and toxin-mediated cell ablation approaches to this transgenic line. We showed that the development of exocrine pancreas is Islet-1 dependent. Injection of the diphtheria toxin A (DTA) construct under the elastaseA promoter resulted in selective ablation of exocrine cells while the endocrine cells and other endodermal derivatives (liver and intestine) were not affected. Thus, our works demonstrated the new transgenic line provided a useful experimental tool in analyzing exocrine pancreas development

  5. Distinct and overlapping functions of ptpn11 genes in Zebrafish development.

    Directory of Open Access Journals (Sweden)

    Monica Bonetti

    Full Text Available The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11 gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5-6 days post fertilization (dpf. Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.

  6. Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

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    Schwend Tyler

    2009-11-01

    Full Text Available Abstract Background The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC. Genetic studies in zebrafish and mice have established that the Hedgehog (Hh-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia 12. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1 for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1. Results con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to

  7. Toxicity of effluents from gasoline stations oil-water separators to early life stages of zebrafish Danio rerio.

    Science.gov (United States)

    Alves, Romulo Nepomuceno; Mariz, Célio Freire; Paulo, Driele Ventura de; Carvalho, Paulo S M

    2017-07-01

    Used petroleum hydrocarbons and gasoline stations runoff are significant sources of polycyclic aromatic hydrocarbons (PAHs) to aquatic ecosystems. Samples of the final effluent of oil-water-separators were collected at gasoline stations in the metropolitan region of Recife, Brazil, before release to sewage or rainwater systems. Effluent soluble fractions (ESF) were prepared and bioassays were performed according to the Fish Embryo Toxicity Test. The test involved exposing zebrafish Danio rerio embryos to dilutions of the ESFs for 96 h, with daily examination of lethality and sublethal morphological effects integrated through the General Morphology Score (GMS), based on the achievement of developmental hallmarks. Frequencies of abnormalities were recorded after exposures. ESF LC50-96h (lethal concentration to 50% of exposed embryos) in the most toxic effluent achieved 8.9% (v/v), equivalent to 11 μg phenanthrene equivalents L -1 . GMS scores indicated significantly delayed embryo-larval development at ESF dilutions of 10% and 20% from effluents of all gas stations. Major abnormalities detected after the 96 h exposure included the presence of a yolk sac not fully absorbed coupled with the lack of an inflated swim bladder, lack of both pectoral fins, and the failure to develop a protruding mouth. Effective equivalent PAH concentrations that induce a 50% frequency of larvae without an inflated swim bladder (EC50) were 4.9 μg phenanthrene L -1 , 21.8 μg naphthalene L -1 , and 34.1 μg chrysene L -1 . This study shows that PAHs in ESFs from gas stations oil water separators are toxic to zebrafish, contributing to the toxicity of urban storm waters. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. The Zebrafish Model Organism Database (ZFIN)

    Data.gov (United States)

    U.S. Department of Health & Human Services — ZFIN serves as the zebrafish model organism database. It aims to: a) be the community database resource for the laboratory use of zebrafish, b) develop and support...

  9. 4D blood flow mapping using SPIM-microPIV in the developing zebrafish heart

    Science.gov (United States)

    Zickus, Vytautas; Taylor, Jonathan M.

    2018-02-01

    Fluid-structure interaction in the developing heart is an active area of research in developmental biology. However, investigation of heart dynamics is mostly limited to computational uid dynamics simulations using heart wall structure information only, or single plane blood ow information - so there is a need for 3D + time resolved data to fully understand cardiac function. We present an imaging platform combining selective plane illumination microscopy (SPIM) with micro particle image velocimetry (μPIV) to enable 3D-resolved flow mapping in a microscopic environment, free from many of the sources of error and bias present in traditional epi uorescence-based μPIV systems. By using our new system in conjunction with optical heart beat synchronization, we demonstrate the ability obtain non-invasive 3D + time resolved blood flow measurements in the heart of a living zebrafish embryo.

  10. Transcriptomic and phenotypic profiling in developing zebrafish exposed to thyroid hormone receptor agonists

    Energy Technology Data Exchange (ETDEWEB)

    Haggard, Derik E.; Noyes, Pamela D.; Waters, Katrina M.; Tanguay, Robert L.

    2018-04-01

    There is a need to develop novel, high-throughput screening and prioritization methods to identify chemicals with adverse estrogen, androgen, and thyroid activity to protect human health and the environment and is of interest to the Endocrine Disruptor Screening Program. The current aim is to explore the utility of zebrafish as a testing paradigm to classify endocrine activity using phenotypically anchored transcriptome profiling. Transcriptome analysis was conducted on embryos exposed to 25 estrogen-, androgen-, or thyroid-active chemicals at a concentration that elicited adverse malformations or mortality at 120 hours post-fertilization in 80% of the animals exposed. Analysis of the top 1000 significant differentially expressed transcripts across all treatments identified a unique transcriptional and phenotypic profile for thyroid hormone receptor agonists, which can be used as a biomarker screen for potential thyroid hormone agonists.

  11. Heart-specific expression of laminopathic mutations in transgenic zebrafish.

    Science.gov (United States)

    Verma, Ajay D; Parnaik, Veena K

    2017-07-01

    Lamins are key determinants of nuclear organization and function in the metazoan nucleus. Mutations in human lamin A cause a spectrum of genetic diseases that affect cardiac muscle and skeletal muscle as well as other tissues. A few laminopathies have been modeled using the mouse. As zebrafish is a well established model for the study of cardiac development and disease, we have investigated the effects of heart-specific lamin A mutations in transgenic zebrafish. We have developed transgenic lines of zebrafish expressing conserved lamin A mutations that cause cardiac dysfunction in humans. Expression of zlamin A mutations Q291P and M368K in the heart was driven by the zebrafish cardiac troponin T2 promoter. Homozygous mutant embryos displayed nuclear abnormalities in cardiomyocyte nuclei. Expression analysis showed the upregulation of genes involved in heart regeneration in transgenic mutant embryos and a cell proliferation marker was increased in adult heart tissue. At the physiological level, there was deviation of up to 20% from normal heart rate in transgenic embryos expressing mutant lamins. Adult homozygous zebrafish were fertile and did not show signs of early mortality. Our results suggest that transgenic zebrafish models of heart-specific laminopathies show cardiac regeneration and moderate deviations in heart rate during embryonic development. © 2017 International Federation for Cell Biology.

  12. Progranulin modulates zebrafish motoneuron development in vivo and rescues truncation defects associated with knockdown of Survival motor neuron 1

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    Bateman Andrew

    2010-10-01

    Full Text Available Abstract Background Progranulin (PGRN encoded by the GRN gene, is a secreted glycoprotein growth factor that has been implicated in many physiological and pathophysiological processes. PGRN haploinsufficiency caused by autosomal dominant mutations within the GRN gene leads to progressive neuronal atrophy in the form of frontotemporal lobar degeneration (FTLD. This form of the disease is associated with neuronal inclusions that bear the ubiquitinated TAR DNA Binding Protein-43 (TDP-43 molecular signature (FTLD-U. The neurotrophic properties of PGRN in vitro have recently been reported but the role of PGRN in neurons is not well understood. Here we document the neuronal expression and functions of PGRN in spinal cord motoneuron (MN maturation and branching in vivo using zebrafish, a well established model of vertebrate embryonic development. Results Whole-mount in situ hybridization and immunohistochemical analyses of zebrafish embryos revealed that zfPGRN-A is expressed within the peripheral and central nervous systems including the caudal primary (CaP MNs within the spinal cord. Knockdown of zfPGRN-A mRNA translation mediated by antisense morpholino oligonucleotides disrupted normal CaP MN development resulting in both truncated MNs and inappropriate early branching. Ectopic over-expression of zfPGRN-A mRNA resulted in increased MN branching and rescued the truncation defects brought about by knockdown of zfPGRN-A expression. The ability of PGRN to interact with established MN developmental pathways was tested. PGRN over-expression was found to reverse the truncation defect resulting from knockdown of Survival of motor neuron 1 (smn1. This is involved in small ribonucleoprotein biogenesis RNA processing, mutations of which cause Spinal Muscular Atrophy (SMA in humans. It did not reverse the MN defects caused by interfering with the neuronal guidance pathway by knockdown of expression of NRP-1, a semaphorin co-receptor. Conclusions Expression of

  13. β-catenin/Wnt signaling controls progenitor fate in the developing and regenerating zebrafish retina

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    Meyers Jason R

    2012-08-01

    Full Text Available Abstract Background The zebrafish retina maintains two populations of stem cells: first, the germinal zone or ciliary marginal zone (CMZ contains multipotent retinal progenitors that add cells to the retinal periphery as the fish continue to grow; second, radial glia (Müller cells occasionally divide asymmetrically to generate committed progenitors that differentiate into rod photoreceptors, which are added interstitially throughout the retina with growth. Retinal injury stimulates Müller glia to dedifferentiate, re-enter the cell cycle, and generate multipotent retinal progenitors similar to those in the CMZ to replace missing neurons. The specific signals that maintain these two distinct populations of endogenous retinal stem cells are not understood. Results We used genetic and pharmacological manipulation of the β-catenin/Wnt signaling pathway to show that it is required to maintain proliferation in the CMZ and that hyperstimulation of β-catenin/Wnt signaling inhibits normal retinal differentiation and expands the population of proliferative retinal progenitors. To test whether similar effects occur during regeneration, we developed a method for making rapid, selective photoreceptor ablations in larval zebrafish with intense light. We found that dephosphorylated β-catenin accumulates in Müller glia as they re-enter the cell cycle following injury, but not in Müller glia that remain quiescent. Activation of Wnt signaling is required for regenerative proliferation, and hyperstimulation results in loss of Müller glia from the INL as all proliferative cells move into the ONL. Conclusions β-catenin/Wnt signaling is thus required for the maintenance of retinal progenitors during both initial development and lesion-induced regeneration, and is sufficient to prevent differentiation of those progenitors and maintain them in a proliferative state. This suggests that the β-catenin/Wnt cascade is part of the shared molecular circuitry that

  14. Cardiac and Metabolic Physiology of Early Larval Zebrafish (Danio rerio) Reflects Parental Swimming Stamina.

    Science.gov (United States)

    Gore, Matthew; Burggren, Warren W

    2012-01-01

    Swimming stamina in adult fish is heritable, it is unknown if inherited traits that support enhanced swimming stamina in offspring appear only in juveniles and/or adults, or if these traits actually appear earlier in the morphologically quite different larvae. To answer this question, mature adult zebrafish (Danio rerio) were subjected to a swimming performance test that allowed separation into low swimming stamina or high swimming stamina groups. Adults were then bred within their own performance groups. Larval offspring from each of the two groups, designated high (L(HSD)) and low stamina-derived larvae (L(LSD)), were then reared at 27°C in aerated water (21% O(2)). Routine (f(H),r) and active (f(H),a) heart rate, and routine [Formula: see text] and active [Formula: see text] mass-specific oxygen consumption were recorded from 5 days post fertilization (dpf) through 21 dpf, and gross cost of transport and factorial aerobic metabolic scope were derived from [Formula: see text] measurements. Heart rate generally ranged between 150 and 225 bpm in both L(HSD) and L(LSD) populations. However, significant (P stamina in adult parents also appear in their larval offspring well before attainment of juvenile or adult features.

  15. Comparing phototoxicity during the development of a zebrafish craniofacial bone using confocal and light sheet fluorescence microscopy techniques.

    Science.gov (United States)

    Jemielita, Matthew; Taormina, Michael J; Delaurier, April; Kimmel, Charles B; Parthasarathy, Raghuveer

    2013-12-01

    The combination of genetically encoded fluorescent proteins and three-dimensional imaging enables cell-type-specific studies of embryogenesis. Light sheet microscopy, in which fluorescence excitation is provided by a plane of laser light, is an appealing approach to live imaging due to its high speed and efficient use of photons. While the advantages of rapid imaging are apparent from recent work, the importance of low light levels to studies of development is not well established. We examine the zebrafish opercle, a craniofacial bone that exhibits pronounced shape changes at early developmental stages, using both spinning disk confocal and light sheet microscopies of fluorescent osteoblast cells. We find normal and aberrant opercle morphologies for specimens imaged with short time intervals using light sheet and spinning disk confocal microscopies, respectively, under equivalent exposure conditions over developmentally-relevant time scales. Quantification of shapes reveals that the differently imaged specimens travel along distinct trajectories in morphological space. Copyright © 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  16. Involvement of COX2–thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish

    International Nuclear Information System (INIS)

    Teraoka, Hiroki; Okuno, Yuki; Nijoukubo, Daisuke; Yamakoshi, Ayumi; Peterson, Richard E.; Stegeman, John J.; Kitazawa, Takio; Hiraga, Takeo; Kubota, Akira

    2014-01-01

    Highlights: • We establish a new indicator of pericardial edema in developing zebrafish (precardiac edema). • Property of precardiac edema by TCDD is similar to that for conventional pericardial edema. • COX2b (but not COX2a)–thromboxane pathway is involved in precardiac edema by TCDD. - Abstract: The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration–response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b

  17. Involvement of COX2–thromboxane pathway in TCDD-induced precardiac edema in developing zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Teraoka, Hiroki, E-mail: hteraoka@rakuno.ac.jp [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Okuno, Yuki; Nijoukubo, Daisuke; Yamakoshi, Ayumi [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Peterson, Richard E. [School of Pharmacy, University of Wisconsin, Madison, WI (United States); Stegeman, John J. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States); Kitazawa, Takio; Hiraga, Takeo [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Kubota, Akira [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States)

    2014-09-15

    Highlights: • We establish a new indicator of pericardial edema in developing zebrafish (precardiac edema). • Property of precardiac edema by TCDD is similar to that for conventional pericardial edema. • COX2b (but not COX2a)–thromboxane pathway is involved in precardiac edema by TCDD. - Abstract: The cardiovascular system is one of the most characteristic and important targets for developmental toxicity by 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in fish larvae. However, knowledge of the mechanism of TCDD-induced edema after heterodimerization of aryl hydrocarbon receptor type 2 (AHR2) and AHR nuclear translocator type 1 (ARNT1) is still limited. In the present study, microscopic analysis with a high-speed camera revealed that TCDD increased the size of a small cavity between the heart and body wall in early eleutheroembryos, a toxic effect that we designate as precardiac edema. A concentration–response curve for precardiac edema at 2 days post fertilization (dpf) showed close similarity to that for conventional pericardial edema at 3 dpf. Precardiac edema caused by TCDD was reduced by morpholino knockdown of AHR2 and ARNT1, as well as by an antioxidant (ascorbic acid). A selective inhibitor of cyclooxygenase type 2 (COX2), NS398, also markedly inhibited TCDD-induced precardiac edema. A thromboxane receptor (TP) antagonist, ICI-192,605 almost abolished TCDD-induced precardiac edema and this effect was canceled by U46619, a TP agonist, which was not influential in the action of TCDD by itself. Knockdown of COX2b and thromboxane A synthase 1 (TBXS), but not COX2a, strongly reduced TCDD-induced precardiac edema. Knockdown of COX2b was without effect on mesencephalic circulation failure caused by TCDD. The edema by TCDD was also inhibited by knockdown of c-mpl, a thrombopoietin receptor necessary for thromobocyte production. Finally, induction of COX2b, but not COX2a, by TCDD was seen in eleutheroembryos at 3 dpf. These results suggest a role of the COX2b

  18. Two novel COLVI long chains in zebrafish that are essential for muscle development.

    Science.gov (United States)

    Ramanoudjame, Laetitia; Rocancourt, Claire; Lainé, Jeanne; Klein, Arnaud; Joassard, Lucette; Gartioux, Corine; Fleury, Marjory; Lyphout, Laura; Kabashi, Edor; Ciura, Sorana; Cousin, Xavier; Allamand, Valérie

    2015-12-01

    Collagen VI (COLVI), a protein ubiquitously expressed in connective tissues, is crucial for structural integrity, cellular adhesion, migration and survival. Six different genes are recognized in mammalians, encoding six COLVI-chains that assemble as two 'short' (α1, α2) and one 'long' chain (theoretically any one of α3-6). In humans, defects in the most widely expressed heterotrimer (α123), due to mutations in the COL6A1-3 genes, cause a heterogeneous group of neuromuscular disorders, collectively termed COLVI-related muscle disorders. Little is known about the function(s) of the recently described α4-6 chains and no mutations have been detected yet. In this study, we characterized two novel COLVI long chains in zebrafish that are most homologous to the mammalian α4 chain; therefore, we named the corresponding genes col6a4a and col6a4b. These orthologues represent ancestors of the mammalian Col6a4-6 genes. By in situ hybridization and RT-qPCR, we unveiled a distinctive expression kinetics for col6a4b, compared with the other col6a genes. Using morpholino antisense oligonucleotides targeting col6a4a, col6a4b and col6a2, we modelled partial and complete COLVI deficiency, respectively. All morphant embryos presented altered muscle structure and impaired motility. While apoptosis was not drastically increased, autophagy induction was defective in all morphants. Furthermore, motoneuron axon growth was abnormal in these morphants. Importantly, some phenotypical differences emerged between col6a4a and col6a4b morphants, suggesting only partial functional redundancy. Overall, our results further confirm the importance of COLVI in zebrafish muscle development and may provide important clues for potential human phenotypes associated with deficiency of the recently described COLVI-chains. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  19. Zebrafish neurofibromatosis type 1 genes have redundant functions in tumorigenesis and embryonic development

    Directory of Open Access Journals (Sweden)

    Jimann Shin

    2012-11-01

    Neurofibromatosis type 1 (NF1 is a common, dominantly inherited genetic disorder that results from mutations in the neurofibromin 1 (NF1 gene. Affected individuals demonstrate abnormalities in neural-crest-derived tissues that include hyperpigmented skin lesions and benign peripheral nerve sheath tumors. NF1 patients also have a predisposition to malignancies including juvenile myelomonocytic leukemia (JMML, optic glioma, glioblastoma, schwannoma and malignant peripheral nerve sheath tumors (MPNSTs. In an effort to better define the molecular and cellular determinants of NF1 disease pathogenesis in vivo, we employed targeted mutagenesis strategies to generate zebrafish harboring stable germline mutations in nf1a and nf1b, orthologues of NF1. Animals homozygous for loss-of-function alleles of nf1a or nf1b alone are phenotypically normal and viable. Homozygous loss of both alleles in combination generates larval phenotypes that resemble aspects of the human disease and results in larval lethality between 7 and 10 days post fertilization. nf1-null larvae demonstrate significant central and peripheral nervous system defects. These include aberrant proliferation and differentiation of oligodendrocyte progenitor cells (OPCs, dysmorphic myelin sheaths and hyperplasia of Schwann cells. Loss of nf1 contributes to tumorigenesis as demonstrated by an accelerated onset and increased penetrance of high-grade gliomas and MPNSTs in adult nf1a+/−; nf1b−/−; p53e7/e7 animals. nf1-null larvae also demonstrate significant motor and learning defects. Importantly, we identify and quantitatively analyze a novel melanophore phenotype in nf1-null larvae, providing the first animal model of the pathognomonic pigmentation lesions of NF1. Together, these findings support a role for nf1a and nf1b as potent tumor suppressor genes that also function in the development of both central and peripheral glial cells as well as melanophores in zebrafish.

  20. Identifying small RNAs derived from maternal- and somatic-type rRNAs in zebrafish development.

    Science.gov (United States)

    Locati, Mauro D; Pagano, Johanna F B; Abdullah, Farah; Ensink, Wim A; van Olst, Marina; van Leeuwen, Selina; Nehrdich, Ulrike; Spaink, Herman P; Rauwerda, Han; Jonker, Martijs J; Dekker, Rob J; Breit, Timo M

    2018-02-09

    rRNAs are non-coding RNAs present in all prokaryotes and eukaryotes. In eukaryotes there are four rRNAs: 18S, 5.8S, 28S, originating from a common precursor (45S), and 5S. We have recently discovered the existence of two distinct developmental types of rRNA: a maternal-type, present in eggs and a somatic-type, expressed in adult tissues. Lately, next-generation sequencing has allowed the discovery of new small-RNAs deriving from longer non-coding RNAs, including small-RNAs from rRNAs (srRNAs). Here, we systemically investigated srRNAs of maternal- or somatic-type 18S, 5.8S, 28S, with small-RNAseq from many zebrafish developmental stages. We identified new srRNAs for each rRNA. For 5.8S, we found srRNA consisting of the 5' or 3' halves, with only the latter having different sequence for the maternal- and somatic-types. For 18S, we discovered 21 nt srRNA from the 5' end of the 18S rRNA with a striking resemblance to microRNAs; as it is likely processed from a stem-loop precursor and present in human and mouse Argonaute-complexed small-RNA. For 28S, an abundant 80 nt srRNA from the 3' end of the 28S rRNA was found. The expression levels during embryogenesis of these srRNA indicate they are not generated from rRNA degradation and might have a role in the zebrafish development.

  1. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Shi, Yanan; Liu, Xiaochun [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y. [School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Cheng, Shuk Han [Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong (China); Li, Shuisheng; Zhang, Yong [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); Cheng, Christopher H.K., E-mail: chkcheng@cuhk.edu.hk [School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, New Territories, Hong Kong (China); Lin, Haoran, E-mail: lsslhr@mail.sysu.edu.cn [State Key Laboratory of Biocontrol, Institute of Aquatic Economic Animals and Guangdong Provincial Key Laboratory for Aquatic Economic Animals, School of Life Sciences, Sun Yat-Sen University, Guangzhou 510275 (China); College of Ocean, Hainan University, Haikou 570228, Hainan (China)

    2013-05-24

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons.

  2. G-protein-coupled estrogen receptor 1 is involved in brain development during zebrafish (Danio rerio) embryogenesis

    International Nuclear Information System (INIS)

    Shi, Yanan; Liu, Xiaochun; Zhu, Pei; Li, Jianzhen; Sham, Kathy W.Y.; Cheng, Shuk Han; Li, Shuisheng; Zhang, Yong; Cheng, Christopher H.K.; Lin, Haoran

    2013-01-01

    Highlights: •The Gper expression was detected in the developing brain of zebrafish. •Gper morpholino knockdown induced apoptosis of brain cells. •Gper morpholino knockdown reduced expression in neuron markers. •Zebrafish Gper may be involved in neuronal development. -- Abstract: G-protein-coupled estrogen receptor 1 (Gper, formerly known as GPR30) is found to be a trophic and protective factor in mediating action of estrogen in adult brain, while its role in developing brain remains to be elucidated. Here we present the expression pattern of Gper and its functions during embryogenesis in zebrafish. Both the mRNA and protein of Gper were detected throughout embryogenesis. Whole mount in situ hybridization (WISH) revealed a wide distribution of gper mRNAs in various regions of the developing brain. Gper knockdown by specific morpholinos resulted in growth retardation in embryos and morphological defects in the developing brain. In addition, induced apoptosis, decreased proliferation of the brain cells and maldevelopment of sensory and motor neurons were also found in the morphants. Our results provide novel insights into Gper functions in the developing brain, revealing that Gper can maintain the survival of the brain cells, and formation and/or differentiation of the sensory and motor neurons

  3. Development and regeneration of the zebrafish maxillary barbel: a novel study system for vertebrate tissue growth and repair.

    Science.gov (United States)

    LeClair, Elizabeth E; Topczewski, Jacek

    2010-01-15

    Barbels are integumentary sense organs found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops paired nasal and maxillary barbels approximately one month post fertilization. Small in diameter and optically clear, these adult appendages offer a window on the development, maintenance and function of multiple cell types including skin cells, neural-crest derived pigment cells, circulatory vessels, taste buds and sensory nerves. Importantly, barbels in other otophysan fishes (e.g., catfish) are known to regenerate; however, this capacity has not been tested in zebrafish. We describe the development of the maxillary barbel in a staged series of wild type and transgenic zebrafish using light microscopy, histology and immunohistochemistry. By imaging transgenic zebrafish containing fluorescently labeled endothelial cells (Tg(fli1a:EGFP)), we demonstrate that the barbel contains a long ( approximately 2-3 mm) closed-end vessel that we interpret as a large lymphatic. The identity of this vessel was further supported by live imaging of the barbel circulation, extending recent descriptions of the lymphatic system in zebrafish. The maxillary barbel can be induced to regenerate by proximal amputation. After more than 750 experimental surgeries in which approximately 85% of the barbel's length was removed, we find that wound healing is complete within hours, followed by blastema formation ( approximately 3 days), epithelial redifferentiation (3-5 days) and appendage elongation. Maximum regrowth occurs within 2 weeks of injury. Although superficially normal, the regenerates are shorter and thicker than the contralateral controls, have abnormally organized mesenchymal cells and extracellular matrix, and contain prominent connective tissue "stumps" at the plane of section--a mode of regeneration more typical of mammalian scarring than other zebrafish appendages. Finally, we show that the maxillary barbel can regenerate after repeated injury and also in

  4. Development and regeneration of the zebrafish maxillary barbel: a novel study system for vertebrate tissue growth and repair.

    Directory of Open Access Journals (Sweden)

    Elizabeth E LeClair

    2010-01-01

    Full Text Available Barbels are integumentary sense organs found in fishes, reptiles and amphibians. The zebrafish, Danio rerio, develops paired nasal and maxillary barbels approximately one month post fertilization. Small in diameter and optically clear, these adult appendages offer a window on the development, maintenance and function of multiple cell types including skin cells, neural-crest derived pigment cells, circulatory vessels, taste buds and sensory nerves. Importantly, barbels in other otophysan fishes (e.g., catfish are known to regenerate; however, this capacity has not been tested in zebrafish.We describe the development of the maxillary barbel in a staged series of wild type and transgenic zebrafish using light microscopy, histology and immunohistochemistry. By imaging transgenic zebrafish containing fluorescently labeled endothelial cells (Tg(fli1a:EGFP, we demonstrate that the barbel contains a long ( approximately 2-3 mm closed-end vessel that we interpret as a large lymphatic. The identity of this vessel was further supported by live imaging of the barbel circulation, extending recent descriptions of the lymphatic system in zebrafish. The maxillary barbel can be induced to regenerate by proximal amputation. After more than 750 experimental surgeries in which approximately 85% of the barbel's length was removed, we find that wound healing is complete within hours, followed by blastema formation ( approximately 3 days, epithelial redifferentiation (3-5 days and appendage elongation. Maximum regrowth occurs within 2 weeks of injury. Although superficially normal, the regenerates are shorter and thicker than the contralateral controls, have abnormally organized mesenchymal cells and extracellular matrix, and contain prominent connective tissue "stumps" at the plane of section--a mode of regeneration more typical of mammalian scarring than other zebrafish appendages. Finally, we show that the maxillary barbel can regenerate after repeated injury and

  5. Polybrominated diphenyl ethers affect the reproduction and development, and alter the sex ratio of zebrafish (Danio rerio)

    International Nuclear Information System (INIS)

    Han, X.B.; Yuen, Karen W.Y.; Wu, Rudolf S.S.

    2013-01-01

    Polybrominated diphenyl ethers (PBDEs) have been commonly used as flame retardants and now become ubiquitous in the global environment. Using zebrafish as a model, we tested the hypothesis that PBDEs may affect the reproduction and development of fish. Zebrafish were exposed to environmentally relevant concentrations of DE-71 (a congener of PBDE commonly found in the environment) throughout their whole life cycle, and the effects of DE-71 on gonadal development, gamete quality, fertilization success, hatching success, embryonic development and sex ratio were investigated. Despite gonadal development was enhanced, reductions in spawning, fertilization success, hatching success and larval survival rate were evident, while significant increases in malformation and percentage of male were also observed in the F1 generation. Our laboratory results suggest that PBDEs may pose a risk to reproductive success and alter the sex ratio of fish in environments highly contaminated with PBDEs. -- Highlights: •Zebrafish were exposed to PBDE from eggs to adults. •An increase in Gonadal-Somatic Index and enhanced gonadal development was enhanced. •Fertilization and hatching successes were reduced, while malformation was increased. •PBDE alters sex differentiation, leading to a male biased F1 population. •Environmental relevant concentrations of PBDE threaten natural fish populations. -- PBDE reduces fertilization and hatching successes, causes malformation and leads to a male biased F1 generation in fish

  6. Cardiac and metabolic physiology of early larval zebrafish (Danio rerio reflects parental swimming stamina

    Directory of Open Access Journals (Sweden)

    Warren W Burggren

    2012-02-01

    Full Text Available Swimming stamina in adult fish is heritable, it is unknown if inherited traits that support enhanced swimming stamina in offspring appear only in juveniles and/or adults, or if these traits actually appear earlier in the morphologically quite different larvae. To answer this question, mature adult zebrafish (Danio rerio were subjected to a swimming performance test that allowed separation into low swimming stamina or high swimming stamina groups. Adults were then bred within their own performance groups. Larval offspring from each of the two groups, designated high (LHSD and low stamina-derived larvae (LLSD, were then reared at 27°C in aerated water (21% O2. Routine (fH,r and active (fH,a heart rate, and routine (M.O2,r and active (M.O2,a mass-specific oxygen consumption were recorded from 5 days post fertilization (dpf through 21 dpf, and gross cost of transport and factorial aerobic metabolic scope were derived from M.O2 measurements. Heart rate generally ranged between 150 and 225 b•min-1 in both LHSD and LLSD populations. However, significant (P<0.05 differences existed between the LLSD and LHSD populations at 5 and 14 dpf in fH,r and at days 10 and 15 dpf in fH,a. M.O2,r was 0.04 to 0.32 μmol•mg-1•hr-1, while M.O2,a was 0.2 to 1.2 μmol•mg-1•hr-1. Significant (P<0.05 differences between the LLSD and LHSD populations in M.O2,r occurred at 7, 10 and 21 dpf and in M.O2,a at 7 dpf. Gross cost of transport was ~6-10 µmol O2 . µg-1 . m-1 at 5 dpf, peaking at 14-19 µmol O2 . µg-1 . m-1 at 7-10 dpf, before falling again to 5-6 µmol O2 . µg-1 . m-1 at 21 dpf, with gross cost of transport significantly higher in the LLSD population at 7 dpf. Collectively, these data indicate that inherited physiological differences contributing to enhanced stamina in adult parents appear in their larval offspring well before attainment of juvenile or adult features.

  7. Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Kraugerud, Marianne, E-mail: Marianne.Kraugerud@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Doughty, Richard William, E-mail: vetrwdoughty@yahoo.co.uk [Sundveien 22, 2015 Leirsund (Norway); Lyche, Jan L., E-mail: Jan.Lyche@nvh.no [Dept. of Food Safety and Infection Biology, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Berg, Vidar, E-mail: Vidar.Berg@nvh.no [Dept. of Food Safety and Infection Biology, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Tremoen, Nina H., E-mail: Nina.Hardnes@nvh.no [Dept. of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Alestrom, Peter, E-mail: Peter.Alestrom@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Aleksandersen, Mona, E-mail: Mona.Aleksandersen@nvh.no [Dept. of Basic Sciences and Aquatic Medicine, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway); Ropstad, Erik, E-mail: Erik.Ropstad@nvh.no [Dept. of Production Animal Clinical Sciences, Norwegian School of Veterinary Science, POB 8146 Dep., 0033 Oslo (Norway)

    2012-07-15

    Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjosa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjosa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjosa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjosa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjosa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjosa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

  8. Development of a quantitative morphological assessment of toxicant-treated zebrafish larvae using brightfield imaging and high-content analysis.

    Science.gov (United States)

    Deal, Samantha; Wambaugh, John; Judson, Richard; Mosher, Shad; Radio, Nick; Houck, Keith; Padilla, Stephanie

    2016-09-01

    One of the rate-limiting procedures in a developmental zebrafish screen is the morphological assessment of each larva. Most researchers opt for a time-consuming, structured visual assessment by trained human observer(s). The present studies were designed to develop a more objective, accurate and rapid method for screening zebrafish for dysmorphology. Instead of the very detailed human assessment, we have developed the computational malformation index, which combines the use of high-content imaging with a very brief human visual assessment. Each larva was quickly assessed by a human observer (basic visual assessment), killed, fixed and assessed for dysmorphology with the Zebratox V4 BioApplication using the Cellomics® ArrayScan® V(TI) high-content image analysis platform. The basic visual assessment adds in-life parameters, and the high-content analysis assesses each individual larva for various features (total area, width, spine length, head-tail length, length-width ratio, perimeter-area ratio). In developing the computational malformation index, a training set of hundreds of embryos treated with hundreds of chemicals were visually assessed using the basic or detailed method. In the second phase, we assessed both the stability of these high-content measurements and its performance using a test set of zebrafish treated with a dose range of two reference chemicals (trans-retinoic acid or cadmium). We found the measures were stable for at least 1 week and comparison of these automated measures to detailed visual inspection of the larvae showed excellent congruence. Our computational malformation index provides an objective manner for rapid phenotypic brightfield assessment of individual larva in a developmental zebrafish assay. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  9. Carbendazim has the potential to induce oxidative stress, apoptosis, immunotoxicity and endocrine disruption during zebrafish larvae development.

    Science.gov (United States)

    Jiang, Jinhua; Wu, Shenggan; Wang, Yanhua; An, Xuehua; Cai, Leiming; Zhao, Xueping; Wu, Changxing

    2015-10-01

    Increasing evidence have suggested deleterious effects of carbendazim on reproduction, apoptosis, immunotoxicity and endocrine disruption in mice and rats, however, the developmental toxicity of carbendazim to aquatic organisms remains obscure. In the present study, we utilized zebrafish as an environmental monitoring model to characterize the effects of carbendazim on expression of genes related to oxidative stress, apoptosis, immunotoxicity and endocrine disruption during larval development. Different trends in gene expression were observed upon exposing the larvae to 4, 20, 100, and 500 μg/L carbendazim for 4 and 8d. The mRNA levels of catalase, glutathione peroxidase and manganese superoxide dismutase (CAT, GPX, and Mn/SOD) were up-regulated after exposure to different concentrations of carbendazim for 4 or 8d. The up-regulation of p53, Apaf1, Cas8 and the down-regulation of Bcl2, Mdm2, Cas3 in the apoptosis pathway, as well as the increased expression of cytokines and chemokines, including CXCL-C1C, CCL1, IL-1b, IFN, IL-8, and TNFα, suggested carbendazim might trigger apoptosis and immune response during zebrafish larval development. In addition, the alteration of mRNA expression of VTG, ERα, ERβ1, ERβ2, TRα, TRβ, Dio1, and Dio2 indicated the potential of carbendazim to induce endocrine disruption in zebrafish larvae. These data suggested that carbendazim could simultaneously induce multiple responses during zebrafish larval development, and bidirectional interactions among oxidative stress, apoptosis pathway, immune and endocrine systems might be present. Copyright © 2015 Elsevier Ltd. All rights reserved.

  10. Shaped 3D Singular Spectrum Analysis for Quantifying Gene Expression, with Application to the Early Zebrafish Embryo

    Directory of Open Access Journals (Sweden)

    Alex Shlemov

    2015-01-01

    Full Text Available Recent progress in microscopy technologies, biological markers, and automated processing methods is making possible the development of gene expression atlases at cellular-level resolution over whole embryos. Raw data on gene expression is usually very noisy. This noise comes from both experimental (technical/methodological and true biological sources (from stochastic biochemical processes. In addition, the cells or nuclei being imaged are irregularly arranged in 3D space. This makes the processing, extraction, and study of expression signals and intrinsic biological noise a serious challenge for 3D data, requiring new computational approaches. Here, we present a new approach for studying gene expression in nuclei located in a thick layer around a spherical surface. The method includes depth equalization on the sphere, flattening, interpolation to a regular grid, pattern extraction by Shaped 3D singular spectrum analysis (SSA, and interpolation back to original nuclear positions. The approach is demonstrated on several examples of gene expression in the zebrafish egg (a model system in vertebrate development. The method is tested on several different data geometries (e.g., nuclear positions and different forms of gene expression patterns. Fully 3D datasets for developmental gene expression are becoming increasingly available; we discuss the prospects of applying 3D-SSA to data processing and analysis in this growing field.

  11. Zebrafish IGF genes: gene duplication, conservation and divergence, and novel roles in midline and notochord development.

    Directory of Open Access Journals (Sweden)

    Shuming Zou

    Full Text Available Insulin-like growth factors (IGFs are key regulators of development, growth, and longevity. In most vertebrate species including humans, there is one IGF-1 gene and one IGF-2 gene. Here we report the identification and functional characterization of 4 distinct IGF genes (termed as igf-1a, -1b, -2a, and -2b in zebrafish. These genes encode 4 structurally distinct and functional IGF peptides. IGF-1a and IGF-2a mRNAs were detected in multiple tissues in adult fish. IGF-1b mRNA was detected only in the gonad and IGF-2b mRNA only in the liver. Functional analysis showed that all 4 IGFs caused similar developmental defects but with different potencies. Many of these embryos had fully or partially duplicated notochords, suggesting that an excess of IGF signaling causes defects in the midline formation and an expansion of the notochord. IGF-2a, the most potent IGF, was analyzed in depth. IGF-2a expression caused defects in the midline formation and expansion of the notochord but it did not alter the anterior neural patterning. These results not only provide new insights into the functional conservation and divergence of the multiple igf genes but also reveal a novel role of IGF signaling in midline formation and notochord development in a vertebrate model.

  12. Effect of titanium dioxide nanoparticles on zebrafish embryos and developing retina

    Directory of Open Access Journals (Sweden)

    Ya-Jie Wang

    2014-12-01

    Full Text Available AIM:To investigate the impact of titanium dioxide nanoparticles (TiO2 NPs on embryonic development and retinal neurogenesis. METHODS:The agglomeration and sedimentation of TiO2 NPs solutions at different dilutions were observed, and the ultraviolet-visible spectra of their supernatants were measured. Zebrafish embryos were experimentally exposed to TiO2 NPs until 72h postfertilization (hpf. The retinal neurogenesis and distribution of the microglia were analyzed by immunohistochemistry and whole mount in situ hybridization. RESULTS: The1 mg/L was determined to be an appropriate exposure dose. Embryos exposed to TiO2 NPs had a normal phenotype. The neurogenesis was initiated on time, and ganglion cells, cones and rods were well differentiated at 72 hpf. The expression of fms mRNA and the 4C4 antibody, which were specific to microglia in the central nervous system (CNS, closely resembled their endogenous profile. CONCLUSION:These data demonstrate that short-term exposure to TiO2 NPs at a low dose does not lead to delayed embryonic development or retinal neurotoxicity.

  13. A zebrafish model of congenital disorders of glycosylation with phosphomannose isomerase deficiency reveals an early opportunity for corrective mannose supplementation

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    Jaime Chu

    2013-01-01

    Individuals with congenital disorders of glycosylation (CDG have recessive mutations in genes required for protein N-glycosylation, resulting in multi-systemic disease. Despite the well-characterized biochemical consequences in these individuals, the underlying cellular defects that contribute to CDG are not well understood. Synthesis of the lipid-linked oligosaccharide (LLO, which serves as the sugar donor for the N-glycosylation of secretory proteins, requires conversion of fructose-6-phosphate to mannose-6-phosphate via the phosphomannose isomerase (MPI enzyme. Individuals who are deficient in MPI present with bleeding, diarrhea, edema, gastrointestinal bleeding and liver fibrosis. MPI-CDG patients can be treated with oral mannose supplements, which is converted to mannose-6-phosphate through a minor complementary metabolic pathway, restoring protein glycosylation and ameliorating most symptoms, although liver disease continues to progress. Because Mpi deletion in mice causes early embryonic lethality and thus is difficult to study, we used zebrafish to establish a model of MPI-CDG. We used a morpholino to block mpi mRNA translation and established a concentration that consistently yielded 13% residual Mpi enzyme activity at 4 days post-fertilization (dpf, which is within the range of MPI activity detected in fibroblasts from MPI-CDG patients. Fluorophore-assisted carbohydrate electrophoresis detected decreased LLO and N-glycans in mpi morphants. These deficiencies resulted in 50% embryonic lethality by 4 dpf. Multi-systemic abnormalities, including small eyes, dysmorphic jaws, pericardial edema, a small liver and curled tails, occurred in 82% of the surviving larvae. Importantly, these phenotypes could be rescued with mannose supplementation. Thus, parallel processes in fish and humans contribute to the phenotypes caused by Mpi depletion. Interestingly, mannose was only effective if provided prior to 24 hpf. These data provide insight into treatment efficacy

  14. Low Doses of Gamma-Irradiation Induce an Early Bystander Effect in Zebrafish Cells Which Is Sufficient to Radioprotect Cells

    Science.gov (United States)

    Pereira, Sandrine; Malard, Véronique; Ravanat, Jean-Luc; Davin, Anne-Hélène; Armengaud, Jean; Foray, Nicolas; Adam-Guillermin, Christelle

    2014-01-01

    The term “bystander effect” is used to describe an effect in which cells that have not been exposed to radiation are affected by irradiated cells though various intracellular signaling mechanisms. In this study we analyzed the kinetics and mechanisms of bystander effect and radioadaptation in embryonic zebrafish cells (ZF4) exposed to chronic low dose of gamma rays. ZF4 cells were irradiated for 4 hours with total doses of gamma irradiation ranging from 0.01–0.1 Gy. In two experimental conditions, the transfer of irradiated cells or culture medium from irradiated cells results in the occurrence of DNA double strand breaks in non-irradiated cells (assessed by the number of γ-H2AX foci) that are repaired at 24 hours post-irradiation whatever the dose. At low total irradiation doses the bystander effect observed does not affect DNA repair mechanisms in targeted and bystander cells. An increase in global methylation of ZF4 cells was observed in irradiated cells and bystander cells compared to control cells. We observed that pre-irradiated cells which are then irradiated for a second time with the same doses contained significantly less γ-H2AX foci than in 24 h gamma-irradiated control cells. We also showed that bystander cells that have been in contact with the pre-irradiated cells and then irradiated alone present less γ-H2AX foci compared to the control cells. This radioadaptation effect is significantly more pronounced at the highest doses. To determine the factors involved in the early events of the bystander effect, we performed an extensive comparative proteomic study of the ZF4 secretomes upon irradiation. In the experimental conditions assayed here, we showed that the early events of bystander effect are probably not due to the secretion of specific proteins neither the oxidation of these secreted proteins. These results suggest that early bystander effect may be due probably to a combination of multiple factors. PMID:24667817

  15. Zebrafish P54 RNA helicases are cytoplasmic granule residents that are required for development and stress resilience

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    Cecilia Zampedri

    2016-10-01

    Full Text Available Stress granules are cytoplasmic foci that directly respond to the protein synthesis status of the cell. Various environmental insults, such as oxidative stress or extreme heat, block protein synthesis; consequently, mRNA will stall in translation, and stress granules will immediately form and become enriched with mRNAs. P54 DEAD box RNA helicases are components of RNA granules such as P-bodies and stress granules. We studied the expression, in cytoplasmic foci, of both zebrafish P54 RNA helicases (P54a and P54b during development and found that they are expressed in cytoplasmic granules under both normal conditions and stress conditions. In zebrafish embryos exposed to heat shock, some proportion of P54a and P54b helicases move to larger granules that exhibit the properties of genuine stress granules. Knockdown of P54a and/or P54b in zebrafish embryos produces developmental abnormalities restricted to the posterior trunk; further, these embryos do not form stress granules, and their survival upon exposure to heat-shock conditions is compromised. Our observations fit the model that cells lacking stress granules have no resilience or ability to recover once the stress has ended, indicating that stress granules play an essential role in the way organisms adapt to a changing environment.

  16. Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish.

    Science.gov (United States)

    Housley, Michael P; Njaine, Brian; Ricciardi, Filomena; Stone, Oliver A; Hölper, Soraya; Krüger, Marcus; Kostin, Sawa; Stainier, Didier Y R

    2016-06-01

    Skeletal muscles provide metazoans with the ability to feed, reproduce and avoid predators. In humans, a heterogeneous group of genetic diseases, termed muscular dystrophies (MD), lead to skeletal muscle dysfunction. Mutations in the gene encoding Caveolin-3, a principal component of the membrane micro-domains known as caveolae, cause defects in muscle maintenance and function; however it remains unclear how caveolae dysfunction underlies MD pathology. The Cavin family of caveolar proteins can form membrane remodeling oligomers and thus may also impact skeletal muscle function. Changes in the distribution and function of Cavin4/Murc, which is predominantly expressed in striated muscles, have been reported to alter caveolae structure through interaction with Caveolin-3. Here, we report the generation and phenotypic analysis of murcb mutant zebrafish, which display impaired swimming capacity, skeletal muscle fibrosis and T-tubule abnormalities during development. To understand the mechanistic importance of Murc loss of function, we assessed Caveolin-1 and 3 localization and found it to be abnormal. We further identified an in vivo function for Murc in Erk signaling. These data link Murc with developmental defects in T-tubule formation and progressive muscle dysfunction, thereby providing a new candidate for the etiology of muscular dystrophy.

  17. Cavin4b/Murcb Is Required for Skeletal Muscle Development and Function in Zebrafish.

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    Michael P Housley

    2016-06-01

    Full Text Available Skeletal muscles provide metazoans with the ability to feed, reproduce and avoid predators. In humans, a heterogeneous group of genetic diseases, termed muscular dystrophies (MD, lead to skeletal muscle dysfunction. Mutations in the gene encoding Caveolin-3, a principal component of the membrane micro-domains known as caveolae, cause defects in muscle maintenance and function; however it remains unclear how caveolae dysfunction underlies MD pathology. The Cavin family of caveolar proteins can form membrane remodeling oligomers and thus may also impact skeletal muscle function. Changes in the distribution and function of Cavin4/Murc, which is predominantly expressed in striated muscles, have been reported to alter caveolae structure through interaction with Caveolin-3. Here, we report the generation and phenotypic analysis of murcb mutant zebrafish, which display impaired swimming capacity, skeletal muscle fibrosis and T-tubule abnormalities during development. To understand the mechanistic importance of Murc loss of function, we assessed Caveolin-1 and 3 localization and found it to be abnormal. We further identified an in vivo function for Murc in Erk signaling. These data link Murc with developmental defects in T-tubule formation and progressive muscle dysfunction, thereby providing a new candidate for the etiology of muscular dystrophy.

  18. Fog1 is required for cardiac looping in zebrafish

    OpenAIRE

    Walton, R. Zaak; Bruce, Ashley E.E.; Olivey, Harold E.; Najib, Khalid; Johnson, Vanitha; Earley, Judy U.; Ho, Robert K.; Svensson, Eric C.

    2006-01-01

    To further our understanding of FOG gene function during cardiac development, we utilized zebrafish to examine FOG’s role in the early steps of heart morphogenesis. We identified fragments of three fog genes in the zebrafish genomic database and isolated full-length coding sequences for each of these genes by using a combination of RT-PCR and 5′-RACE. One gene was similar to murine FOG-1 (fog1), while the remaining two were similar to murine FOG-2 (fog2a and fog2b). All Fog proteins were able...

  19. Phenothiourea sensitizes zebrafish cranial neural crest and extraocular muscle development to changes in retinoic acid and IGF signaling.

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    Brenda L Bohnsack

    Full Text Available 1-Phenyl 2-thiourea (PTU is a tyrosinase inhibitor commonly used to block pigmentation and aid visualization of zebrafish development. At the standard concentration of 0.003% (200 µM, PTU inhibits melanogenesis and reportedly has minimal other effects on zebrafish embryogenesis. We found that 0.003% PTU altered retinoic acid and insulin-like growth factor (IGF regulation of neural crest and mesodermal components of craniofacial development. Reduction of retinoic acid synthesis by the pan-aldehyde dehydrogenase inhibitor diethylbenzaldehyde, only when combined with 0.003% PTU, resulted in extraocular muscle disorganization. PTU also decreased retinoic acid-induced teratogenic effects on pharyngeal arch and jaw cartilage despite morphologically normal appearing PTU-treated controls. Furthermore, 0.003% PTU in combination with inhibition of IGF signaling through either morpholino knockdown or pharmacologic inhibition of tyrosine kinase receptor phosphorylation, disrupted jaw development and extraocular muscle organization. PTU in and of itself inhibited neural crest development at higher concentrations (0.03% and had the greatest inhibitory effect when added prior to 22 hours post fertilization (hpf. Addition of 0.003% PTU between 4 and 20 hpf decreased thyroxine (T4 in thyroid follicles in the nasopharynx of 96 hpf embryos. Treatment with exogenous triiodothyronine (T3 and T4 improved, but did not completely rescue, PTU-induced neural crest defects. Thus, PTU should be used with caution when studying zebrafish embryogenesis as it alters the threshold of different signaling pathways important during craniofacial development. The effects of PTU on neural crest development are partially caused by thyroid hormone signaling.

  20. Influence of carbon nanotube length on toxicity to zebrafish embryos

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    Cheng J

    2012-07-01

    Full Text Available Jinping Cheng,1,2 Shuk Han Cheng11Department of Biology and Chemistry, City University of Hong Kong, Hong Kong; 2State Key Laboratory of Estuarine and Coastal Research, East China Normal University, Shanghai, ChinaAbstract: There is currently a large difference of opinion in nanotoxicology studies of nanomaterials. There is concern about why some studies have indicated that there is strong toxicity, while others have not. In this study, the length of carbon nanotubes greatly affected their toxicity in zebrafish embryos. Multiwalled carbon nanotubes (MWCNTs were sonicated in a nitric acid solution for 24 hours and 48 hours. The modified MWCNTs were tested in early developing zebrafish embryo. MWCNTs prepared with the longer sonication time resulted in severe developmental toxicity; however, the shorter sonication time did not induce any obvious toxicity in the tested developing zebrafish embryos. The cellular and molecular changes of the affected zebrafish embryos were studied and the observed phenotypes scored. This study suggests that length plays an important role in the in vivo toxicity of functionalized CNTs. This study will help in furthering the understanding on current differences in toxicity studies of nanomaterials.Keywords: length, carbon nanotubes, sonication, developmental toxicity, zebrafish

  1. Noonan and LEOPARD syndrome in zebrafish : molecular mechanisms and cardiac development

    NARCIS (Netherlands)

    Bonetti, Monica; Paardekooper Overman, Jeroen

    2014-01-01

    This thesis describes the use of zebrafish to study Noonan-(NS) and LEOPARD syndromes (LS), two autosomal dominant disorders with overlapping symptoms, caused by mutations in protein-tyrosine phosphatase, non-receptor type 11 (PTPN11). Intriguingly, while NS mutations result in a more ‘active’ state

  2. Elucidation of possible molecular mechanisms underlying the estrogen-induced disruption of cartilage development in zebrafish larvae.

    Science.gov (United States)

    He, Hanliang; Wang, Chunqing; Tang, Qifeng; Yang, Fan; Xu, Youjia

    2018-06-01

    Estrogen can affect the cartilage development of zebrafish; however, the mechanism underlying its effects is not completely understood. Four-day-old zebrafish larvae were treated with 0.8 μM estrogen, the 5 days post fertilization (dpf) zebrafish larvae did not demonstrate obvious abnormalities during development; however, the 6 dpf and 7 dpf larvae exhibited abnormal craniofacial bone development along with craniofacial bone degradation. RNA deep sequencing was performed to elucidate the mechanism involved. Gene Ontology functional and KEGG pathway enrichment analysis of differentially expressed genes (DEGs) showed that the extracellular matrix (ECM), extracellular region, ECM-interaction receptor, focal adhesion, cell cycle, apoptosis, and bone-related signaling pathways were disrupted. In these signaling pathways, the expressions of key genes, such as collagen encoded (col19a1a, col7a1, col7al, col18a1, and col9a3), MAPK signaling pathway (fgf19, fgf6a), TGF-beta signaling pathway (tgfbr1), and cell cycle (cdnk1a) genes were altered. The qRT-PCR results showed that after treatment with 0.8 μM 17-β estradiol (E2), col19a1a, col7a1, col7al, col18a1, col9a3, fgf6a, cdkn1a were downregulated, and fgf19, tgfr1 were upregulated, which were consistent with deep sequencing analysis. Therefore, the effect of estrogen on cartilage development might occur via multiple mechanisms. The study results demonstrate the mechanism underlying the effect of estrogen on cartilage development. Copyright © 2018 Elsevier B.V. All rights reserved.

  3. CERKL knockdown causes retinal degeneration in zebrafish.

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    Marina Riera

    Full Text Available The human CERKL gene is responsible for common and severe forms of retinal dystrophies. Despite intense in vitro studies at the molecular and cellular level and in vivo analyses of the retina of murine knockout models, CERKL function remains unknown. In this study, we aimed to approach the developmental and functional features of cerkl in Danio rerio within an Evo-Devo framework. We show that gene expression increases from early developmental stages until the formation of the retina in the optic cup. Unlike the high mRNA-CERKL isoform multiplicity shown in mammals, the moderate transcriptional complexity in fish facilitates phenotypic studies derived from gene silencing. Moreover, of relevance to pathogenicity, teleost CERKL shares the two main human protein isoforms. Morpholino injection has been used to generate a cerkl knockdown zebrafish model. The morphant phenotype results in abnormal eye development with lamination defects, failure to develop photoreceptor outer segments, increased apoptosis of retinal cells and small eyes. Our data support that zebrafish Cerkl does not interfere with proliferation and neural differentiation during early developmental stages but is relevant for survival and protection of the retinal tissue. Overall, we propose that this zebrafish model is a powerful tool to unveil CERKL contribution to human retinal degeneration.

  4. Tritiated water exposure disrupts myofibril structure and induces mis-regulation of eye opacity and DNA repair genes in zebrafish early life stages.

    Science.gov (United States)

    Arcanjo, Caroline; Armant, Olivier; Floriani, Magali; Cavalie, Isabelle; Camilleri, Virginie; Simon, Olivier; Orjollet, Daniel; Adam-Guillermin, Christelle; Gagnaire, Béatrice

    2018-04-27

    Tritium ( 3 H) is a radioactive isotope of hydrogen. In the environment, the most common form of tritium is tritiated water (HTO). The present study aimed to identify early biomarkers of HTO contamination through the use of an aquatic model, the zebrafish (Danio rerio). We used the zebrafish embryo-larvae model to investigate the modes of action of HTO exposure at dose rates of 0.4 and 4 mGy/h, dose rates expected to induce deleterious effects on fish. Zebrafish were exposed to HTO from 3 hpf (hours post fertilization) to 96 hpf. The transcriptomic effects were investigated 24 h and 96 h after the beginning of the contamination, using mRNAseq. Results suggested an impact of HTO contamination, regardless of the dose rate, on genes involved in muscle contraction (tnnt2d, tnni2a.4, slc6a1a or atp2a1l) and eye opacity (crygm2d9, crygmxl1, mipb or lim2.3) after 24 h of contamination. Interestingly, an opposite differential expression was highlighted in genes playing a role in muscle contraction and eye opacity in 24 hpf embryos when comparing dose rates, suggesting an onset of DNA protective mechanisms. The expression of h2afx and ddb2 involved in DNA repair was enhanced in response to HTO exposure. The entrainment of circadian clock and the response to H 2 O 2 signalling pathways were enriched at 96 hpf at 0.4 mGy/h and in both stages after 4 mGy/h. Genes involved in ROS scavenging were differentially expressed only after 24 h of exposure for the lowest dose rate, suggesting the onset of early protective mechanisms against oxidative stress. Effects highlighted on muscle at the molecular scale were confirmed at a higher biological scale, as electron microscopy observations revealed sarcomere impairments in 96 hpf larvae for both dose rates. Together with other studies, the present work provides useful data to better understand modes of action of tritium on zebrafish embryos-larvae. Copyright © 2018 Elsevier B.V. All rights reserved.

  5. Disruption of the folate pathway in zebrafish causes developmental defects

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    Lee Marina S

    2012-04-01

    Full Text Available Abstract Background Folic acid supplementation reduces the risk of neural tube defects and congenital heart defects. The biological mechanisms through which folate prevents birth defects are not well understood. We explore the use of zebrafish as a model system to investigate the role of folate metabolism during development. Results We first identified zebrafish orthologs of 12 human folate metabolic genes. RT-PCR and in situ analysis indicated maternal transcripts supply the embryo with mRNA so that the embryo has an intact folate pathway. To perturb folate metabolism we exposed zebrafish embryos to methotrexate (MTX, a potent inhibitor of dihydrofolate reductase (Dhfr an essential enzyme in the folate metabolic pathway. Embryos exposed to high doses of MTX exhibited developmental arrest prior to early segmentation. Lower doses of MTX resulted in embryos with a shortened anterior-posterior axis and cardiac defects: linear heart tubes or incomplete cardiac looping. Inhibition of dhfr mRNA with antisense morpholino oligonucleotides resulted in embryonic lethality. One function of the folate pathway is to provide essential one-carbon units for dTMP synthesis, a rate-limiting step of DNA synthesis. After 24 hours of exposure to high levels of MTX, mutant embryos continue to incorporate the thymidine analog BrdU. However, additional experiments indicate that these embryos have fewer mitotic cells, as assayed with phospho-histone H3 antibodies, and that treated embryos have perturbed cell cycles. Conclusions Our studies demonstrate that human and zebrafish utilize similar one-carbon pathways. Our data indicate that folate metabolism is essential for early zebrafish development. Zebrafish studies of the folate pathway and its deficiencies could provide insight into the underlying etiology of human birth defects and the natural role of folate in development.

  6. Effects of metal exposure on motor neuron development, neuromasts and the escape response of zebrafish embryos.

    Science.gov (United States)

    Sonnack, Laura; Kampe, Sebastian; Muth-Köhne, Elke; Erdinger, Lothar; Henny, Nicole; Hollert, Henner; Schäfers, Christoph; Fenske, Martina

    2015-01-01

    Low level metal contaminations are a prevalent issue with often unknown consequences for health and the environment. Effect-based, multifactorial test systems with zebrafish embryos to assess in particular developmental toxicity are beneficial but rarely used in this context. We therefore exposed wild-type embryos to the metals copper (CuSO4), cadmium (CdCl2) and cobalt (CoSO4) for 72 h to determine lethal as well as sublethal morphological effects. Motor neuron damage was investigated by immunofluorescence staining of primary motor neurons (PMNs) and secondary motor neurons (SMNs). In vivo stainings using the vital dye DASPEI were used to quantify neuromast development and damage. The consequences of metal toxicity were also assessed functionally, by testing fish behavior following tactile stimulation. The median effective concentration (EC50) values for morphological effects 72 h post fertilization (hpf) were 14.6 mg/L for cadmium and 0.018 mg/L for copper, whereas embryos exposed up to 45.8 mg/L cobalt showed no morphological effects. All three metals caused a concentration-dependent reduction in the numbers of normal PMNs and SMNs, and in the fluorescence intensity of neuromasts. The results for motor neuron damage and behavior were coincident for all three metals. Even the lowest metal concentrations (cadmium 2mg/L, copper 0.01 mg/L and cobalt 0.8 mg/L) resulted in neuromast damage. The results demonstrate that the neuromast cells were more sensitive to metal exposure than morphological traits or the response to tactile stimulation and motor neuron damage. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Harmonin (Ush1c is required in zebrafish Müller glial cells for photoreceptor synaptic development and function

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    Jennifer B. Phillips

    2011-11-01

    Usher syndrome is the most prevalent cause of hereditary deaf-blindness, characterized by congenital sensorineural hearing impairment and progressive photoreceptor degeneration beginning in childhood or adolescence. Diagnosis and management of this disease are complex, and the molecular changes underlying sensory cell impairment remain poorly understood. Here we characterize two zebrafish models for a severe form of Usher syndrome, Usher syndrome type 1C (USH1C: one model is a mutant with a newly identified ush1c nonsense mutation, and the other is a morpholino knockdown of ush1c. Both have defects in hearing, balance and visual function from the first week of life. Histological analyses reveal specific defects in sensory cell structure that are consistent with these behavioral phenotypes and could implicate Müller glia in the retinal pathology of Usher syndrome. This study shows that visual defects associated with loss of ush1c function in zebrafish can be detected from the onset of vision, and thus could be applicable to early diagnosis for USH1C patients.

  8. Effects of dietary exposure to brominated flame retardant BDE-47 on thyroid condition, gonadal development and growth of zebrafish

    Science.gov (United States)

    Torres, Leticia; Orazio, Carl E.; Peterman, Paul H.; Patino, Reynaldo

    2013-01-01

    Little is known about the effects of brominated flame retardants in teleosts and some of the information currently available is inconsistent. This study examined effects of dietary exposure to 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) on thyroid condition, body mass and size, and gonadal development of zebrafish. Pubertal, 49-day-old (posthatch) fish were fed diets without BDE-47 (control) or with 1, 5 or 25 μg/g BDE-47/diet. Treatments were conducted in triplicate 30-L tanks each containing 50 zebrafish, and 15 fish per treatment (5 per tank) were sampled at days 40, 80 and 120 of exposure. Measurements were taken of body mass, standard length, head depth and head length. Sex (at 40–120 days of exposure), germ cell stage (at 40 days) and thyroid condition (at 120 days; follicular cell height, colloid depletion, angiogenesis) were histologically determined. Whole-body BDE-47 levels at study completion were within the high end of levels reported in environmentally exposed (wild) fishes. Analysis of variance was used to determine differences among treatments at each sampling time. No effects were observed on thyroid condition or germ cell stage in either sex. Reduced head length was observed in females exposed to BDE-47 at 80 days but not at 40 or 120 days. In males, no apparent effects of BDE-47 were observed at 40 and 80 days, but fish exposed to 25 μg/g had lower body mass at 120 days compared to control fish. These observations suggest that BDE-47 at environmentally relevant whole-body concentrations does not affect thyroid condition or pubertal development of zebrafish but does affect growth during the juvenile-to-adult transition, especially in males.

  9. A genetic screen for vascular mutants in zebrafish reveals dynamic roles for Vegf/Plcg1 signaling during artery development.

    Science.gov (United States)

    Covassin, L D; Siekmann, A F; Kacergis, M C; Laver, E; Moore, J C; Villefranc, J A; Weinstein, B M; Lawson, N D

    2009-05-15

    In this work we describe a forward genetic approach to identify mutations that affect blood vessel development in the zebrafish. By applying a haploid screening strategy in a transgenic background that allows direct visualization of blood vessels, it was possible to identify several classes of mutant vascular phenotypes. Subsequent characterization of mutant lines revealed that defects in Vascular endothelial growth factor (Vegf) signaling specifically affected artery development. Comparison of phenotypes associated with different mutations within a functional zebrafish Vegf receptor-2 ortholog (referred to as kdr-like, kdrl) revealed surprisingly varied effects on vascular development. In parallel, we identified an allelic series of mutations in phospholipase c gamma 1 (plcg1). Together with in vivo structure-function analysis, our results suggest a requirement for Plcg1 catalytic activity downstream of receptor tyrosine kinases. We further find that embryos lacking both maternal and zygotic plcg1 display more severe defects in artery differentiation but are otherwise similar to zygotic mutants. Finally, we demonstrate through mosaic analysis that plcg1 functions autonomously in endothelial cells. Together our genetic analyses suggest that Vegf/Plcg1 signaling acts at multiple time points and in different signaling contexts to mediate distinct aspects of artery development.

  10. Natural mixtures of persistent organic pollutants (POPs) suppress ovarian follicle development, liver vitellogenin immunostaining and hepatocyte proliferation in female zebrafish (Danio rerio)

    International Nuclear Information System (INIS)

    Kraugerud, Marianne; Doughty, Richard William; Lyche, Jan L.; Berg, Vidar; Tremoen, Nina H.; Alestrøm, Peter; Aleksandersen, Mona; Ropstad, Erik

    2012-01-01

    Persistent organic pollutants such as polychlorinated biphenyls (PCBs), polybrominated diphenyl ethers (PBDEs) and dichlorodiphenyltrichloroethane (DDT) are present in high concentrations in livers of burbot (Lota lota) in Lake Mjøsa, Norway. In order to assess effects of such pollutants on fish gonadal morphology, female zebrafish were exposed in two generations by food to mixtures of pollutants extracted from livers of burbot from Lake Mjøsa (high and low dose) and Lake Losna, which represents background pollution, and compared to a control group. Ovarian follicle counts detected a significant decrease in late vitellogenic follicle stages in fish exposed to the Losna and the high concentrations of Mjøsa mixtures in fish from the first generation. In addition, proliferation of granulosa cells, visualized by immunohistochemistry against proliferating cell nuclear antigen (PCNA), was decreased in all exposure groups in either early or late vitellogenic follicle stages compared to control. This was accompanied by increased apoptosis of granulosa cells. There was a decrease in proliferation of liver hepatocytes with exposure to both Mjøsa mixtures. In addition, immunopositivity for vitellogenin in the liver was significantly lower in the Mjøsa high group than in the control group. When analysing effects of parental exposure, fish with parents exposed to Mjøsa high mixture had significantly higher numbers of perinucleolar follicles than fish with control parents. We conclude that long-term exposure of a real-life mixture of pollutants containing high- and background levels of chemicals supress ovarian follicle development, liver vitellogenin immunostaining intensity and hepatocyte proliferation in the zebrafish model.

  11. Effects of titanium dioxide nanoparticles on lead bioconcentration and toxicity on thyroid endocrine system and neuronal development in zebrafish larvae.

    Science.gov (United States)

    Miao, Wei; Zhu, Biran; Xiao, Xiaohong; Li, Ying; Dirbaba, Niguse Bekele; Zhou, Bingsheng; Wu, Hongjuan

    2015-04-01

    Nanoparticles (NPs) have attracted considerable attention because of their wide range of applications. Interactions between heavy metals (e.g., Pb) and NPs in aquatic environments may modify the bioavailability and toxicity of heavy metals. Therefore, this study investigated the influence of NPs (e.g., nano-TiO2) on the bioavailability and toxicity of Pb and its effects in the thyroid endocrine and nervous systems of zebrafish (Danio rerio) larvae. Zebrafish embryos (2-h post-fertilization) were exposed to five concentrations of Pb alone (0, 5, 10, 20, and 30μg/L) or in combination with nano-TiO2 (0.1mg/L) until 6 days post-fertilization. Results showed that the bioconcentration of Pb was significantly enhanced when combined with nano-TiO2 than when used alone. Zebrafish exposure to Pb alone at 30μg/L significantly decreased the thyroid hormone levels (T4 and T3), whereas nano-TiO2 treatment alone did not produce detectable changes. The levels of T4 and T3 were further decreased when Pb was combined with nano-TiO2 than when used alone. The transcription of the thyroid hormone-related factor tg gene was remarkably down-regulated by Pb treatment alone but up-regulated when Pb was combined with nano-TiO2. The significant up-regulation of tshβ gene and the down-regulation of TTR gene expression in the hypothalamic-pituitary-thyroid were observed in Pb with or without nano-TiO2 treatment groups. In addition, the transcription of genes involved in central nervous system (CNS) development (α-tubulin, mbp, gfap and shha) were significantly down-regulated by Pb and nano-TiO2 co-exposure as compared with Pb exposure alone. The locomotion activity analyzes confirmed that nano-TiO2 might enhance the toxicity of Pb to CNS development. These results suggest that nano-TiO2 increase bioconcentration of lead, which lead to the disruption of thyroid endocrine and neuronal system in zebrafish larvae. Copyright © 2015 Elsevier B.V. All rights reserved.

  12. Telomere lengthening early in development.

    Science.gov (United States)

    Liu, Lin; Bailey, Susan M; Okuka, Maja; Muñoz, Purificación; Li, Chao; Zhou, Lingjun; Wu, Chao; Czerwiec, Eva; Sandler, Laurel; Seyfang, Andreas; Blasco, Maria A; Keefe, David L

    2007-12-01

    Stem cells and cancer cells maintain telomere length mostly through telomerase. Telomerase activity is high in male germ line and stem cells, but is low or absent in mature oocytes and cleavage stage embryos, and then high again in blastocysts. How early embryos reset telomere length remains poorly understood. Here, we show that oocytes actually have shorter telomeres than somatic cells, but their telomeres lengthen remarkably during early cleavage development. Moreover, parthenogenetically activated oocytes also lengthen their telomeres, thus the capacity to elongate telomeres must reside within oocytes themselves. Notably, telomeres also elongate in the early cleavage embryos of telomerase-null mice, demonstrating that telomerase is unlikely to be responsible for the abrupt lengthening of telomeres in these cells. Coincident with telomere lengthening, extensive telomere sister-chromatid exchange (T-SCE) and colocalization of the DNA recombination proteins Rad50 and TRF1 were observed in early cleavage embryos. Both T-SCE and DNA recombination proteins decrease in blastocyst stage embryos, whereas telomerase activity increases and telomeres elongate only slowly. We suggest that telomeres lengthen during the early cleavage cycles following fertilization through a recombination-based mechanism, and that from the blastocyst stage onwards, telomerase only maintains the telomere length established by this alternative mechanism.

  13. Toxic effects of {sup 56}Fe ion radiation on the zebrafish (Danio rerio) embryonic development

    Energy Technology Data Exchange (ETDEWEB)

    Si, Jing; Zhou, Rong [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Song, Jing’e [Hospital of Stomatology, Lanzhou University, Lanzhou 730000 (China); Gan, Lu; Zhou, Xin; Di, Cuixia; Liu, Yang; Mao, Aihong; Zhao, Qiuyue; Wang, Yupei [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Zhang, Hong, E-mail: zhangh@impcas.ac.cn [Department of Radiation Medicine, Institute of Modern Physics, Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Heavy Ion Radiation Biology and Medicine of Chinese Academy of Sciences, Lanzhou 730000 (China); Key Laboratory of Basic Research on Heavy Ion Radiation Application in Medicine, Gansu Province, Lanzhou 730000 (China); Gansu Wuwei Institute of Medical Sciences, Wuwei 733000 (China)

    2017-05-15

    Highlights: • Iron ion radiation induced developmental toxicity and apoptosis in zebrafish embryos. • The mRNA expression levels of apoptosis-related genes displayed more sensitivity than the developmental toxicity. • Iron ion radiation induced apoptosis in zebrafish embryos potentially due to DNA damage and mitochondrial dysfunction. - Abstract: All living organisms and ecosystems are permanently exposed to ionizing radiation. Of all the types of ionizing radiation, heavy ions such as {sup 56}Fe have the potential to cause the most severe biological effects. We therefore examined the effects and potential mechanisms of iron ion irradiation on the induction of developmental toxicity and apoptosis in zebrafish embryos. Zebrafish embryos at 4 h post-fertilization (hpf) were divided into five groups: a control group; and four groups irradiated with 0.5, 1, 2, and 4 Gy radiation, respectively. Mortality and teratogenesis were significantly increased, and spontaneous movement, heart rate, and swimming distance were decreased in the irradiated groups, accompanied by increased apoptosis. mRNA levels of genes involved in the apoptotic pathway, including p53, bax, bcl-2, and caspase-3, were significantly affected by radiation exposure. Moreover, protein expression levels of P53 and Bcl-2 changed in accordance with the corresponding mRNA expression levels. In addition, we detected the protein expression levels of γ-H2AX, which is a biomarker for radiation-induced DNA double-strand breaks, and found that γ-H2AX protein levels were significantly increased in the irradiated groups. Overall, the results of this study improve our understanding of the mechanisms of iron ion radiation-induced developmental toxicity and apoptosis, potentially involving the induction of DNA damage and mitochondrial dysfunction. The findings of this study may aid future impact assessment of environmental radioactivity in fish.

  14. Induction of cytochrome P450 1 genes and stress response genes in developing zebrafish exposed to ultraviolet radiation

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    Behrendt, Lars [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Joensson, Maria E. [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Department of Environmental Toxicology, Uppsala University (Sweden); Goldstone, Jared V. [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Redfield 352 MS-32, Woods Hole Oceanographic Institution, 45 Water Street, Woods Hole, MA 02543 (United States)

    2010-06-01

    Ultraviolet (UV) radiation damages cell molecules, and has been suggested to up-regulate mammalian cytochrome P4501 (CYP1) genes through an aryl hydrocarbon receptor (AHR) mediated mechanism. In this study, embryos and larvae of zebrafish (Danio rerio) were exposed to UV to determine the effects on expression of CYP1 and stress response genes in vivo in these fish. Zebrafish embryos were exposed for varying times to UV on two consecutive days, with exposure beginning at 24 and 48 h post-fertilization (hpf). Embryos exposed for 2, 4 or 6 h twice over 2 days to UVB (0.62 W/m{sup 2}; 8.9-26.7 kJ/m{sup 2}) plus UVA (2.05 W/m{sup 2}; 29.5-144.6 kJ/m{sup 2}) had moderately (2.4 {+-} 0.8-fold) but significantly up-regulated levels of CYP1A. UVA alone had no effect on CYP1A expression. Proliferating cellular nuclear antigen (PCNA) and Cu-Zn superoxide dismutase (SOD1) transcript levels were induced (2.1 {+-} 0.2 and 2.3 {+-} 0.5-fold, respectively) in embryos exposed to two 6-h pulses of 0.62 W/m{sup 2} UVB (26.8 kJ/m{sup 2}). CYP1A was induced also in embryos exposed to higher intensity UVB (0.93 W/m{sup 2}) for two 3-h or two 4-h pulses (20.1 or 26.8 kJ/m{sup 2}). CYP1B1, SOD1 and PCNA expression was induced by the two 3-h pulses of the higher intensity UVB, but not after two 4-h pulses of the higher intensity UVB, possibly due to impaired condition of surviving embryos, reflected in a mortality of 34% at that UVB dose. A single 8-h long exposure of zebrafish larvae (8 dpf) to UVB at 0.93 W/m{sup 2} (26.8 kJ/m{sup 2}) significantly induced CYP1A and CYP1B1 expression, but other CYP1 genes (CYP1C1, CYP1C2 and CYP1D1) showed no significant increase. The results show that UVB can induce expression of CYP1 genes as well stress response genes in developing zebrafish, and that UVB intensity and duration influence the responses.

  15. Zebrafish msxB, msxC and msxE function together to refine the neural-nonneural border and regulate cranial placodes and neural crest development.

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    Phillips, Bryan T; Kwon, Hye-Joo; Melton, Colt; Houghtaling, Paul; Fritz, Andreas; Riley, Bruce B

    2006-06-15

    The zebrafish muscle segment homeobox genes msxB, msxC and msxE are expressed in partially overlapping domains in the neural crest and preplacodal ectoderm. We examined the roles of these msx genes in early development. Disrupting individual msx genes causes modest variable defects, whereas disrupting all three produces a reproducible severe phenotype, suggesting functional redundancy. Neural crest differentiation is blocked at an early stage. Preplacodal development begins normally, but placodes arising from the msx expression domain later show elevated apoptosis and are reduced in size. Cell proliferation is normal in these tissues. Unexpectedly, Msx-deficient embryos become ventralized by late gastrulation whereas misexpression of msxB dorsalizes the embryo. These effects appear to involve Distal-less (Dlx) protein activity, as loss of dlx3b and dlx4b suppresses ventralization in Msx-depleted embryos. At the same time, Msx-depletion restores normal preplacodal gene expression to dlx3b-dlx4b mutants. These data suggest that mutual antagonism between Msx and Dlx proteins achieves a balance of function required for normal preplacodal differentiation and placement of the neural-nonneural border.

  16. Reprimo tissue-specific expression pattern is conserved between zebrafish and human.

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    Ricardo J Figueroa

    Full Text Available Reprimo (RPRM, a member of the RPRM gene family, is a tumor-suppressor gene involved in the regulation of the p53-mediated cell cycle arrest at G2/M. RPRM has been associated with malignant tumor progression and proposed as a potential biomarker for early cancer detection. However, the expression and role of RPRM, as well as its family, are poorly understood and their physiology is as yet unstudied. In this scenario, a model system like the zebrafish could serve to dissect the role of the RPRM family members in vivo. Phylogenetic analysis reveals that RPRM and RPRML have been differentially retained by most species throughout vertebrate evolution, yet RPRM3 has been retained only in a small group of distantly related species, including zebrafish. Herein, we characterized the spatiotemporal expression of RPRM (present in zebrafish as an infraclass duplication rprma/rprmb, RPRML and RPRM3 in the zebrafish. By whole-mount in situ hybridization (WISH and fluorescent in situ hybridization (FISH, we demonstrate that rprm (rprma/rprmb and rprml show a similar spatiotemporal expression profile during zebrafish development. At early developmental stages rprmb is expressed in somites. After one day post-fertilization, rprm (rprma/rprmb and rprml are expressed in the notochord, brain, blood vessels and digestive tube. On the other hand, rprm3 shows the most unique expression profile, being expressed only in the central nervous system (CNS. We assessed the expression patterns of RPRM gene transcripts in adult zebrafish and human RPRM protein product in tissue samples by RT-qPCR and immunohistochemistry (IHC staining, respectively. Strikingly, tissue-specific expression patterns of the RPRM transcripts and protein are conserved between zebrafish and humans. We propose the zebrafish as a powerful tool to elucidate the both physiological and pathological roles of the RPRM gene family.

  17. N-acetylcysteine prevents ketamine-induced adverse effects on development, heart rate and monoaminergic neurons in zebrafish.

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    Robinson, Bonnie; Dumas, Melanie; Gu, Qiang; Kanungo, Jyotshna

    2018-06-08

    N-acetylcysteine, a precursor molecule of glutathione, is an antioxidant. Ketamine, a pediatric anesthetic, has been implicated in cardiotoxicity and neurotoxicity including modulation of monoaminergic systems in mammals and zebrafish. Here, we show that N-acetylcysteine prevents ketamine's adverse effects on development and monoaminergic neurons in zebrafish embryos. The effects of ketamine and N-acetylcysteine alone or in combination were measured on the heart rate, body length, brain serotonergic neurons and tyrosine hydroxylase-immunoreactive (TH-IR) neurons. In the absence of N-acetylcysteine, a concentration of ketamine that produces an internal embryo exposure level comparable to human anesthetic plasma concentrations significantly reduced heart rate and body length and those effects were prevented by N-acetylcysteine co-treatment. Ketamine also reduced the areas occupied by serotonergic neurons in the brain, whereas N-acetylcysteine co-exposure counteracted this effect. TH-IR neurons in the embryo brain and TH-IR cells in the trunk were significantly reduced with ketamine treatment, but not in the presence of N-acetylcysteine. In our continued search for compounds that can prevent ketamine toxicity, this study using specific endpoints of developmental toxicity, cardiotoxicity and neurotoxicity, demonstrates protective effects of N-acetylcysteine against ketamine's adverse effects. This is the first study that shows the protective effects of N-acetylcysteine on ketamine-induced developmental defects of monoaminergic neurons as observed in a whole organism. Published by Elsevier B.V.

  18. Differential maturation of rhythmic clock gene expression during early development in medaka (Oryzias latipes).

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    Cuesta, Ines H; Lahiri, Kajori; Lopez-Olmeda, Jose Fernando; Loosli, Felix; Foulkes, Nicholas S; Vallone, Daniela

    2014-05-01

    One key challenge for the field of chronobiology is to identify how circadian clock function emerges during early embryonic development. Teleosts such as the zebrafish are ideal models for studying circadian clock ontogeny since the entire process of development occurs ex utero in an optically transparent chorion. Medaka (Oryzias latipes) represents another powerful fish model for exploring early clock function with, like the zebrafish, many tools available for detailed genetic analysis. However, to date there have been no reports documenting circadian clock gene expression during medaka development. Here we have characterized the expression of key clock genes in various developmental stages and in adult tissues of medaka. As previously reported for other fish, light dark cycles are required for the emergence of clock gene expression rhythms in this species. While rhythmic expression of per and cry genes is detected very early during development and seems to be light driven, rhythmic clock and bmal expression appears much later around hatching time. Furthermore, the maturation of clock function seems to correlate with the appearance of rhythmic expression of these positive elements of the clock feedback loop. By accelerating development through elevated temperatures or by artificially removing the chorion, we show an earlier onset of rhythmicity in clock and bmal expression. Thus, differential maturation of key elements of the medaka clock mechanism depends on the developmental stage and the presence of the chorion.

  19. CRISPR/Cas9-Mediated Zebrafish Knock-in as a Novel Strategy to Study Midbrain-Hindbrain Boundary Development.

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    Kesavan, Gokul; Chekuru, Avinash; Machate, Anja; Brand, Michael

    2017-01-01

    The midbrain-hindbrain boundary (MHB) acts as an organizer and controls the fate of neighboring cells to develop into either mesencephalic (midbrain) or metencephalic (hindbrain) cells by secreting signaling molecules like Wnt1 and Fgf8. The zebrafish is an excellent vertebrate model for studying MHB development due to the ease of gene manipulation and the possibility of following cellular dynamics and morphogenetic processes using live imaging. Currently, only very few reporter and/or Cre-driver lines are available to study gene expression at the MHB, hampering the understanding of MHB development, and traditional transgenic technologies using promoter/enhancer fragments or bacterial artificial chromosome (BAC)-mediated transgenesis often do not faithfully recapitulate endogenous expression patterns. In contrast, CRISPR/Cas9-mediated genome editing technology now provides a great opportunity to efficiently knock-in or knock-out genes. We have generated four CRISPR/Cas9-based knock-in fluorescent reporter lines for two crucial genes involved in MHB development, namely otx2 and pax2a . The coding sequences of the reporters were knocked-in upstream of the corresponding ATG and are, thus, under the control of the endogenous promoter/enhancer elements. Interestingly, this strategy does not disturb endogenous gene expression. Using the fast maturing fluorescent protein reporter, Venus, enabled us to follow MHB development using cell tracking and live imaging. In addition, we show that these reporter lines label various neuronal and glial cell types in the adult zebrafish brain, making them highly suitable for investigating embryonic and adult midbrain, hindbrain, and MHB development.

  20. Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

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    Seiler, Christoph; Gebhart, Nichole; Zhang, Yong; Shinton, Susan A; Li, Yue-sheng; Ross, Nicola L; Liu, Xingjun; Li, Qin; Bilbee, Alison N; Varshney, Gaurav K; LaFave, Matthew C; Burgess, Shawn M; Balciuniene, Jorune; Balciunas, Darius; Hardy, Richard R; Kappes, Dietmar J; Wiest, David L; Rhodes, Jennifer

    2015-01-01

    Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain) genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP) during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

  1. Mutagenesis Screen Identifies agtpbp1 and eps15L1 as Essential for T lymphocyte Development in Zebrafish.

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    Christoph Seiler

    Full Text Available Genetic screens are a powerful tool to discover genes that are important in immune cell development and function. The evolutionarily conserved development of lymphoid cells paired with the genetic tractability of zebrafish make this a powerful model system for this purpose. We used a Tol2-based gene-breaking transposon to induce mutations in the zebrafish (Danio rerio, AB strain genome, which served the dual purpose of fluorescently tagging cells and tissues that express the disrupted gene and provided a means of identifying the disrupted gene. We identified 12 lines in which hematopoietic tissues expressed green fluorescent protein (GFP during embryonic development, as detected by microscopy. Subsequent analysis of young adult fish, using a novel approach in which single cell suspensions of whole fish were analyzed by flow cytometry, revealed that 8 of these lines also exhibited GFP expression in young adult cells. An additional 15 lines that did not have embryonic GFP+ hematopoietic tissue by microscopy, nevertheless exhibited GFP+ cells in young adults. RT-PCR analysis of purified GFP+ populations for expression of T and B cell-specific markers identified 18 lines in which T and/or B cells were fluorescently tagged at 6 weeks of age. As transposon insertion is expected to cause gene disruption, these lines can be used to assess the requirement for the disrupted genes in immune cell development. Focusing on the lines with embryonic GFP+ hematopoietic tissue, we identified three lines in which homozygous mutants exhibited impaired T cell development at 6 days of age. In two of the lines we identified the disrupted genes, agtpbp1 and eps15L1. Morpholino-mediated knockdown of these genes mimicked the T cell defects in the corresponding mutant embryos, demonstrating the previously unrecognized, essential roles of agtpbp1 and eps15L1 in T cell development.

  2. Cognitive Development in Early Readers.

    Science.gov (United States)

    Briggs, Chari; Elkind, David

    Some studies of early readers are discussed. It is pointed out that study of early readers has relevance for practical and theoretical issues in psychology and education. Of interest in this document are the following questions: (1) Are there any special talents or traits distinguishing early from non-early readers? (2) Do children who read early…

  3. Modeling tuberculous meningitis in zebrafish using Mycobacterium marinum

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    Lisanne M. van Leeuwen

    2014-09-01

    Full Text Available Tuberculous meningitis (TBM is one of the most severe extrapulmonary manifestations of tuberculosis, with a high morbidity and mortality. Characteristic pathological features of TBM are Rich foci, i.e. brain- and spinal-cord-specific granulomas formed after hematogenous spread of pulmonary tuberculosis. Little is known about the early pathogenesis of TBM and the role of Rich foci. We have adapted the zebrafish model of Mycobacterium marinum infection (zebrafish–M. marinum model to study TBM. First, we analyzed whether TBM occurs in adult zebrafish and showed that intraperitoneal infection resulted in granuloma formation in the meninges in 20% of the cases, with occasional brain parenchyma involvement. In zebrafish embryos, bacterial infiltration and clustering of infected phagocytes was observed after infection at three different inoculation sites: parenchyma, hindbrain ventricle and caudal vein. Infection via the bloodstream resulted in the formation of early granulomas in brain tissue in 70% of the cases. In these zebrafish embryos, infiltrates were located in the proximity of blood vessels. Interestingly, no differences were observed when embryos were infected before or after early formation of the blood-brain barrier (BBB, indicating that bacteria are able to cross this barrier with relatively high efficiency. In agreement with this observation, infected zebrafish larvae also showed infiltration of the brain tissue. Upon infection of embryos with an M. marinum ESX-1 mutant, only small clusters and scattered isolated phagocytes with high bacterial loads were present in the brain tissue. In conclusion, our adapted zebrafish–M. marinum infection model for studying granuloma formation in the brain will allow for the detailed analysis of both bacterial and host factors involved in TBM. It will help solve longstanding questions on the role of Rich foci and potentially contribute to the development of better diagnostic tools and therapeutics.

  4. Analysis of Zebrafish Kidney Development with Time-lapse Imaging Using a Dissecting Microscope Equipped for Optical Sectioning.

    Science.gov (United States)

    Perner, Birgit; Schnerwitzki, Danny; Graf, Michael; Englert, Christoph

    2016-04-07

    In order to understand organogenesis, the spatial and temporal alterations that occur during development of tissues need to be recorded. The method described here allows time-lapse analysis of normal and impaired kidney development in zebrafish embryos by using a fluorescence dissecting microscope equipped for structured illumination and z-stack acquisition. To visualize nephrogenesis, transgenic zebrafish (Tg(wt1b:GFP)) with fluorescently labeled kidney structures were used. Renal defects were triggered by injection of an antisense morpholino oligonucleotide against the Wilms tumor gene wt1a, a factor known to be crucial for kidney development. The advantage of the experimental setup is the combination of a zoom microscope with simple strategies for re-adjusting movements in x, y or z direction without additional equipment. To circumvent focal drift that is induced by temperature variations and mechanical vibrations, an autofocus strategy was applied instead of utilizing a usually required environmental chamber. In order to re-adjust the positional changes due to a xy-drift, imaging chambers with imprinted relocation grids were employed. In comparison to more complex setups for time-lapse recording with optical sectioning such as confocal laser scanning or light sheet microscopes, a zoom microscope is easy to handle. Besides, it offers dissecting microscope-specific benefits such as high depth of field and an extended working distance. The method to study organogenesis presented here can also be used with fluorescence stereo microscopes not capable of optical sectioning. Although limited for high-throughput, this technique offers an alternative to more complex equipment that is normally used for time-lapse recording of developing tissues and organ dynamics.

  5. A method to quantify mechanobiologic forces during zebrafish cardiac development using 4-D light sheet imaging and computational modeling.

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    Vijay Vedula

    2017-10-01

    Full Text Available Blood flow and mechanical forces in the ventricle are implicated in cardiac development and trabeculation. However, the mechanisms of mechanotransduction remain elusive. This is due in part to the challenges associated with accurately quantifying mechanical forces in the developing heart. We present a novel computational framework to simulate cardiac hemodynamics in developing zebrafish embryos by coupling 4-D light sheet imaging with a stabilized finite element flow solver, and extract time-dependent mechanical stimuli data. We employ deformable image registration methods to segment the motion of the ventricle from high resolution 4-D light sheet image data. This results in a robust and efficient workflow, as segmentation need only be performed at one cardiac phase, while wall position in the other cardiac phases is found by image registration. Ventricular hemodynamics are then quantified by numerically solving the Navier-Stokes equations in the moving wall domain with our validated flow solver. We demonstrate the applicability of the workflow in wild type zebrafish and three treated fish types that disrupt trabeculation: (a chemical treatment using AG1478, an ErbB2 signaling inhibitor that inhibits proliferation and differentiation of cardiac trabeculation; (b injection of gata1a morpholino oligomer (gata1aMO suppressing hematopoiesis and resulting in attenuated trabeculation; (c weak-atriumm58 mutant (wea with inhibited atrial contraction leading to a highly undeveloped ventricle and poor cardiac function. Our simulations reveal elevated wall shear stress (WSS in wild type and AG1478 compared to gata1aMO and wea. High oscillatory shear index (OSI in the grooves between trabeculae, compared to lower values on the ridges, in the wild type suggest oscillatory forces as a possible regulatory mechanism of cardiac trabeculation development. The framework has broad applicability for future cardiac developmental studies focused on quantitatively

  6. Regulation of bone morphogenetic proteins in early embryonic development

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    Yamamoto, Yukiyo; Oelgeschläger, Michael

    2004-11-01

    Bone morphogenetic proteins (BMPs), a large subgroup of the TGF-β family of secreted growth factors, control fundamental events in early embryonic development, organogenesis and adult tissue homeostasis. The plethora of dose-dependent cellular processes regulated by BMP signalling demand a tight regulation of BMP activity. Over the last decade, a number of proteins have been identified that bind BMPs in the extracellular space and regulate the interaction of BMPs with their cognate receptors, including the secreted BMP antagonist Chordin. In the early vertebrate embryo, the localized secretion of BMP antagonists from the dorsal blastopore lip establishes a functional BMP signalling gradient that is required for the determination of the dorsoventral or back to belly body axis. In particular, inhibition of BMP activity is essential for the formation of neural tissue in the development of vertebrate and invertebrate embryos. Here we review recent studies that have provided new insight into the regulation of BMP signalling in the extracellular space. In particular, we discuss the recently identified Twisted gastrulation protein that modulates, in concert with metalloproteinases of the Tolloid family, the interaction of Chordin with BMP and a family of proteins that share structural similarities with Chordin in the respective BMP binding domains. In addition, genetic and functional studies in zebrafish and frog provide compelling evidence that the secreted protein Sizzled functionally interacts with the Chd BMP pathway, despite being expressed ventrally in the early gastrula-stage embryo. These intriguing discoveries may have important implications, not only for our current concept of early embryonic patterning, but also for the regulation of BMP activity at later developmental stages and tissue homeostasis in the adult.

  7. Cardiac development in zebrafish and human embryonic stem cells is inhibited by exposure to tobacco cigarettes and e-cigarettes.

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    Nathan J Palpant

    Full Text Available Maternal smoking is a risk factor for low birth weight and other adverse developmental outcomes.We sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo.Zebrafish (Danio rerio were used to assess developmental effects in vivo and cardiac differentiation of human embryonic stem cells (hESCs was used as a model for in vitro cardiac development.In zebrafish, exposure to both types of cigarettes results in broad, dose-dependent developmental defects coupled with severe heart malformation, pericardial edema and reduced heart function. Tobacco cigarettes are more toxic than e-cigarettes at comparable nicotine concentrations. During cardiac differentiation of hESCs, tobacco smoke exposure results in a delayed transition through mesoderm. Both types of cigarettes decrease expression of cardiac transcription factors in cardiac progenitor cells, suggesting a persistent delay in differentiation. In definitive human cardiomyocytes, both e-cigarette- and tobacco cigarette-treated samples showed reduced expression of sarcomeric genes such as MLC2v and MYL6. Furthermore, tobacco cigarette-treated samples had delayed onset of beating and showed low levels and aberrant localization of N-cadherin, reduced myofilament content with significantly reduced sarcomere length, and increased expression of the immature cardiac marker smooth muscle alpha-actin.These data indicate a negative effect of both tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Tobacco cigarettes are more toxic than E-cigarettes and exhibit a broader spectrum of cardiac developmental defects.

  8. Glucocorticoid receptor, but not mineralocorticoid receptor, mediates cortisol regulation of epidermal ionocyte development and ion transport in zebrafish (danio rerio.

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    Shelly Abad Cruz

    Full Text Available Cortisol is the major endogenous glucocorticoid (GC both in human and fish, mediated by corticosteroid receptors. Due to the absence of aldosterone production in teleost fish, cortisol is also traditionally accepted to function as mineralocorticoid (MC; but whether it acts through the glucocorticoid receptor (GR or the mineralocorticoid receptor (MR remains a subject of debate. Here, we used loss-of-function and rescue assays to determine whether cortisol affects zebrafish epidermal ionocyte development and function via the GR and/or the MR. GR knockdown morphants displayed a significant decrease in the major ionocytes, namely Na(+-K(+-ATPase-rich cells (NaRCs and H(+-ATPase-rich cells (HRCs, as well as other cells, including epidermal stem cells (ESCs, keratinocytes, and mucus cells; conversely, cell numbers were unaffected in MR knockdown morphants. In agreement, GR morphants, but not MR morphants, exhibited decreased NaRC-mediated Ca(2+ uptake and HRC-mediated H(+ secretion. Rescue via GR capped mRNA injection or exogenous cortisol incubation normalized the number of epidermal ionocytes in GR morphants. We also provide evidence for GR localization in epidermal cells. At the transcript level, GR mRNA is ubiquitously expressed in gill sections and present in both NaRCs and HRCs, supporting the knockdown and functional assay results in embryo. Altogether, we have provided solid molecular evidence that GR is indeed present on ionocytes, where it mediates the effects of cortisol on ionocyte development and function. Hence, cortisol-GR axis performs the roles of both GC and MC in zebrafish skin and gills.

  9. The Early Stages of Heart Development: Insights from Chicken Embryos

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    Johannes G. Wittig

    2016-04-01

    Full Text Available The heart is the first functioning organ in the developing embryo and a detailed understanding of the molecular and cellular mechanisms involved in its formation provides insights into congenital malformations affecting its function and therefore the survival of the organism. Because many developmental mechanisms are highly conserved, it is possible to extrapolate from observations made in invertebrate and vertebrate model organisms to humans. This review will highlight the contributions made through studying heart development in avian embryos, particularly the chicken. The major advantage of chick embryos is their accessibility for surgical manipulation and functional interference approaches, both gain- and loss-of-function. In addition to experiments performed in ovo, the dissection of tissues for ex vivo culture, genomic, or biochemical approaches is straightforward. Furthermore, embryos can be cultured for time-lapse imaging, which enables tracking of fluorescently labeled cells and detailed analysis of tissue morphogenesis. Owing to these features, investigations in chick embryos have led to important discoveries, often complementing genetic studies in mice and zebrafish. As well as including some historical aspects, we cover here some of the crucial advances made in understanding early heart development using the chicken model.

  10. Toxicogenomic analysis in the combined effect of tributyltin and benzo[a]pyrene on the development of zebrafish embryos.

    Science.gov (United States)

    Huang, Lixing; Zuo, Zhenghong; Zhang, Youyu; Wang, Chonggang

    2015-01-01

    There is a growing recognition that the toxic effects of chemical mixtures are been an important issue in toxicological sciences. Tributyltin (TBT) and benzo[a]pyrene (BaP) are widespread pollutants that occur simultaneously in the aquatic environments. This study was designed to examine comprehensively the combined effects of TBT and BaP on zebrafish (Danio rerio) embryos using toxicogenomic approach combined with biochemical detection and morphological analysis, and tried to gain insight into the mechanisms underlying the combined effects of TBT and BaP. The results of toxicogenomic data indicated that: (1) TBT cotreatment rescued the embryos from decreased hatching ratio caused by BaP alone, while the alteration of gene expression (in this article the phrase gene expression is used as a synonym to gene transcription, although in is acknowledged that gene expression can also be regulated by, e.g., translation and mRNA or protein stability) relative to zebrafish hatching in the BaP groups was resumed by the cotreatment with TBT; (2) BaP cotreatment decreased TBT-mediated dorsal curvature, and alleviated the perturbation of Notch pathway caused by TBT alone; (3) cotreatment with TBT decreased BaP-mediated bradycardia, which might be due to that TBT cotreatment alleviated the perturbation in expression of genes related to cardiac muscle cell development and calcium handling caused by BaP alone; 4) TBT cotreatment brought an antagonistic effect on the BaP-mediated oxidative stress and DNA damage. These results suggested that toxicogenomic approach was available for analyzing combined toxicity with high sensitivity and accuracy, which might improve our understanding and predictability for the combined effects of chemicals. Copyright © 2014 Elsevier B.V. All rights reserved.

  11. Skin too thin? The developing utility of zebrafish skin (neuro)pharmacology for CNS drug discovery research.

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    Nguyen, Michael; Poudel, Manoj K; Stewart, Adam Michael; Kalueff, Allan V

    2013-09-01

    Skin coloration can be affected by many genetic, environmental and pharmacological factors. Zebrafish (Danio rerio) are a useful and versatile model organism in biomedical research due to their genetic tractability, physiological homology to mammals, low cost, reproducibility and high throughput. Zebrafish coloration is mediated by chromatophores - the skin color pigment cells largely controlled by endocrine and neural mechanisms. The characteristic darkening of zebrafish skin is caused by the dispersion (and paling - by aggregation) of melanosomes (pigment-containing organelles), which show high homology to mammalian structures. Various pharmacological agents potently affect zebrafish coloration - the phenotype that often accompanies behavioral effects of the drugs, and may be used for drug discovery. Although zebrafish behavior and skin responses are usually not directly related, they share common regulatory (neural, endocrine) mechanisms, and therefore may be assessed in parallel during psychotropic drug screening. For example, some psychoactive drugs can potently affect zebrafish skin coloration. Can we use this knowledge to refine phenotype-driven psychotropic drug discovery? Here, we present current models using zebrafish skin coloration assays, and discuss how these models may be applied to enhance in vivo CNS drug discovery. Copyright © 2013 Elsevier Inc. All rights reserved.

  12. Automated processing of zebrafish imaging data: a survey.

    Science.gov (United States)

    Mikut, Ralf; Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A; Kausler, Bernhard X; Ledesma-Carbayo, María J; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-09-01

    Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines.

  13. Automated Processing of Zebrafish Imaging Data: A Survey

    Science.gov (United States)

    Dickmeis, Thomas; Driever, Wolfgang; Geurts, Pierre; Hamprecht, Fred A.; Kausler, Bernhard X.; Ledesma-Carbayo, María J.; Marée, Raphaël; Mikula, Karol; Pantazis, Periklis; Ronneberger, Olaf; Santos, Andres; Stotzka, Rainer; Strähle, Uwe; Peyriéras, Nadine

    2013-01-01

    Abstract Due to the relative transparency of its embryos and larvae, the zebrafish is an ideal model organism for bioimaging approaches in vertebrates. Novel microscope technologies allow the imaging of developmental processes in unprecedented detail, and they enable the use of complex image-based read-outs for high-throughput/high-content screening. Such applications can easily generate Terabytes of image data, the handling and analysis of which becomes a major bottleneck in extracting the targeted information. Here, we describe the current state of the art in computational image analysis in the zebrafish system. We discuss the challenges encountered when handling high-content image data, especially with regard to data quality, annotation, and storage. We survey methods for preprocessing image data for further analysis, and describe selected examples of automated image analysis, including the tracking of cells during embryogenesis, heartbeat detection, identification of dead embryos, recognition of tissues and anatomical landmarks, and quantification of behavioral patterns of adult fish. We review recent examples for applications using such methods, such as the comprehensive analysis of cell lineages during early development, the generation of a three-dimensional brain atlas of zebrafish larvae, and high-throughput drug screens based on movement patterns. Finally, we identify future challenges for the zebrafish image analysis community, notably those concerning the compatibility of algorithms and data formats for the assembly of modular analysis pipelines. PMID:23758125

  14. The zebrafish spi1 promoter drives myeloid-specific expression in stable transgenic fish

    NARCIS (Netherlands)

    Ward, AC; McPhee, DO; Condron, MM; Varma, S; Cody, SH; Onnebo, SMN; Paw, BH; Zon, LI; Lieschke, GJ

    2003-01-01

    The spi1 (pu.1) gene has recently been identified as a useful marker of early myeloid cells in zebrafish. To enhance the versatility of this organism as a model for studying myeloid development, the promoter of this gene has been isolated and characterized. Transient transgenesis revealed that a 5.3

  15. Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formation

    Directory of Open Access Journals (Sweden)

    Bensimon-Brito Anabela

    2012-10-01

    Full Text Available Abstract Background In chondrichthyans, basal osteichthyans and tetrapods, vertebral bodies have cartilaginous anlagen that subsequently mineralize (chondrichthyans or ossify (osteichthyans. Chondrocytes that form the vertebral centra derive from somites. In teleost fish, vertebral centrum formation starts in the absence of cartilage, through direct mineralization of the notochord sheath. In a second step, the notochord is surrounded by somite-derived intramembranous bone. In several small teleost species, including zebrafish (Danio rerio, even haemal and neural arches form directly as intramembranous bone and only modified caudalmost arches remain cartilaginous. This study compares initial patterns of mineralization in different regions of the vertebral column in zebrafish. We ask if the absence or presence of cartilaginous arches influences the pattern of notochord sheath mineralization. Results To reveal which cells are involved in mineralization of the notochord sheath we identify proliferating cells, we trace mineralization on the histological level and we analyze cell ultrastructure by TEM. Moreover, we localize proteins and genes that are typically expressed by skeletogenic cells such as Collagen type II, Alkaline phosphatase (ALP and Osteocalcin (Oc. Mineralization of abdominal and caudal vertebrae starts with a complete ring within the notochord sheath and prior to the formation of the bony arches. In contrast, notochord mineralization of caudal fin centra starts with a broad ventral mineral deposition, associated with the bases of the modified cartilaginous arches. Similar, arch-related, patterns of mineralization occur in teleosts that maintain cartilaginous arches throughout the spine. Throughout the entire vertebral column, we were able to co-localize ALP-positive signal with chordacentrum mineralization sites, as well as Collagen II and Oc protein accumulation in the mineralizing notochord sheath. In the caudal fin region, ALP and

  16. Distinct patterns of notochord mineralization in zebrafish coincide with the localization of Osteocalcin isoform 1 during early vertebral centra formation.

    Science.gov (United States)

    Bensimon-Brito, Anabela; Cardeira, João; Cancela, Maria Leonor; Huysseune, Ann; Witten, Paul Eckhard

    2012-10-09

    In chondrichthyans, basal osteichthyans and tetrapods, vertebral bodies have cartilaginous anlagen that subsequently mineralize (chondrichthyans) or ossify (osteichthyans). Chondrocytes that form the vertebral centra derive from somites. In teleost fish, vertebral centrum formation starts in the absence of cartilage, through direct mineralization of the notochord sheath. In a second step, the notochord is surrounded by somite-derived intramembranous bone. In several small teleost species, including zebrafish (Danio rerio), even haemal and neural arches form directly as intramembranous bone and only modified caudalmost arches remain cartilaginous. This study compares initial patterns of mineralization in different regions of the vertebral column in zebrafish. We ask if the absence or presence of cartilaginous arches influences the pattern of notochord sheath mineralization. To reveal which cells are involved in mineralization of the notochord sheath we identify proliferating cells, we trace mineralization on the histological level and we analyze cell ultrastructure by TEM. Moreover, we localize proteins and genes that are typically expressed by skeletogenic cells such as Collagen type II, Alkaline phosphatase (ALP) and Osteocalcin (Oc). Mineralization of abdominal and caudal vertebrae starts with a complete ring within the notochord sheath and prior to the formation of the bony arches. In contrast, notochord mineralization of caudal fin centra starts with a broad ventral mineral deposition, associated with the bases of the modified cartilaginous arches. Similar, arch-related, patterns of mineralization occur in teleosts that maintain cartilaginous arches throughout the spine.Throughout the entire vertebral column, we were able to co-localize ALP-positive signal with chordacentrum mineralization sites, as well as Collagen II and Oc protein accumulation in the mineralizing notochord sheath. In the caudal fin region, ALP and Oc signals were clearly produced both by the

  17. Multiple zebrafish atoh1 genes specify a diversity of neuronal types in the zebrafish cerebellum.

    Science.gov (United States)

    Kidwell, Chelsea U; Su, Chen-Ying; Hibi, Masahiko; Moens, Cecilia B

    2018-06-01

    A single Atoh1 basic-helix-loop-helix transcription factor specifies multiple neuron types in the mammalian cerebellum and anterior hindbrain. The zebrafish genome encodes three paralagous atoh1 genes whose functions in cerebellum and anterior hindbrain development we explore here. With use of a transgenic reporter, we report that zebrafish atoh1c-expressing cells are organized in two distinct domains that are separated both by space and developmental time. An early isthmic expression domain gives rise to an extracerebellar population in rhombomere 1 and an upper rhombic lip domain gives rise to granule cell progenitors that migrate to populate all four granule cell territories of the fish cerebellum. Using genetic mutants we find that of the three zebrafish atoh1 paralogs, atoh1c and atoh1a are required for the full complement of granule neurons. Surprisingly, the two genes are expressed in non-overlapping granule cell progenitor populations, indicating that fish use duplicate atoh1 genes to generate granule cell diversity that is not detected in mammals. Finally, live imaging of granule cell migration in wildtype and atoh1c mutant embryos reveals that while atoh1c is not required for granule cell specification per se, it is required for granule cells to delaminate and migrate away from the rhombic lip. Copyright © 2018 Elsevier Inc. All rights reserved.

  18. Structurally distinct polycyclic aromatic hydrocarbons induce differential transcriptional responses in developing zebrafish

    International Nuclear Information System (INIS)

    Goodale, Britton C.; Tilton, Susan C.; Corvi, Margaret M.; Wilson, Glenn R.; Janszen, Derek B.; Anderson, Kim A.; Waters, Katrina M.; Tanguay, Robert L.

    2013-01-01

    Polycyclic aromatic hydrocarbons (PAHs) are ubiquitous in the environment as components of fossil fuels and by-products of combustion. These multi-ring chemicals differentially activate the aryl hydrocarbon receptor (AHR) in a structurally dependent manner, and induce toxicity via both AHR-dependent and -independent mechanisms. PAH exposure is known to induce developmental malformations in zebrafish embryos, and recent studies have shown cardiac toxicity induced by compounds with low AHR affinity. Unraveling the potentially diverse molecular mechanisms of PAH toxicity is essential for understanding the hazard posed by complex PAH mixtures present in the environment. We analyzed transcriptional responses to PAH exposure in zebrafish embryos exposed to benz(a)anthracene (BAA), dibenzothiophene (DBT) and pyrene (PYR) at concentrations that induced developmental malformations by 120 h post-fertilization (hpf). Whole genome microarray analysis of mRNA expression at 24 and 48 hpf identified genes that were differentially regulated over time and in response to the three PAH structures. PAH body burdens were analyzed at both time points using GC–MS, and demonstrated differences in PAH uptake into the embryos. This was important for discerning dose-related differences from those that represented unique molecular mechanisms. While BAA misregulated the least number of transcripts, it caused strong induction of cyp1a and other genes known to be downstream of the AHR, which were not induced by the other two PAHs. Analysis of functional roles of misregulated genes and their predicted regulatory transcription factors also distinguished the BAA response from regulatory networks disrupted by DBT and PYR exposure. These results indicate that systems approaches can be used to classify the toxicity of PAHs based on the networks perturbed following exposure, and may provide a path for unraveling the toxicity of complex PAH mixtures. - Highlights: • Defined global mRNA expression

  19. Distinct functional and temporal requirements for zebrafish Hdac1 during neural crest-derived craniofacial and peripheral neuron development.

    Science.gov (United States)

    Ignatius, Myron S; Unal Eroglu, Arife; Malireddy, Smitha; Gallagher, Glen; Nambiar, Roopa M; Henion, Paul D

    2013-01-01

    The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In hdac1(b382) mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer hoxb3a, dlx2 and dlx3-expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in hdac1(b382) mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in hdac1(b382) mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce hdac1(b382) defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish hdac1 during neural crest-derived craniofacial and peripheral neuron development.

  20. Distinct functional and temporal requirements for zebrafish Hdac1 during neural crest-derived craniofacial and peripheral neuron development.

    Directory of Open Access Journals (Sweden)

    Myron S Ignatius

    Full Text Available The regulation of gene expression is accomplished by both genetic and epigenetic means and is required for the precise control of the development of the neural crest. In hdac1(b382 mutants, craniofacial cartilage development is defective in two distinct ways. First, fewer hoxb3a, dlx2 and dlx3-expressing posterior branchial arch precursors are specified and many of those that are consequently undergo apoptosis. Second, in contrast, normal numbers of progenitors are present in the anterior mandibular and hyoid arches, but chondrocyte precursors fail to terminally differentiate. In the peripheral nervous system, there is a disruption of enteric, DRG and sympathetic neuron differentiation in hdac1(b382 mutants compared to wildtype embryos. Specifically, enteric and DRG-precursors differentiate into neurons in the anterior gut and trunk respectively, while enteric and DRG neurons are rarely present in the posterior gut and tail. Sympathetic neuron precursors are specified in hdac1(b382 mutants and they undergo generic neuronal differentiation but fail to undergo noradrenergic differentiation. Using the HDAC inhibitor TSA, we isolated enzyme activity and temporal requirements for HDAC function that reproduce hdac1(b382 defects in craniofacial and sympathetic neuron development. Our study reveals distinct functional and temporal requirements for zebrafish hdac1 during neural crest-derived craniofacial and peripheral neuron development.

  1. Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Gao, Ming [Tianjin University, Department of Pharmaceutical Engineering, Tianjin 300072 (China); Beijing Institute of Radiation Medicine, Beijing 100850 (China); Yan, Hui [Beijing Institute of Pharmacology and Toxicology, Beijing 100850 (China); Yin, Rong-Hua [Beijing Institute of Radiation Medicine, Beijing 100850 (China); State Key Laboratory of Proteomics, Beijing 100850 (China); Wang, Qiang [Institute of Zoology, Chinese Academy of Sciences, Beijing 100101 (China); Zhan, Yi-Qun; Yu, Miao; Ge, Chang-Hui; Li, Chang-Yan; Wang, Xiao-Hui [Beijing Institute of Radiation Medicine, Beijing 100850 (China); State Key Laboratory of Proteomics, Beijing 100850 (China); Ge, Zhi-Qiang [Tianjin University, Department of Pharmaceutical Engineering, Tianjin 300072 (China); Yang, Xiao-Ming, E-mail: xiaomingyang@sina.com [Tianjin University, Department of Pharmaceutical Engineering, Tianjin 300072 (China); Beijing Institute of Radiation Medicine, Beijing 100850 (China); State Key Laboratory of Proteomics, Beijing 100850 (China)

    2015-07-31

    Background & aims: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and results: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. Conclusions: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis. - Highlights: • HPS is enriched in zebrafish liver and has strong similarities with other species. • Knocking down HPS with MOs results in small liver phenotype. • HPS depletion regulates liver outgrowth but not liver specification and budding. • HPS depletion causes hepatocyte proliferation arrest but not apoptosis induction.

  2. Hepassocin is required for hepatic outgrowth during zebrafish hepatogenesis

    International Nuclear Information System (INIS)

    Gao, Ming; Yan, Hui; Yin, Rong-Hua; Wang, Qiang; Zhan, Yi-Qun; Yu, Miao; Ge, Chang-Hui; Li, Chang-Yan; Wang, Xiao-Hui; Ge, Zhi-Qiang; Yang, Xiao-Ming

    2015-01-01

    Background & aims: Hepassocin (HPS) is a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage. In this paper, zebrafish were used to investigate the role of HPS in liver development. Methods and results: During zebrafish development, HPS expression is enriched in liver throughout hepatogenesis. Knockdown of HPS using its specific morpholino leads to a smaller liver phenotype. Further results showed that the HPS knockdown has no effect on the expression of the early endoderm marker gata6 and early hepatic marker hhex. In addition, results showed that the smaller-liver phenotype in HPS morphants was caused by suppression of cell proliferation, not induction of cell apoptosis. Conclusions: Current findings indicated that HPS is essential to the later stages of development in vertebrate liver organogenesis. - Highlights: • HPS is enriched in zebrafish liver and has strong similarities with other species. • Knocking down HPS with MOs results in small liver phenotype. • HPS depletion regulates liver outgrowth but not liver specification and budding. • HPS depletion causes hepatocyte proliferation arrest but not apoptosis induction

  3. Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

    Science.gov (United States)

    Eom, Dae Seok; Inoue, Shinya; Patterson, Larissa B; Gordon, Tiffany N; Slingwine, Rebecca; Kondo, Shigeru; Watanabe, Masakatsu; Parichy, David M

    2012-01-01

    The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11). We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

  4. Melanophore migration and survival during zebrafish adult pigment stripe development require the immunoglobulin superfamily adhesion molecule Igsf11.

    Directory of Open Access Journals (Sweden)

    Dae Seok Eom

    Full Text Available The zebrafish adult pigment pattern has emerged as a useful model for understanding the development and evolution of adult form as well as pattern-forming mechanisms more generally. In this species, a series of horizontal melanophore stripes arises during the larval-to-adult transformation, but the genetic and cellular bases for stripe formation remain largely unknown. Here, we show that the seurat mutant phenotype, consisting of an irregular spotted pattern, arises from lesions in the gene encoding Immunoglobulin superfamily member 11 (Igsf11. We find that Igsf11 is expressed by melanophores and their precursors, and we demonstrate by cell transplantation and genetic rescue that igsf11 functions autonomously to this lineage in promoting adult stripe development. Further analyses of cell behaviors in vitro, in vivo, and in explant cultures ex vivo demonstrate that Igsf11 mediates adhesive interactions and that mutants for igsf11 exhibit defects in both the migration and survival of melanophores and their precursors. These findings identify the first in vivo requirements for igsf11 as well as the first instance of an immunoglobulin superfamily member functioning in pigment cell development and patterning. Our results provide new insights into adult pigment pattern morphogenesis and how cellular interactions mediate pattern formation.

  5. Kupffer's vesicle is a ciliated organ of asymmetry in the zebrafish embryo that initiates left-right development of the brain, heart and gut.

    Science.gov (United States)

    Essner, Jeffrey J; Amack, Jeffrey D; Nyholm, Molly K; Harris, Erin B; Yost, H Joseph

    2005-03-01

    Monocilia have been proposed to establish the left-right (LR) body axis in vertebrate embryos by creating a directional fluid flow that triggers asymmetric gene expression. In zebrafish, dorsal forerunner cells (DFCs) express a conserved ciliary dynein gene (left-right dynein-related1, lrdr1) and form a ciliated epithelium inside a fluid-filled organ called Kupffer's vesicle (KV). Here, videomicroscopy demonstrates that cilia inside KV are motile and create a directional fluid flow just prior to the onset of asymmetric gene expression in lateral cells. Laser ablation of DFCs and surgical disruption of KV provide direct evidence that ciliated KV cells are required during early somitogenesis for subsequent LR patterning in the brain, heart and gut. Antisense morpholinos against lrdr1 disrupt KV fluid flow and perturb LR development. Furthermore, lrdr1 morpholinos targeted to DFC/KV cells demonstrate that Lrdr1 functions in these ciliated cells to control LR patterning. This provides the first direct evidence, in any vertebrate, that impairing cilia function in derivatives of the dorsal organizer, and not in other cells that express ciliogenic genes, alters LR development. Finally, genetic analysis reveals novel roles for the T-box transcription factor no tail and the Nodal signaling pathway as upstream regulators of lrdr1 expression and KV morphogenesis. We propose that KV is a transient embryonic 'organ of asymmetry' that directs LR development by establishing a directional fluid flow. These results suggest that cilia are an essential component of a conserved mechanism that controls the transition from bilateral symmetry to LR asymmetry in vertebrates.

  6. Non-invasive imaging of zebrafish with spinal deformities using optical coherence tomography: a preliminary study

    Science.gov (United States)

    Bernstein, Liane; Beaudette, Kathy; Patten, Kessen; Beaulieu-Ouellet, Émilie; Strupler, Mathias; Moldovan, Florina; Boudoux, Caroline

    2013-03-01

    A zebrafish model has recently been introduced to study various genetic mutations that could lead to spinal deformities such as scoliosis. However, current imaging techniques make it difficult to perform longitudinal studies of this condition in zebrafish, especially in the early stages of development. The goal of this project is to determine whether optical coherence tomography (OCT) is a viable non-invasive method to image zebrafish exhibiting spinal deformities. Images of both live and fixed malformed zebrafish (5 to 21 days postfertilization) as well as wild-type fish (5 to 29 days postfertilization) were acquired non-invasively using a commercial SD-OCT system, with a laser source centered at 930nm (λ=100nm), permitting axial and lateral resolutions of 7 and 8μm respectively. Using two-dimensional images and three-dimensional reconstructions, it was possible to identify the malformed notochord as well as deformities in other major organs at different stages of formation. Visualization of the notochord was facilitated with the development of a segmentation algorithm. OCT images were compared to HE histological sections and images obtained by calcein staining. Because of the possibility of performing longitudinal studies on a same fish and reducing image processing time as compared with staining techniques and histology, the use of OCT could facilitate phenotypic characterization in studying genetic factors leading to spinal deformities in zebrafish and could eventually contribute to the identification of the genetic causes of spinal deformities such as scoliosis.

  7. Integrated in silico and in vivo approaches to investigate effects of BDE-99 mediated by the nuclear receptors on developing zebrafish.

    Science.gov (United States)

    Zhang, Li; Jin, Yaru; Han, Zhihua; Liu, Hongling; Shi, Laihao; Hua, Xiaoxue; Doering, Jon A; Tang, Song; Giesy, John P; Yu, Hongxia

    2018-03-01

    One of the most abundant polybrominated diphenyl ethers (PBDEs) is 2,2',4,4',5-pentabromodiphenyl ether (BDE-99), which persists and potentially bioaccumulates in aquatic wildlife. Previous studies in mammals have shown that BDE-99 affects development and disrupts certain endocrine functions through signaling pathways mediated by nuclear receptors. However, fewer studies have investigated the potential of BDE-99 to interact with nuclear receptors in aquatic vertebrates such as fish. In the present study, interactions between BDE-99 and nuclear receptors were investigated by in silico and in vivo approaches. This PBDE was able to dock into the ligand-binding domain of zebrafish aryl hydrocarbon receptor 2 (AhR2) and pregnane X receptor (PXR). It had a significant effect on the transcriptional profiles of genes associated with AhR or PXR. Based on the developed cytoscape of all zebrafish genes, it was also inferred that AhR and PXR could interact via cross-talk. In addition, both the in silico and in vivo approaches found that BDE-99 affected peroxisome proliferator-activated receptor alpha (PPARα), glucocorticoid receptor, and thyroid receptor. Collectively, our results demonstrate for the first time detailed in silico evidence that BDE-99 can bind to and interact with zebrafish AhR and PXR. These findings can be used to elaborate the molecular mechanism of BDE-99 and guide more objective environmental risk assessments. Environ Toxicol Chem 2018;37:780-787. © 2017 SETAC. © 2017 SETAC.

  8. The importance of Zebrafish in biomedical research.

    Science.gov (United States)

    Tavares, Bárbara; Santos Lopes, Susana

    2013-01-01

    Zebrafish (Danio rerio) is an ideal model organism for the study of vertebrate development. This is due to the large clutches that each couple produces, with up to 200 embryos every 7 days, and to the fact that the embryos and larvae are small, transparent and undergo rapid external development. Using scientific literature research tools available online and the keywords Zebrafish, biomedical research, human disease, and drug screening, we reviewed original studies and reviews indexed in PubMed. In this review we summarized work conducted with this model for the advancement of our knowledge related to several human diseases. We also focused on the biomedical research being performed in Portugal with the zebrafish model. Powerful live imaging and genetic tools are currently available for zebrafish making it a valuable model in biomedical research. The combination of these properties with the optimization of automated systems for drug screening has transformed the zebrafish into "a top model" in biomedical research, drug discovery and toxicity testing. Furthermore, with the optimization of xenografts technology it will be possible to use zebrafish to aide in the choice of the best therapy for each patient. Zebrafish is an excellent model organism in biomedical research, drug development and in clinical therapy.

  9. Pharmacologic modeling of primary mitochondrial respiratory chain dysfunction in zebrafish.

    Science.gov (United States)

    Byrnes, James; Ganetzky, Rebecca; Lightfoot, Richard; Tzeng, Michael; Nakamaru-Ogiso, Eiko; Seiler, Christoph; Falk, Marni J

    2017-07-18

    Mitochondrial respiratory chain (RC) disease is a heterogeneous and highly morbid group of energy deficiency disorders for which no proven effective therapies exist. Robust vertebrate animal models of primary RC dysfunction are needed to explore the effects of variation in RC disease subtypes, tissue-specific manifestations, and major pathogenic factors contributing to each disorder, as well as their pre-clinical response to therapeutic candidates. We have developed a series of zebrafish (Danio rerio) models that inhibit, to variable degrees, distinct aspects of RC function, and enable quantification of animal development, survival, behaviors, and organ-level treatment effects as well as effects on mitochondrial biochemistry and physiology. Here, we characterize four pharmacologic inhibitor models of mitochondrial RC dysfunction in early larval zebrafish, including rotenone (complex I inhibitor), azide (complex IV inhibitor), oligomycin (complex V inhibitor), and chloramphenicol (mitochondrial translation inhibitor that leads to multiple RC complex dysfunction). A range of concentrations and exposure times of each RC inhibitor were systematically evaluated on early larval development, animal survival, integrated behaviors (touch and startle responses), organ physiology (brain death, neurologic tone, heart rate), and fluorescence-based analyses of mitochondrial physiology in zebrafish skeletal muscle. Pharmacologic RC inhibitor effects were validated by spectrophotometric analysis of Complex I, II and IV enzyme activities, or relative quantitation of ATP levels in larvae. Outcomes were prioritized that utilize in vivo animal imaging and quantitative behavioral assessments, as may optimally inform the translational potential of pre-clinical drug screens for future clinical study in human mitochondrial disease subjects. The RC complex inhibitors each delayed early embryo development, with short-term exposures of these three agents or chloramphenicol from 5 to 7 days

  10. Hypoxia-induced metastasis model in embryonic zebrafish

    DEFF Research Database (Denmark)

    Rouhi, Pegah; Jensen, Lasse D.; Cao, Ziquan

    2010-01-01

    Hypoxia facilitates tumor invasion and metastasis by promoting neovascularization and co-option of tumor cells in the peritumoral vasculature, leading to dissemination of tumor cells into the circulation. However, until recently, animal models and imaging technology did not enable monitoring...... of the early events of tumor cell invasion and dissemination in living animals. We recently developed a zebrafish metastasis model to dissect the detailed events of hypoxia-induced tumor cell invasion and metastasis in association with angiogenesis at the single-cell level. In this model, fluorescent Di......I-labeled human or mouse tumor cells are implanted into the perivitelline cavity of 48-h-old zebrafish embryos, which are subsequently placed in hypoxic water for 3 d. Tumor cell invasion, metastasis and pathological angiogenesis are detected under fluorescent microscopy in the living fish. The average...

  11. Optogenetics: a new enlightenment age for zebrafish neurobiology.

    Science.gov (United States)

    Del Bene, Filippo; Wyart, Claire

    2012-03-01

    Zebrafish became a model of choice for neurobiology because of the transparency of its brain and because of its amenability to genetic manipulation. In particular, at early stages of development the intact larva is an ideal system to apply optical techniques for deep imaging in the nervous system, as well as genetically encoded tools for targeting subsets of neurons and monitoring and manipulating their activity. For these applications,new genetically encoded optical tools, fluorescent sensors, and light-gated channels have been generated,creating the field of "optogenetics." It is now possible to monitor and control neuronal activity with minimal perturbation and unprecedented spatio-temporal resolution.We describe here the main achievements that have occurred in the last decade in imaging and manipulating neuronal activity in intact zebrafish larvae. We provide also examples of functional dissection of neuronal circuits achieved with the applications of these techniques in the visual and locomotor systems.

  12. Zebrafish kidney phagocytes utilize macropinocytosis and Ca+-dependent endocytic mechanisms.

    Directory of Open Access Journals (Sweden)

    Claudia Hohn

    Full Text Available BACKGROUND: The innate immune response constitutes the first line of defense against invading pathogens and consists of a variety of immune defense mechanisms including active endocytosis by macrophages and granulocytes. Endocytosis can be used as a reliable measure of selective and non-selective mechanisms of antigen uptake in the early phase of an immune response. Numerous assays have been developed to measure this response in a variety of mammalian and fish species. The small size of the zebrafish has prevented the large-scale collection of monocytes/macrophages and granulocytes for these endocytic assays. METHODOLOGY/PRINCIPAL FINDINGS: Pooled zebrafish kidney hematopoietic tissues were used as a source of phagocytic cells for flow-cytometry based endocytic assays. FITC-Dextran, Lucifer Yellow and FITC-Edwardsiella ictaluri were used to evaluate selective and non-selective mechanisms of uptake in zebrafish phagocytes. CONCLUSIONS/SIGNIFICANCE: Zebrafish kidney phagocytes characterized as monocytes/macrophages, neutrophils and lymphocytes utilize macropinocytosis and Ca(2+-dependant endocytosis mechanisms of antigen uptake. These cells do not appear to utilize a mannose receptor. Heat-killed Edwardsiella ictaluri induces cytoskeletal interactions for internalization in zebrafish kidney monocytes/macrophages and granulocytes. The proposed method is easy to implement and should prove especially useful in immunological, toxicological and epidemiological research.

  13. Epilepsy, Behavioral Abnormalities, and Physiological Comorbidities in Syntaxin-Binding Protein 1 (STXBP1 Mutant Zebrafish.

    Directory of Open Access Journals (Sweden)

    Brian P Grone

    Full Text Available Mutations in the synaptic machinery gene syntaxin-binding protein 1, STXBP1 (also known as MUNC18-1, are linked to childhood epilepsies and other neurodevelopmental disorders. Zebrafish STXBP1 homologs (stxbp1a and stxbp1b have highly conserved sequence and are prominently expressed in the larval zebrafish brain. To understand the functions of stxbp1a and stxbp1b, we generated loss-of-function mutations using CRISPR/Cas9 gene editing and studied brain electrical activity, behavior, development, heart physiology, metabolism, and survival in larval zebrafish. Homozygous stxbp1a mutants exhibited a profound lack of movement, low electrical brain activity, low heart rate, decreased glucose and mitochondrial metabolism, and early fatality compared to controls. On the other hand, homozygous stxbp1b mutants had spontaneous electrographic seizures, and reduced locomotor activity response to a movement-inducing "dark-flash" visual stimulus, despite showing normal metabolism, heart rate, survival, and baseline locomotor activity. Our findings in these newly generated mutant lines of zebrafish suggest that zebrafish recapitulate clinical phenotypes associated with human syntaxin-binding protein 1 mutations.

  14. A review of monoaminergic neuropsychopharmacology in zebrafish.

    Science.gov (United States)

    Maximino, Caio; Herculano, Anderson Manoel

    2010-12-01

    Monoamine neurotransmitters are the major regulatory mechanisms in the vertebrate brain, involved in the adjustment of motivation, emotion, and cognition. The chemical anatomy of these systems is thought to be highly conserved in the brain of all vertebrates, including zebrafish. Recently, the development of behavioral assays in zebrafish allowed the neuropsychopharmacological investigation of these circuits and its functions. Here we review neuroanatomical, genetic, neurochemical, and psychopharmacological evidence regarding the roles of histaminergic, dopaminergic, noradrenergic, serotonergic, and melatonergic systems in this species. We conclude that, in spite of species differences, zebrafish are suitable for the investigation of neuropsychopharmacology of drugs that affect theses systems; nonetheless, more thorough validation of behavioral methods is still needed.

  15. Differential modulation of expression of nuclear receptor mediated genes by tris(2-butoxyethyl) phosphate (TBOEP) on early life stages of zebrafish (Danio rerio)

    Energy Technology Data Exchange (ETDEWEB)

    Ma, Zhiyuan, E-mail: zhiyuan_nju@163.com [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Yu, Yijun, E-mail: yjun.yu@gmail.com [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Tang, Song [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Liu, Hongling, E-mail: hlliu@nju.edu.cn [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Su, Guanyong; Xie, Yuwei [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Giesy, John P. [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Veterinary Biomedical Sciences, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Department of Biology and Chemistry, City University of Hong Kong, Kowloon, Hong Kong Special Administrative Region (Hong Kong); Hecker, Markus [School of Environment and Sustainability, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Toxicology Centre, University of Saskatchewan, Saskatoon, SK S7N 5B3 (Canada); Yu, Hongxia [State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing, Jiangsu 210023 (China)

    2015-12-15

    Highlights: • Effects of TBOEP on expression of genes of several nuclear hormone receptors and their relationship with adverse effect pathways in zebrafish. • TBOEP was neither an agonist nor antagonist of AR or AhR as determined by use of in vitro mammalian cell-based receptor transactivation assays. • Modulation of ER- and MR-dependent pathways allowed for development of feasible receptor-mediated, critical mechanisms of toxic action. - Abstract: As one substitute for phased-out brominated flame retardants (BFRs), tris(2-butoxyethyl) phosphate (TBOEP) is frequently detected in aquatic organisms. However, knowledge about endocrine disrupting mechanisms associated with nuclear receptors caused by TBOEP remained restricted to results from in vitro studies with mammalian cells. In the study, results of which are presented here, embryos/larvae of zebrafish (Danio rerio) were exposed to 0.02, 0.1 or 0.5 μM TBOEP to investigate expression of genes under control of several nuclear hormone receptors (estrogen receptors (ERs), androgen receptor (AR), thyroid hormone receptor alpha (TRα), mineralocorticoid receptor (MR), glucocorticoid receptor (GR), aryl hydrocarbon (AhR), peroxisome proliferator-activated receptor alpha (PPARα), and pregnane × receptor (P × R)) pathways at 120 hpf. Exposure to 0.5 μM TBOEP significantly (p < 0.05, one-way analysis of variance) up-regulated expression of estrogen receptors (ERs, er1, er2a, and er2b) genes and ER-associated genes (vtg4, vtg5, pgr, ncor, and ncoa3), indicating TBOEP modulates the ER pathway. In contrast, expression of most genes (mr, 11βhsd, ube2i,and adrb2b) associated with the mineralocorticoid receptor (MR) pathway were significantly down-regulated. Furthermore, in vitro mammalian cell-based (MDA-kb2 and H4IIE-luc) receptor transactivation assays, were also conducted to investigate possible agonistic or antagonistic effects on AR- and AhR-mediated pathways. In mammalian cells, none of these pathways were

  16. Functional analysis of the zebrafish ortholog of HMGCS1 reveals independent functions for cholesterol and isoprenoids in craniofacial development.

    Directory of Open Access Journals (Sweden)

    Anita M Quintana

    Full Text Available There are 8 different human syndromes caused by mutations in the cholesterol synthesis pathway. A subset of these disorders such as Smith-Lemli-Opitz disorder, are associated with facial dysmorphia. However, the molecular and cellular mechanisms underlying such facial deficits are not fully understood, primarily because of the diverse functions associated with the cholesterol synthesis pathway. Recent evidence has demonstrated that mutation of the zebrafish ortholog of HMGCR results in orofacial clefts. Here we sought to expand upon these data, by deciphering the cholesterol dependent functions of the cholesterol synthesis pathway from the cholesterol independent functions. Moreover, we utilized loss of function analysis and pharmacological inhibition to determine the extent of sonic hedgehog (Shh signaling in animals with aberrant cholesterol and/or isoprenoid synthesis. Our analysis confirmed that mutation of hmgcs1, which encodes the first enzyme in the cholesterol synthesis pathway, results in craniofacial abnormalities via defects in cranial neural crest cell differentiation. Furthermore targeted pharmacological inhibition of the cholesterol synthesis pathway revealed a novel function for isoprenoid synthesis during vertebrate craniofacial development. Mutation of hmgcs1 had no effect on Shh signaling at 2 and 3 days post fertilization (dpf, but did result in a decrease in the expression of gli1, a known Shh target gene, at 4 dpf, after morphological deficits in craniofacial development and chondrocyte differentiation were observed in hmgcs1 mutants. These data raise the possibility that deficiencies in cholesterol modulate chondrocyte differentiation by a combination of Shh independent and Shh dependent mechanisms. Moreover, our results describe a novel function for isoprenoids in facial development and collectively suggest that cholesterol regulates craniofacial development through versatile mechanisms.

  17. R-spondin 3 regulates dorsoventral and anteroposterior patterning by antagonizing Wnt/β-catenin signaling in zebrafish embryos.

    Directory of Open Access Journals (Sweden)

    Xiaozhi Rong

    Full Text Available The Wnt/β-catenin or canonical Wnt signaling pathway plays fundamental roles in early development and in maintaining adult tissue homeostasis. R-spondin 3 (Rspo3 is a secreted protein that has been implicated in activating the Wnt/β-catenin signaling in amphibians and mammals. Here we report that zebrafish Rspo3 plays a negative role in regulating the zygotic Wnt/β-catenin signaling. Zebrafish Rspo3 has a unique domain structure. It contains a third furin-like (FU3 domain. This FU3 is present in other four ray-finned fish species studied but not in elephant shark. In zebrafish, rspo3 mRNA is maternally deposited and has a ubiquitous expression in early embryonic stages. After 12 hpf, its expression becomes tissue-specific. Forced expression of rspo3 promotes dorsoanterior patterning and increases the expression of dorsal and anterior marker genes. Knockdown of rspo3 increases ventral-posterior development and stimulates ventral and posterior marker genes expression. Forced expression of rspo3 abolishes exogenous Wnt3a action and reduces the endogenous Wnt signaling activity. Knockdown of rspo3 results in increased Wnt/β-catenin signaling activity. Further analyses indicate that Rspo3 does not promote maternal Wnt signaling. Human RSPO3 has similar action when tested in zebrafish embryos. These results suggest that Rspo3 regulates dorsoventral and anteroposterior patterning by negatively regulating the zygotic Wnt/β-catenin signaling in zebrafish embryos.

  18. Detection of vitellogenin incorporation into zebrafish oocytes by FITC fluorescence

    Directory of Open Access Journals (Sweden)

    Yokoi Hayato

    2011-04-01

    Full Text Available Abstract Background Large volumes of lymph can be collected from the eye-sacs of bubble-eye goldfish. We attempted to induce vitellogenin (Vtg in the eye-sac lymph of bubble-eye goldfish and develop a method for visualizing Vtg incorporation by zebrafish oocytes using FITC-labeling. Methods Estrogen efficiently induced Vtg in the eye-sac lymph of goldfish. After FITC-labeled Vtg was prepared, it was injected into mature female zebrafish. Results Incorporation of FITC-labeled Vtg by zebrafish oocytes was detected in in vivo and in vitro experiments. The embryos obtained from zebrafish females injected with FITC-labeled Vtg emitted FITC fluorescence from the yolk sac and developed normally. Conclusion This method for achieving Vtg incorporation by zebrafish oocytes could be useful in experiments related to the development and endocrinology of zebrafish oocytes.

  19. Regulation of gonadal sex ratios and pubertal development by the thyroid endocrine system in zebrafish (Danio rerio).

    Science.gov (United States)

    Sharma, Prakash; Patiño, Reynaldo

    2013-04-01

    We examined associations between thyroid condition, gonadal sex and pubertal development in zebrafish. Seventy-two-hour postfertilization larvae were reared in untreated medium or in the presence of goitrogens (sodium perchlorate, 0.82 mM; methimazole, 0.15 and 0.3 mM) or thyroxine (1 and 10 nM) for 30 days. Thyrocyte height, gonadal sex and gonadal development were histologically determined at 45 and 60 days postfertilization (dpf). Thyrocyte hypertrophy, an index of hypothyroidism, was observed at 45 and 60 dpf in perchlorate-treated but only at 45 dpf in methimazole-treated fish. Similarly, gonadal sex ratios were biased toward ovaries relative to control animals at 45 and 60 dpf in perchlorate-treated fish but only at 45 dpf in methimazole-treated fish. Gonadal sex ratios were biased toward testes at 45 and 60 dpf in thyroxine-treated fish. Spermatogenesis was delayed in testes from goitrogen-treated fish at 60 dpf relative to control values, but was unaffected in testes from thyroxine-treated individuals. Oogenesis seemed to be nonspecifically delayed in all treatments relative to control at 60 dpf. This study confirmed the previously reported association between hypothyroid condition and ovarian-skewed ratios, and hyperthyroid condition and testicular-skewed ratios, and also showed that male pubertal development is specifically delayed by experimental hypothyroidism. The simultaneous recovery from the hypothyroid and ovary-inducing effects of methimazole by 60 dpf (27 days post-treatment) suggests that the ovary-skewing effect of goitrogens is reversible when thyroid conditions return to basal levels before developmental commitment of gonadal sex. Conversely, the masculinizing effect of hyperthyroidism seems to be stable and perhaps permanent. Published by Elsevier Inc.

  20. Regulation of gonadal sex ratios and pubertal development by the thyroid endocrine system in zebrafish (Danio rerio)

    Science.gov (United States)

    Sharma, Prakash; Patino, Reynaldo

    2013-01-01

    We examined associations between thyroid condition, gonadal sex and pubertal development in zebrafish. Seventy-two-hour postfertilization larvae were reared in untreated medium or in the presence of goitrogens (sodium perchlorate, 0.82 mM; methimazole, 0.15 and 0.3 mM) or thyroxine (1 and 10 nM) for 30 days. Thyrocyte height, gonadal sex and gonadal development were histologically determined at 45 and 60 days postfertilization (dpf). Thyrocyte hypertrophy, an index of hypothyroidism, was observed at 45 and 60 dpf in perchlorate-treated but only at 45 dpf in methimazole-treated fish. Similarly, gonadal sex ratios were biased toward ovaries relative to control animals at 45 and 60 dpf in perchlorate-treated fish but only at 45 dpf in methimazole-treated fish. Gonadal sex ratios were biased toward testes at 45 and 60 dpf in thyroxine-treated fish. Spermatogenesis was delayed in testes from goitrogen-treated fish at 60 dpf relative to control values, but was unaffected in testes from thyroxine-treated individuals. Oogenesis seemed to be nonspecifically delayed in all treatments relative to control at 60 dpf. This study confirmed the previously reported association between hypothyroid condition and ovarian-skewed ratios, and hyperthyroid condition and testicular-skewed ratios, and also showed that male pubertal development is specifically delayed by experimental hypothyroidism. The simultaneous recovery from the hypothyroid and ovary-inducing effects of methimazole by 60 dpf (27 days post-treatment) suggests that the ovary-skewing effect of goitrogens is reversible when thyroid conditions return to basal levels before developmental commitment of gonadal sex. Conversely, the masculinizing effect of hyperthyroidism seems to be stable and perhaps permanent.

  1. Zebrafish swimming in the flow: a particle image velocimetry study

    Directory of Open Access Journals (Sweden)

    Violet Mwaffo

    2017-11-01

    Full Text Available Zebrafish is emerging as a species of choice for the study of a number of biomechanics problems, including balance development, schooling, and neuromuscular transmission. The precise quantification of the flow physics around swimming zebrafish is critical toward a mechanistic understanding of the complex swimming style of this fresh-water species. Although previous studies have elucidated the vortical structures in the wake of zebrafish swimming in placid water, the flow physics of zebrafish swimming against a water current remains unexplored. In an effort to illuminate zebrafish swimming in a dynamic environment reminiscent of its natural habitat, we experimentally investigated the locomotion and hydrodynamics of a single zebrafish swimming in a miniature water tunnel using particle image velocimetry. Our results on zebrafish locomotion detail the role of flow speed on tail beat undulations, heading direction, and swimming speed. Our findings on zebrafish hydrodynamics offer a precise quantification of vortex shedding during zebrafish swimming and demonstrate that locomotory patterns play a central role on the flow physics. This knowledge may help clarify the evolutionary advantage of burst and cruise swimming movements in zebrafish.

  2. Transforming early childhood education for sustainable development

    African Journals Online (AJOL)

    This paper examined the ways in which early childhood education needs to be transformed for sustainable development. These ways include teaching children environmental security through play, personal hygiene, appropriate waste use and disposal, and nature awareness. It was recommended that early childhood ...

  3. Genetic Dissection of Dual Roles for the Transcription Factor six7 in Photoreceptor Development and Patterning in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Mailin Sotolongo-Lopez

    2016-04-01

    Full Text Available The visual system of a particular species is highly adapted to convey detailed ecological and behavioral information essential for survival. The consequences of structural mutations of opsins upon spectral sensitivity and environmental adaptation have been studied in great detail, but lacking is knowledge of the potential influence of alterations in gene regulatory networks upon the diversity of cone subtypes and the variation in the ratio of rods and cones observed in numerous diurnal and nocturnal species. Exploiting photoreceptor patterning in cone-dominated zebrafish, we uncovered two independent mechanisms by which the sine oculis homeobox homolog 7 (six7 regulates photoreceptor development. In a genetic screen, we isolated the lots-of-rods-junior (ljrp23ahub mutation that resulted in an increased number and uniform distribution of rods in otherwise normal appearing larvae. Sequence analysis, genome editing using TALENs and knockdown strategies confirm ljrp23ahub as a hypomorphic allele of six7, a teleost orthologue of six3, with known roles in forebrain patterning and expression of opsins. Based on the lack of predicted protein-coding changes and a deletion of a conserved element upstream of the transcription start site, a cis-regulatory mutation is proposed as the basis of the reduced expression of six7 in ljrp23ahub. Comparison of the phenotypes of the hypomorphic and knock-out alleles provides evidence of two independent roles in photoreceptor development. EdU and PH3 labeling show that the increase in rod number is associated with extended mitosis of photoreceptor progenitors, and TUNEL suggests that the lack of green-sensitive cones is the result of cell death of the cone precursor. These data add six7 to the small but growing list of essential genes for specification and patterning of photoreceptors in non-mammalian vertebrates, and highlight alterations in transcriptional regulation as a potential source of photoreceptor variation

  4. Pregnancy-associated plasma protein-A (PAPP-A) modulates early developmental rate in zebrafish independent of its proteolytic activity

    DEFF Research Database (Denmark)

    Kjær-Sørensen, Kasper; Engholm, Ditte Høyer; Kamei, Hiroyasu

    2013-01-01

    the developmental rate beginning during gastrulation without affecting the normal patterning of the embryo. This phenotype is different from those resulting from deficiency of Igf receptor or ligand in zebrafish, suggesting a function of Papp-a outside the Igf system. Biochemical analysis of recombinant zebrafish...... Papp-a demonstrates conservation of proteolytic activity, specificity, and intrinsic regulatory mechanism. However, in vitro transcribed mRNA, which encodes a proteolytically inactive Papp-a mutant, recues the papp-a knockdown phenotype as efficient as wild-type Papp-a. Thus, the developmental...

  5. Early development of the aplacophoran mollusc Chaetoderma

    DEFF Research Database (Denmark)

    Nielsen, Claus; Haszprunar, Gerhard; Ruthensteiner, Bernhard

    2007-01-01

    The early development of the trochophore larva of the aplacophoran Chaetoderma nitidulum (Mollusca: Caudofoveata = Chaetodermomorpha) is described using scanning and transmission electron microscopy and using fluorescence staining and confocal laser scanning microscopy of the muscle system...

  6. Histological Characterization of the Dicer1 Mutant Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Saeed Akhtar

    2015-01-01

    Full Text Available DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA and RNA-interference (RNAi functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter have an arrest in early growth with significantly smaller eyes and are dead at 14–18 dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE cells are normal with no sign of degeneration at the age of 20 months.

  7. Loss of ribosomal protein L11 affects zebrafish embryonic development through a p53-dependent apoptotic response.

    Directory of Open Access Journals (Sweden)

    Anirban Chakraborty

    Full Text Available Ribosome is responsible for protein synthesis in all organisms and ribosomal proteins (RPs play important roles in the formation of a functional ribosome. L11 was recently shown to regulate p53 activity through a direct binding with MDM2 and abrogating the MDM2-induced p53 degradation in response to ribosomal stress. However, the studies were performed in cell lines and the significance of this tumor suppressor function of L11 has yet to be explored in animal models. To investigate the effects of the deletion of L11 and its physiological relevance to p53 activity, we knocked down the rpl11 gene in zebrafish and analyzed the p53 response. Contrary to the cell line-based results, our data indicate that an L11 deficiency in a model organism activates the p53 pathway. The L11-deficient embryos (morphants displayed developmental abnormalities primarily in the brain, leading to embryonic lethality within 6-7 days post fertilization. Extensive apoptosis was observed in the head region of the morphants, thus correlating the morphological defects with apparent cell death. A decrease in total abundance of genes involved in neural patterning of the brain was observed in the morphants, suggesting a reduction in neural progenitor cells. Upregulation of the genes involved in the p53 pathway were observed in the morphants. Simultaneous knockdown of the p53 gene rescued the developmental defects and apoptosis in the morphants. These results suggest that ribosomal dysfunction due to the loss of L11 activates a p53-dependent checkpoint response to prevent improper embryonic development.

  8. Genome-wide survey and developmental expression mapping of zebrafish SET domain-containing genes.

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    Xiao-Jian Sun

    Full Text Available SET domain-containing proteins represent an evolutionarily conserved family of epigenetic regulators, which are responsible for most histone lysine methylation. Since some of these genes have been revealed to be essential for embryonic development, we propose that the zebrafish, a vertebrate model organism possessing many advantages for developmental studies, can be utilized to study the biological functions of these genes and the related epigenetic mechanisms during early development. To this end, we have performed a genome-wide survey of zebrafish SET domain genes. 58 genes total have been identified. Although gene duplication events give rise to several lineage-specific paralogs, clear reciprocal orthologous relationship reveals high conservation between zebrafish and human SET domain genes. These data were further subject to an evolutionary analysis ranging from yeast to human, leading to the identification of putative clusters of orthologous groups (COGs of this gene family. By means of whole-mount mRNA in situ hybridization strategy, we have also carried out a developmental expression mapping of these genes. A group of maternal SET domain genes, which are implicated in the programming of histone modification states in early development, have been identified and predicted to be responsible for all known sites of SET domain-mediated histone methylation. Furthermore, some genes show specific expression patterns in certain tissues at certain stages, suggesting the involvement of epigenetic mechanisms in the development of these systems. These results provide a global view of zebrafish SET domain histone methyltransferases in evolutionary and developmental dimensions and pave the way for using zebrafish to systematically study the roles of these genes during development.

  9. Extraocular muscle regeneration in zebrafish requires late signals from Insulin-like growth factors.

    Science.gov (United States)

    Saera-Vila, Alfonso; Louie, Ke'ale W; Sha, Cuilee; Kelly, Ryan M; Kish, Phillip E; Kahana, Alon

    2018-01-01

    Insulin-like growth factors (Igfs) are key regulators of key biological processes such as embryonic development, growth, and tissue repair and regeneration. The role of Igf in myogenesis is well documented and, in zebrafish, promotes fin and heart regeneration. However, the mechanism of action of Igf in muscle repair and regeneration is not well understood. Using adult zebrafish extraocular muscle (EOM) regeneration as an experimental model, we show that Igf1 receptor blockage using either chemical inhibitors (BMS754807 and NVP-AEW541) or translation-blocking morpholino oligonucleotides (MOs) reduced EOM regeneration. Zebrafish EOMs regeneration depends on myocyte dedifferentiation, which is driven by early epigenetic reprogramming and requires autophagy activation and cell cycle reentry. Inhibition of Igf signaling had no effect on either autophagy activation or cell proliferation, indicating that Igf signaling was not involved in the early reprogramming steps of regeneration. Instead, blocking Igf signaling produced hypercellularity of regenerating EOMs and diminished myosin expression, resulting in lack of mature differentiated muscle fibers even many days after injury, indicating that Igf was involved in late re-differentiation steps. Although it is considered the main mediator of myogenic Igf actions, Akt activation decreased in regenerating EOMs, suggesting that alternative signaling pathways mediate Igf activity in muscle regeneration. In conclusion, Igf signaling is critical for re-differentiation of reprogrammed myoblasts during late steps of zebrafish EOM regeneration, suggesting a regulatory mechanism for determining regenerated muscle size and timing of differentiation, and a potential target for regenerative therapy.

  10. Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

    International Nuclear Information System (INIS)

    Jönsson, Maria E.; Kubota, Akira; Timme-Laragy, Alicia R.; Woodin, Bruce; Stegeman, John J.

    2012-01-01

    The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC 50 values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells. -- Highlights: ► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox-2.

  11. Ahr2-dependence of PCB126 effects on the swim bladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Jönsson, Maria E., E-mail: maria.jonsson@ebc.uu.se [Dept. of Environmental Toxicology, Evolutionary Biology, Centre, Uppsala University, Norbyvägen 18A, 752 36 Uppsala (Sweden); Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Kubota, Akira, E-mail: akubota@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Timme-Laragy, Alicia R., E-mail: atimmelaragy@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Division of Environmental Health, Department of Public Health, School of Public Health and Health Sciences, University of Massachusetts, Amherst, MA, 01003 (United States); Woodin, Bruce, E-mail: bwoodin@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Redfield 3-42 MS 32, Woods Hole Oceanographic Institution, Woods Hole, MA, 02543 (United States)

    2012-12-01

    The teleost swim bladder is assumed a homolog of the tetrapod lung. Both swim bladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR) agonists; in zebrafish (Danio rerio) the swim bladder fails to inflate with exposure to 3,3′,4,4′,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P450 1 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swim bladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependence of the effect of PCB126 on swim bladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swim bladder inflation. The effects of PCB126 were concentration-dependent with EC{sub 50} values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swim bladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swim bladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos failed to inflate the swim bladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swim bladder. Our results indicate that PCB126 blocks swim bladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swim bladder cells. -- Highlights: ► PCB126 caused cellular changes in the developing swim bladder. ► Swim bladder inflation was not related to expression of CYP1 or cox

  12. WNK1/HSN2 mutation in human peripheral neuropathy deregulates KCC2 expression and posterior lateral line development in zebrafish (Danio rerio.

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    Valérie Bercier

    Full Text Available Hereditary sensory and autonomic neuropathy type 2 (HSNAII is a rare pathology characterized by an early onset of severe sensory loss (all modalities in the distal limbs. It is due to autosomal recessive mutations confined to exon "HSN2" of the WNK1 (with-no-lysine protein kinase 1 serine-threonine kinase. While this kinase is well studied in the kidneys, little is known about its role in the nervous system. We hypothesized that the truncating mutations present in the neural-specific HSN2 exon lead to a loss-of-function of the WNK1 kinase, impairing development of the peripheral sensory system. To investigate the mechanisms by which the loss of WNK1/HSN2 isoform function causes HSANII, we used the embryonic zebrafish model and observed strong expression of WNK1/HSN2 in neuromasts of the peripheral lateral line (PLL system by immunohistochemistry. Knocking down wnk1/hsn2 in embryos using antisense morpholino oligonucleotides led to improper PLL development. We then investigated the reported interaction between the WNK1 kinase and neuronal potassium chloride cotransporter KCC2, as this transporter is a target of WNK1 phosphorylation. In situ hybridization revealed kcc2 expression in mature neuromasts of the PLL and semi-quantitative RT-PCR of wnk1/hsn2 knockdown embryos showed an increased expression of kcc2 mRNA. Furthermore, overexpression of human KCC2 mRNA in embryos replicated the wnk1/hsn2 knockdown phenotype. We validated these results by obtaining double knockdown embryos, both for wnk1/hsn2 and kcc2, which alleviated the PLL defects. Interestingly, overexpression of inactive mutant KCC2-C568A, which does not extrude ions, allowed a phenocopy of the PLL defects. These results suggest a pathway in which WNK1/HSN2 interacts with KCC2, producing a novel regulation of its transcription independent of KCC2's activation, where a loss-of-function mutation in WNK1 induces an overexpression of KCC2 and hinders proper peripheral sensory nerve

  13. Duplication and diversification of the hypoxia-inducible IGFBP-1 gene in zebrafish.

    Directory of Open Access Journals (Sweden)

    Hiroyasu Kamei

    2008-08-01

    Full Text Available Gene duplication is the primary force of new gene evolution. Deciphering whether a pair of duplicated genes has evolved divergent functions is often challenging. The zebrafish is uniquely positioned to provide insight into the process of functional gene evolution due to its amenability to genetic and experimental manipulation and because it possess a large number of duplicated genes.We report the identification and characterization of two hypoxia-inducible genes in zebrafish that are co-ortholgs of human IGF binding protein-1 (IGFBP-1. IGFBP-1 is a secreted protein that binds to IGF and modulates IGF actions in somatic growth, development, and aging. Like their human and mouse counterparts, in adult zebrafish igfbp-1a and igfbp-1b are exclusively expressed in the liver. During embryogenesis, the two genes are expressed in overlapping spatial domains but with distinct temporal patterns. While zebrafish IGFBP-1a mRNA was easily detected throughout embryogenesis, IGFBP-1b mRNA was detectable only in advanced stages. Hypoxia induces igfbp-1a expression in early embryogenesis, but induces the igfbp-1b expression later in embryogenesis. Both IGFBP-1a and -b are capable of IGF binding, but IGFBP-1b has much lower affinities for IGF-I and -II because of greater dissociation rates. Overexpression of IGFBP-1a and -1b in zebrafish embryos caused significant decreases in growth and developmental rates. When tested in cultured zebrafish embryonic cells, IGFBP-1a and -1b both inhibited IGF-1-induced cell proliferation but the activity of IGFBP-1b was significantly weaker.These results indicate subfunction partitioning of the duplicated IGFBP-1 genes at the levels of gene expression, physiological regulation, protein structure, and biological actions. The duplicated IGFBP-1 may provide additional flexibility in fine-tuning IGF signaling activities under hypoxia and other catabolic conditions.

  14. Evaluation of visible implant elastomer tags in zebrafish (Danio rerio

    Directory of Open Access Journals (Sweden)

    Claudia Hohn

    2013-11-01

    The use of the visible implant elastomer (VIE tagging system in zebrafish (Danio rerio was examined. Two tag orientations (horizontal and vertical at the dorsal fin base were tested for tag retention, tag fragmentation and whether VIE tags affected growth and survival of juvenile zebrafish (1–4 month post hatch. Six tag locations (abdomen, anal fin base, caudal peduncle, dorsal fin base, pectoral fin base, isthmus and 5 tag colors (yellow, red, pink, orange, blue were evaluated for ease of VIE tag application and tag visibility in adult zebrafish. Long-term retention (1 year and multiple tagging sites (right and left of dorsal fin and pectoral fin base were examined in adult zebrafish. Lastly, survival of recombination activation gene 1−/− (rag1−/− zebrafish was evaluated after VIE tagging. The best tag location was the dorsal fin base, and the most visible tag color was pink. Growth rate of juvenile zebrafish was not affected by VIE tagging. Horizontal tagging is recommended in early stages of fish growth (1–2 months post hatch. VIE tags were retained for 1 year and tagging did not interfere with long-term growth and survival. There was no mortality associated with VIE tagging in rag1−/− zebrafish. The VIE tagging system is highly suitable for small-sized zebrafish. When familiar with the procedure, 120 adult zebrafish can be tagged in one hour. It does not increase mortality in adult zebrafish or interfere with growth in juvenile or adult zebrafish.

  15. Early development of the human pelvic diaphragm

    NARCIS (Netherlands)

    Koch, Wijnandus Franciscus Robertus Maria

    2006-01-01

    The last decade an increasing interest in the pelvic floor can be observed in medical sciences. The lack of data on the development of the human pelvic floor is striking. The early development of the human pelvic diaphragm was studied. Materials and methodsUse was made of 38 human embryos and

  16. Lessons from early experience in reactor development

    International Nuclear Information System (INIS)

    Allen, W.

    1976-09-01

    This paper deals with several issues in U.S. reactor development and demonstration experience. The focus is on the period between 1946 and 1963 during which the Atomic Energy Commission (AEC) guided early reactor research and development (R and D) and conducted the Power Reactor Demonstration Program

  17. GLUT12 deficiency during early development results in heart failure and a diabetic phenotype in zebrafish

    NARCIS (Netherlands)

    Jiménez-Amilburu, Vanesa; Jong-Raadsen, Susanne; Bakkers, Jeroen; Spaink, Herman P.; Marín-Juez, Rubén

    2015-01-01

    Cardiomyopathies-associated metabolic pathologies (e.g., type 2 diabetes and insulin resistance) are a leading cause of mortality. It is known that the association between these pathologies works in both directions, for which heart failure can lead to metabolic derangements such as insulin

  18. GLUT12 deficiency during early development results in heart failure and a diabetic phenotype in zebrafish

    NARCIS (Netherlands)

    Jiménez-Amilburu, Vanesa; Jong-Raadsen, Susanne; Bakkers, Jeroen; Spaink, Herman P; Marín-Juez, Rubén

    Cardiomyopathies-associated metabolic pathologies (e.g., type 2 diabetes and insulin resistance) are a leading cause of mortality. It is known that the association between these pathologies works in both directions, for which heart failure can lead to metabolic derangements such as insulin

  19. Gene Duplication of the zebrafish kit ligand and partitioning of melanocyte development functions to kit ligand a.

    Directory of Open Access Journals (Sweden)

    Keith A Hultman

    2007-01-01

    Full Text Available The retention of particular genes after the whole genome duplication in zebrafish has given insights into how genes may evolve through partitioning of ancestral functions. We examine the partitioning of expression patterns and functions of two zebrafish kit ligands, kit ligand a (kitla and kit ligand b (kitlb, and discuss their possible coevolution with the duplicated zebrafish kit receptors (kita and kitb. In situ hybridizations show that kitla mRNA is expressed in the trunk adjacent to the notochord in the middle of each somite during stages of melanocyte migration and later expressed in the skin, when the receptor is required for melanocyte survival. kitla is also expressed in other regions complementary to kita receptor expression, including the pineal gland, tail bud, and ear. In contrast, kitlb mRNA is expressed in brain ventricles, ear, and cardinal vein plexus, in regions generally not complementary to either zebrafish kit receptor ortholog. However, like kitla, kitlb is expressed in the skin during stages consistent with melanocyte survival. Thus, it appears that kita and kitla have maintained congruent expression patterns, while kitb and kitlb have evolved divergent expression patterns. We demonstrate the interaction of kita and kitla by morpholino knockdown analysis. kitla morphants, but not kitlb morphants, phenocopy the null allele of kita, with defects for both melanocyte migration and survival. Furthermore, kitla morpholino, but not kitlb morpholino, interacts genetically with a sensitized allele of kita, confirming that kitla is the functional ligand to kita. Last, we examine kitla overexpression in embryos, which results in hyperpigmentation caused by an increase in the number and size of melanocytes. This hyperpigmentation is dependent on kita function. We conclude that following genome duplication, kita and kitla have maintained their receptor-ligand relationship, coevolved complementary expression patterns, and that

  20. Heads and tails of endoderm development and adult tissue homeostasis in zebrafish

    NARCIS (Netherlands)

    Faro, A.

    2010-01-01

    The regulatory signaling pathways crucial during embryonic development seem to play key roles in adult tissues homeostasis and are often deregulated in pathological conditions. The Wnt pathway plays a pivotal role in orchestrating cell fate decisions during embryonic development, organogenesis, and

  1. Identification and characterization of zebrafish thrombocytes.

    Science.gov (United States)

    Jagadeeswaran, P; Sheehan, J P; Craig, F E; Troyer, D

    1999-12-01

    To analyse primary haemostasis in the zebrafish we have identified and characterized the zebrafish thrombocyte by morphologic, immunologic and functional approaches. Novel methods were developed for harvesting zebrafish blood with preservation of thrombocytes, and assaying whole blood adhesion/aggregation responses in microtitre plates. Light and electron microscopy of the thrombocyte illustrated morphological characteristics including the formation of aggregates, pseudopodia, and surface-connected vesicles analagous to the platelet canalicular system. Immunostaining with polyclonal antisera versus human platelet glycoproteins demonstrated the presence of glycoprotein Ib and IIb/IIIa-like complexes on the thrombocyte surface. Whole blood assays for adhesion/aggregation and ATP release showed ristocetin-induced adhesion without ATP release, and platelet agonist (collagen, arachidonic acid) induced aggregation with ATP release. Blood harvested from zebrafish treated with aspirin demonstrated inhibition of arachidonic acid induced aggregation and agonist induced ATP release, consistent with at least partial dependence on an intact cyclo oxygenase pathway. The combined morphologic immunologic and functional evidence suggest that the zebrafish thrombocyte is the haemostatic homologue of the mammalian platelet. Conservation of major haemostatic pathways involved in platelet function and coagulation suggests that the zebrafish is a relevant model for mammalian haemostasis and thrombosis.

  2. Genomic Organization of Zebrafish microRNAs

    Directory of Open Access Journals (Sweden)

    Paydar Ima

    2008-05-01

    Full Text Available Abstract Background microRNAs (miRNAs are small (~22 nt non-coding RNAs that regulate cell movement, specification, and development. Expression of miRNAs is highly regulated, both spatially and temporally. Based on direct cloning, sequence conservation, and predicted secondary structures, a large number of miRNAs have been identified in higher eukaryotic genomes but whether these RNAs are simply a subset of a much larger number of noncoding RNA families is unknown. This is especially true in zebrafish where genome sequencing and annotation is not yet complete. Results We analyzed the zebrafish genome to identify the number and location of proven and predicted miRNAs resulting in the identification of 35 new miRNAs. We then grouped all 415 zebrafish miRNAs into families based on seed sequence identity as a means to identify possible functional redundancy. Based on genomic location and expression analysis, we also identified those miRNAs that are likely to be encoded as part of polycistronic transcripts. Lastly, as a resource, we compiled existing zebrafish miRNA expression data and, where possible, listed all experimentally proven mRNA targets. Conclusion Current analysis indicates the zebrafish genome encodes 415 miRNAs which can be grouped into 44 families. The largest of these families (the miR-430 family contains 72 members largely clustered in two main locations along chromosome 4. Thus far, most zebrafish miRNAs exhibit tissue specific patterns of expression.

  3. Long-term effects of antibiotics, norfloxacin, and sulfamethoxazole, in a partial life-cycle study with zebrafish (Danio rerio): effects on growth, development, and reproduction.

    Science.gov (United States)

    Yan, Zhenhua; Lu, Guanghua; Ye, Qiuxia; Liu, Jianchao

    2016-09-01

    A partial life-cycle study with zebrafish (Danio rerio) was conducted to evaluate the long-term effects of antibiotics, norfloxacin (NOR) and sulfamethoxazole (SMX). A series of bio-endpoints correlated to the growth, development, and reproduction was assessed. The results showed that the body weight and the condition factor were depressed by SMX at 200 μg/L during the growth period. Meanwhile, the activities of metabolic enzyme (ethoxyresorufin O-deethylase, EROD) and antioxidant enzymes (superoxide dismutase, SOD and catalase, CAT) were stimulated in all cases. The consequences of parental exposure to antibiotics for the next generation were also examined. The egg production of parents were depressed by the 200 μg/L NOR and SMX alone or in combination. Similarly, decreased hatching, survival, and enhanced development abnormality of the next generation also occurred after parental exposure to SMX at the highest concentration. The heartbeat however was not altered in all cases. Furthermore, there was no significant difference in the bio-endpoints between the combined and individual treatment in most cases, with the exception of lower EROD activity and egg production in the co-treatment. The results suggest that long-term exposure to NOR and SMX at environmentally relevant concentrations, individually and in a mixture, may not significantly pose a threat to the growth, development, and reproduction of zebrafish, and an adverse effect may be expected at high concentration.

  4. Dietary β-glucan enhances the contents of complement component 3 and factor B in eggs of zebrafish.

    Science.gov (United States)

    Jiang, Chengyan; Wang, Peng; Li, Mengyang; Liu, Shousheng; Zhang, Shicui

    2016-12-01

    β-glucan has been shown to increase non-specific immunity and resistance against infections or pathogenic bacteria in several fish species, but no information is available regarding its trans-generational effects to date. Here we clearly demonstrated that β-glucan enhanced the contents of immune-relevant molecules C3 and Bf in eggs of zebrafish, and the embryos derived from β-1,3 glucan-treated zebrafish were more resistant to bacterial challenge than control embryos. Moreover, the transferred C3 and Bf were directly associated with the antimicrobial defense of early embryos. In addition, feeding female zebrafish with β-glucan had little detrimental effects on the number of spawned eggs and their embryonic development. Collectively, these data show for the first time that β-glucan can be safely used to promote the non-specific immunity in offspring of fishes. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Moderate alcohol exposure during early brain development increases stimulus-response habits in adulthood.

    Science.gov (United States)

    Parker, Matthew O; Evans, Alexandra M-D; Brock, Alistair J; Combe, Fraser J; Teh, Muy-Teck; Brennan, Caroline H

    2016-01-01

    Exposure to alcohol during early central nervous system development has been shown variously to affect aspects of physiological and behavioural development. In extreme cases, this can extend to craniofacial defects, severe developmental delay and mental retardation. At more moderate levels, subtle differences in brain morphology and behaviour have been observed. One clear effect of developmental alcohol exposure is an increase in the propensity to develop alcoholism and other addictions. The mechanisms by which this occurs, however, are not currently understood. In this study, we tested the hypothesis that adult zebrafish chronically exposed to moderate levels of ethanol during early brain ontogenesis would show an increase in conditioned place preference for alcohol and an increased propensity towards habit formation, a key component of drug addiction in humans. We found support for both of these hypotheses and found that the exposed fish had changes in mRNA expression patterns for dopamine receptor, nicotinic acetylcholine receptor and μ-opioid receptor encoding genes. Collectively, these data show an explicit link between the increased proclivity for addiction and addiction-related behaviour following exposure to ethanol during early brain development and alterations in the neural circuits underlying habit learning. © 2014 Society for the Study of Addiction.

  6. Developmental social isolation affects adult behavior, social interaction, and dopamine metabolite levels in zebrafish.

    Science.gov (United States)

    Shams, Soaleha; Amlani, Shahid; Buske, Christine; Chatterjee, Diptendu; Gerlai, Robert

    2018-01-01

    The zebrafish is a social vertebrate and an excellent translational model for a variety of human disorders. Abnormal social behavior is a hallmark of several human brain disorders. Social behavioral problems can arise as a result of adverse early social environment. Little is known about the effects of early social isolation in adult zebrafish. We compared zebrafish that were isolated for either short (7 days) or long duration (180 days) to socially housed zebrafish, testing their behavior across ontogenesis (ages 10, 30, 60, 90, 120, 180 days), and shoal cohesion and whole-brain monoamines and their metabolites in adulthood. Long social isolation increased locomotion and decreased shoal cohesion and anxiety in the open-field in adult. Additionally, both short and long social isolation reduced dopamine metabolite levels in response to social stimuli. Thus, early social isolation has lasting effects in zebrafish, and may be employed to generate zebrafish models of human neuropsychiatric conditions. © 2017 Wiley Periodicals, Inc.

  7. Early executive function predicts reasoning development.

    Science.gov (United States)

    Richland, Lindsey E; Burchinal, Margaret R

    2013-01-01

    Analogical reasoning is a core cognitive skill that distinguishes humans from all other species and contributes to general fluid intelligence, creativity, and adaptive learning capacities. Yet its origins are not well understood. In the study reported here, we analyzed large-scale longitudinal data from the Study of Early Child Care and Youth Development to test predictors of growth in analogical-reasoning skill from third grade to adolescence. Our results suggest an integrative resolution to the theoretical debate regarding contributory factors arising from smaller-scale, cross-sectional experiments on analogy development. Children with greater executive-function skills (both composite and inhibitory control) and vocabulary knowledge in early elementary school displayed higher scores on a verbal analogies task at age 15 years, even after adjusting for key covariates. We posit that knowledge is a prerequisite to analogy performance, but strong executive-functioning resources during early childhood are related to long-term gains in fundamental reasoning skills.

  8. Early Childhood Development and E-Learning in Africa: The Early Childhood Development Virtual University Programme

    Science.gov (United States)

    Pence, Alan

    2007-01-01

    This article explores the development and evaluation of the graduate-level Early Childhood Development Virtual University (ECDVU) programme in Sub-Saharan Africa from 2001 through to 2004. It outlines the history of the ECDVU and the establishing of a Sub-Saharan programme for future leaders in the early childhood field guided by the key principle…

  9. Development of a new screening assay to identify proteratogenic substances using zebrafish danio rerio embryo combined with an exogenous mammalian metabolic activation system (mDarT).

    Science.gov (United States)

    Busquet, François; Nagel, Roland; von Landenberg, Friedrich; Mueller, Stefan O; Huebler, Nicole; Broschard, Thomas H

    2008-07-01

    The assessment of teratogenic effects of chemicals is generally performed using in vivo teratogenicity assays, for example, in rats or rabbits. We have developed an in vitro teratogenicity assay using the zebrafish Danio rerio embryo combined with an exogenous mammalian metabolic activation system (MAS), able to biotransform proteratogenic compounds. Cyclophosphamide (CPA) and ethanol were used as proteratogens to test the efficiency of this assay. Briefly, the zebrafish embryos were cocultured at 2 hpf (hours postfertilization) with the test material at varying concentrations, induced male rat liver microsomes and nicotinamide adenine dinucleotide phosphate (reduced) for 60 min at 32 degrees C under moderate agitation in Tris-buffer. The negative control (test material alone) and the MAS control (MAS alone) were incubated in parallel. For each test group, 20 eggs were used for statistical robustness. Afterward fish embryos were transferred individually into 24-well plates filled with fish medium for 48 h at 26 degrees C with a 12-h light cycle. Teratogenicity was scored after 24 and 48 hpf using morphological endpoints. No teratogenic effects were observed in fish embryos exposed to the proteratogens alone, that is, without metabolic activation. In contrast, CPA and ethanol induced abnormalities in fish embryos when coincubated with microsomes. The severity of malformations increased with increasing concentrations of the proteratogens. We conclude that the application of microsomes will improve and refine the D. rerio teratogenicity assay as a predictive and valuable alternative method to screen teratogenic substances.

  10. Early Phonological Development: Creating an Assessment Test

    Science.gov (United States)

    Stoel-Gammon, Carol; Williams, A. Lynn

    2013-01-01

    This paper describes a new protocol for assessing the phonological systems of two-year-olds with typical development and older children with delays in vocabulary acquisition. The test (Profiles of Early Expressive Phonological Skills ("PEEPS"), Williams & Stoel-Gammon, in preparation) differs from currently available assessments in…

  11. Early and Late Rate of Force Development

    DEFF Research Database (Denmark)

    Andersen, Lars L; Andersen, Jesper L; Zebis, Mette K

    2010-01-01

    The objective of this study is to investigate the potentially opposing influence of qualitative and quantitative muscular adaptations in response to high-intensity resistance training on contractile rate of force development (RFD) in the early (<100 ms) and later phases (>200 ms) of rising muscle...

  12. Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio)

    OpenAIRE

    Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V.; Bobe, Julien

    2017-01-01

    Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chr...

  13. A bioenergetic model for zebrafish Danio rerio (Hamilton)

    Science.gov (United States)

    Chizinski, C.J.; Sharma, Bibek; Pope, K.L.; Patino, R.

    2008-01-01

    A bioenergetics model was developed from observed consumption, respiration and growth rates for zebrafish Danio rerio across a range (18-32?? C) of water temperatures, and evaluated with a 50 day laboratory trial at 28?? C. No significant bias in variable estimates was found during the validation trial; namely, predicted zebrafish mass generally agreed with observed mass. ?? 2008 The Authors.

  14. Primary Spinal OPC Culture System from Adult Zebrafish to Study Oligodendrocyte Differentiation In Vitro

    Directory of Open Access Journals (Sweden)

    Volker Kroehne

    2017-09-01

    Full Text Available Endogenous oligodendrocyte progenitor cells (OPCs are a promising target to improve functional recovery after spinal cord injury (SCI by remyelinating denuded, and therefore vulnerable, axons. Demyelination is the result of a primary insult and secondary injury, leading to conduction blocks and long-term degeneration of the axons, which subsequently can lead to the loss of their neurons. In response to SCI, dormant OPCs can be activated and subsequently start to proliferate and differentiate into mature myelinating oligodendrocytes (OLs. Therefore, researchers strive to control OPC responses, and utilize small molecule screening approaches in order to identify mechanisms of OPC activation, proliferation, migration and differentiation. In zebrafish, OPCs remyelinate axons of the optic tract after lysophosphatidylcholine (LPC-induced demyelination back to full thickness myelin sheaths. In contrast to zebrafish, mammalian OPCs are highly vulnerable to excitotoxic stress, a cause of secondary injury, and remyelination remains insufficient. Generally, injury induced remyelination leads to shorter internodes and thinner myelin sheaths in mammals. In this study, we show that myelin sheaths are lost early after a complete spinal transection injury, but are re-established within 14 days after lesion. We introduce a novel, easy-to-use, inexpensive and highly reproducible OPC culture system based on dormant spinal OPCs from adult zebrafish that enables in vitro analysis. Zebrafish OPCs are robust, can easily be purified with high viability and taken into cell culture. This method enables to examine why zebrafish OPCs remyelinate better than their mammalian counterparts, identify cell intrinsic responses, which could lead to pro-proliferating or pro-differentiating strategies, and to test small molecule approaches. In this methodology paper, we show efficient isolation of OPCs from adult zebrafish spinal cord and describe culture conditions that enable

  15. Network analysis reveals stage-specific changes in zebrafish embryo development using time course whole transcriptome profiling and prior biological knowledge.

    Science.gov (United States)

    Zhang, Yuji

    2015-01-01

    Molecular networks act as the backbone of molecular activities within cells, offering a unique opportunity to better understand the mechanism of diseases. While network data usually constitute only static network maps, integrating them with time course gene expression information can provide clues to the dynamic features of these networks and unravel the mechanistic driver genes characterizing cellular responses. Time course gene expression data allow us to broadly "watch" the dynamics of the system. However, one challenge in the analysis of such data is to establish and characterize the interplay among genes that are altered at different time points in the context of a biological process or functional category. Integrative analysis of these data sources will lead us a more complete understanding of how biological entities (e.g., genes and proteins) coordinately perform their biological functions in biological systems. In this paper, we introduced a novel network-based approach to extract functional knowledge from time-dependent biological processes at a system level using time course mRNA sequencing data in zebrafish embryo development. The proposed method was applied to investigate 1α, 25(OH)2D3-altered mechanisms in zebrafish embryo development. We applied the proposed method to a public zebrafish time course mRNA-Seq dataset, containing two different treatments along four time points. We constructed networks between gene ontology biological process categories, which were enriched in differential expressed genes between consecutive time points and different conditions. The temporal propagation of 1α, 25-Dihydroxyvitamin D3-altered transcriptional changes started from a few genes that were altered initially at earlier stage, to large groups of biological coherent genes at later stages. The most notable biological processes included neuronal and retinal development and generalized stress response. In addition, we also investigated the relationship among

  16. Modeling Myeloid Malignancies Using Zebrafish

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    Kathryn S. Potts

    2017-12-01

    Full Text Available Human myeloid malignancies represent a substantial disease burden to individuals, with significant morbidity and death. The genetic underpinnings of disease formation and progression remain incompletely understood. Large-scale human population studies have identified a high frequency of potential driver mutations in spliceosomal and epigenetic regulators that contribute to malignancies, such as myelodysplastic syndromes (MDS and leukemias. The high conservation of cell types and genes between humans and model organisms permits the investigation of the underlying mechanisms of leukemic development and potential therapeutic testing in genetically pliable pre-clinical systems. Due to the many technical advantages, such as large-scale screening, lineage-tracing studies, tumor transplantation, and high-throughput drug screening approaches, zebrafish is emerging as a model system for myeloid malignancies. In this review, we discuss recent advances in MDS and leukemia using the zebrafish model.

  17. In Vivo Quantitative Study of Sized-Dependent Transport and Toxicity of Single Silver Nanoparticles Using Zebrafish Embryos

    Science.gov (United States)

    Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Desai, Tanvi; Cherukui, Pavan K.; Xu, Xiao-Hong Nancy

    2012-01-01

    Nanomaterials possess distinctive physicochemical properties (e.g., small sizes, high surface area-to-volume ratios) and promise a wide variety of applications, ranging from design of high quality consumer products to effective disease diagnosis and therapy. These properties can lead to toxic effects, potentially hindering advance in nanotechnology. In this study, we have synthesized and characterized purified and stable (non-aggregation) silver nanoparticles (Ag NPs, 41.6±9.1 nm in average diameters), and utilized early-developing (cleavage-stage) zebrafish embryos (critical aquatic and eco- species) as in vivo model organisms to probe diffusion and toxicity of Ag NPs. We found that single Ag NPs (30–72 nm diameters) passively diffused into the embryos through chorionic pores via random Brownian motion and stayed inside the embryos throughout their entire development (120 hours-post-fertilization, hpf). Dose and size dependent toxic effects of the NPs on embryonic development were observed, showing the possibility of tuning biocompatibility and toxicity of the NPs. At lower concentrations of the NPs (≤ 0.02 nM), 75–91% of embryos developed to normal zebrafish. At the higher concentrations of NPs (≥ 0.20 nM), 100% of embryos became dead. At the concentrations in between (0.02–0.2 nM), embryos developed to various deformed zebrafish. Number and sizes of individual Ag NPs embedded in tissues of normal and deformed zebrafish at 120 hpf were quantitatively analyzed, showing deformed zebrafish with higher number of larger NPs than normal zebrafish, and size-dependent nanotoxicity. By comparing with our previous studies of smaller Ag NPs (11.6±3.5 nm), the results further demonstrate striking size-dependent nanotoxicity that, at the same molar concentration, the larger Ag NPs (41.6±9.1 nm) are more toxic than the smaller Ag NPs (11.6±3.5 nm). PMID:22486336

  18. Evolution of the osteoblast: skeletogenesis in gar and zebrafish

    Directory of Open Access Journals (Sweden)

    Eames B Frank

    2012-03-01

    surprising finding that the "chondrogenic" transcription factor sox9 is expressed in developing osteoblasts of both zebrafish and gar can help explain the expression of chondrocyte genes in osteoblasts of ray-finned fish. More broadly, our data suggest that the molecular fingerprint of the osteoblast, which largely is constrained among land animals, was not fixed during early vertebrate evolution.

  19. Evaluating human cancer cell metastasis in zebrafish

    International Nuclear Information System (INIS)

    Teng, Yong; Xie, Xiayang; Walker, Steven; White, David T; Mumm, Jeff S; Cowell, John K

    2013-01-01

    In vivo metastasis assays have traditionally been performed in mice, but the process is inefficient and costly. However, since zebrafish do not develop an adaptive immune system until 14 days post-fertilization, human cancer cells can survive and metastasize when transplanted into zebrafish larvae. Despite isolated reports, there has been no systematic evaluation of the robustness of this system to date. Individual cell lines were stained with CM-Dil and injected into the perivitelline space of 2-day old zebrafish larvae. After 2-4 days fish were imaged using confocal microscopy and the number of metastatic cells was determined using Fiji software. To determine whether zebrafish can faithfully report metastatic potential in human cancer cells, we injected a series of cells with different metastatic potential into the perivitelline space of 2 day old embryos. Using cells from breast, prostate, colon and pancreas we demonstrated that the degree of cell metastasis in fish is proportional to their invasion potential in vitro. Highly metastatic cells such as MDA231, DU145, SW620 and ASPC-1 are seen in the vasculature and throughout the body of the fish after only 24–48 hours. Importantly, cells that are not invasive in vitro such as T47D, LNCaP and HT29 do not metastasize in fish. Inactivation of JAK1/2 in fibrosarcoma cells leads to loss of invasion in vitro and metastasis in vivo, and in zebrafish these cells show limited spread throughout the zebrafish body compared with the highly metastatic parental cells. Further, knockdown of WASF3 in DU145 cells which leads to loss of invasion in vitro and metastasis in vivo also results in suppression of metastasis in zebrafish. In a cancer progression model involving normal MCF10A breast epithelial cells, the degree of invasion/metastasis in vitro and in mice is mirrored in zebrafish. Using a modified version of Fiji software, it is possible to quantify individual metastatic cells in the transparent larvae to correlate with

  20. In Vivo Testing of MicroRNA-Mediated Gene Knockdown in Zebrafish

    Directory of Open Access Journals (Sweden)

    Ivone Un San Leong

    2012-01-01

    Full Text Available The zebrafish (Danio rerio has become an attractive model for human disease modeling as there are a large number of orthologous genes that encode similar proteins to those found in humans. The number of tools available to manipulate the zebrafish genome is limited and many currently used techniques are only effective during early development (such as morpholino-based antisense technology or it is phenotypically driven and does not offer targeted gene knockdown (such as chemical mutagenesis. The use of RNA interference has been met with controversy as off-target effects can make interpreting phenotypic outcomes difficult; however, this has been resolved by creating zebrafish lines that contain stably integrated miRNA constructs that target the desired gene of interest. In this study, we show that a commercially available miRNA vector system with a mouse-derived miRNA backbone is functional in zebrafish and is effective in causing eGFP knockdown in a transient in vivo eGFP sensor assay system. We chose to apply this system to the knockdown of transcripts that are implicated in the human cardiac disorder, Long QT syndrome.

  1. A category approach to predicting the developmental (neuro) toxicity of organotin compounds: The value of the zebrafish (Danio rerio) embryotoxicity test (ZET)

    NARCIS (Netherlands)

    Beker van Woudenberg, A.; Wolterbeek, A.; Brake, L. te; Snel, C.; Menke, A.; Rubingh, C.; Groot, D. de; Kroese, D.

    2013-01-01

    Zebrafish embryos were exposed to different organotin compounds during very early development (<100. h post fertilization). Morphology, histopathology and swimming activity (in a motor activity test) were the endpoints analyzed. DBTC was, by far, the most embryotoxic compound at all time points and

  2. Macrophage–Microbe Interactions: Lessons from the Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Nagisa Yoshida

    2017-12-01

    Full Text Available Macrophages provide front line defense against infections. The study of macrophage–microbe interplay is thus crucial for understanding pathogenesis and infection control. Zebrafish (Danio rerio larvae provide a unique platform to study macrophage–microbe interactions in vivo, from the level of the single cell to the whole organism. Studies using zebrafish allow non-invasive, real-time visualization of macrophage recruitment and phagocytosis. Furthermore, the chemical and genetic tractability of zebrafish has been central to decipher the complex role of macrophages during infection. Here, we discuss the latest developments using zebrafish models of bacterial and fungal infection. We also review novel aspects of macrophage biology revealed by zebrafish, which can potentiate development of new therapeutic strategies for humans.

  3. Generation and characterization of Kctd15 mutations in zebrafish.

    Directory of Open Access Journals (Sweden)

    Alison Heffer

    Full Text Available Potassium channel tetramerization domain containing 15 (Kctd15 was previously found to have a role in early neural crest (NC patterning, specifically delimiting the region where NC markers are expressed via repression of transcription factor AP-2a and inhibition of Wnt signaling. We used transcription activator-like effector nucleases (TALENs to generate null mutations in zebrafish kctd15a and kctd15b paralogs to study the in vivo role of Kctd15. We found that while deletions producing frame-shift mutations in each paralog showed no apparent phenotype, kctd15a/b double mutant zebrafish are smaller in size and show several phenotypes including some affecting the NC, such as expansion of the early NC domain, increased pigmentation, and craniofacial defects. Both melanophore and xanthophore pigment cell numbers and early markers are up-regulated in the double mutants. While we find no embryonic craniofacial defects, adult mutants have a deformed maxillary segment and missing barbels. By confocal imaging of mutant larval brains we found that the torus lateralis (TLa, a region implicated in gustatory networks in other fish, is absent. Ablation of this brain tissue in wild type larvae mimics some aspects of the mutant growth phenotype. Thus kctd15 mutants show deficits in the development of both neural crest derivatives, and specific regions within the central nervous system, leading to a strong reduction in normal growth rates.

  4. FishNet: an online database of zebrafish anatomy

    Directory of Open Access Journals (Sweden)

    Gibson Abigail J

    2007-08-01

    Full Text Available Abstract Background Over the last two decades, zebrafish have been established as a genetically versatile model system for investigating many different aspects of vertebrate developmental biology. With the credentials of zebrafish as a developmental model now well recognized, the emerging new opportunity is the wider application of zebrafish biology to aspects of human disease modelling. This rapidly increasing use of zebrafish as a model for human disease has necessarily generated interest in the anatomy of later developmental phases such as the larval, juvenile, and adult stages, during which many of the key aspects of organ morphogenesis and maturation take place. Anatomical resources and references that encompass these stages are non-existent in zebrafish and there is therefore an urgent need to understand how different organ systems and anatomical structures develop throughout the life of the fish. Results To overcome this deficit we have utilized the technique of optical projection tomography to produce three-dimensional (3D models of larval fish. In order to view and display these models we have created FishNet http://www.fishnet.org.au, an interactive reference of zebrafish anatomy spanning the range of zebrafish development from 24 h until adulthood. Conclusion FishNet contains more than 36 000 images of larval zebrafish, with more than 1 500 of these being annotated. The 3D models can be manipulated on screen or virtually sectioned. This resource represents the first complete embryo to adult atlas for any species in 3D.

  5. Nurturing care: promoting early childhood development.

    Science.gov (United States)

    Britto, Pia R; Lye, Stephen J; Proulx, Kerrie; Yousafzai, Aisha K; Matthews, Stephen G; Vaivada, Tyler; Perez-Escamilla, Rafael; Rao, Nirmala; Ip, Patrick; Fernald, Lia C H; MacMillan, Harriet; Hanson, Mark; Wachs, Theodore D; Yao, Haogen; Yoshikawa, Hirokazu; Cerezo, Adrian; Leckman, James F; Bhutta, Zulfiqar A

    2017-01-07

    The UN Sustainable Development Goals provide a historic opportunity to implement interventions, at scale, to promote early childhood development. Although the evidence base for the importance of early childhood development has grown, the research is distributed across sectors, populations, and settings, with diversity noted in both scope and focus. We provide a comprehensive updated analysis of early childhood development interventions across the five sectors of health, nutrition, education, child protection, and social protection. Our review concludes that to make interventions successful, smart, and sustainable, they need to be implemented as multi-sectoral intervention packages anchored in nurturing care. The recommendations emphasise that intervention packages should be applied at developmentally appropriate times during the life course, target multiple risks, and build on existing delivery platforms for feasibility of scale-up. While interventions will continue to improve with the growth of developmental science, the evidence now strongly suggests that parents, caregivers, and families need to be supported in providing nurturing care and protection in order for young children to achieve their developmental potential. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Immobilization of Dystrophin and Laminin α2-Chain Deficient Zebrafish Larvae In Vivo Prevents the Development of Muscular Dystrophy.

    Directory of Open Access Journals (Sweden)

    Mei Li

    Full Text Available Muscular dystrophies are often caused by genetic alterations in the dystrophin-dystroglycan complex or its extracellular ligands. These structures are associated with the cell membrane and provide mechanical links between the cytoskeleton and the matrix. Mechanical stress is considered a pathological mechanism and muscle immobilization has been shown to be beneficial in some mouse models of muscular dystrophy. The zebrafish enables novel and less complex models to examine the effects of extended immobilization or muscle relaxation in vivo in different dystrophy models. We have examined effects of immobilization in larvae from two zebrafish strains with muscular dystrophy, the Sapje dystrophin-deficient and the Candyfloss laminin α2-chain-deficient strains. Larvae (4 days post fertilization, dpf of both mutants have significantly lower active force in vitro, alterations in the muscle structure with gaps between muscle fibers and altered birefringence patterns compared to their normal siblings. Complete immobilization (18 hrs to 4 dpf was achieved using a small molecular inhibitor of actin-myosin interaction (BTS, 50 μM. This treatment resulted in a significantly weaker active contraction at 4 dpf in both mutated larvae and normal siblings, most likely reflecting a general effect of immobilization on myofibrillogenesis. The immobilization also significantly reduced the structural damage in the mutated strains, showing that muscle activity is an important pathological mechanism. Following one-day washout of BTS, muscle tension partly recovered in the Candyfloss siblings and caused structural damage in these mutants, indicating activity-induced muscle recovery and damage, respectively.

  7. QCD development in the early universe

    Energy Technology Data Exchange (ETDEWEB)

    Gromov, N. A., E-mail: gromov@dm.komisc.ru [Komi Science Center of the Ural Division of the Russian Academy of Sciences, Department of Mathematics (Russian Federation)

    2017-03-15

    The high-energy limit of Quantum Chromodynamics is generated by the contraction of its gauge groups. Contraction parameters are taken identical with those of the Electroweak Model and tend to zero when energy increases. At the infinite energy limit all quarks lose masses and have only one color degree of freedom. The limit model represents the development of Quantum Chromodynamics in the early Universe from the Big Bang up to the end of several milliseconds.

  8. Transient exposure to ethanol during zebrafish embryogenesis results in defects in neuronal differentiation: an alternative model system to study FASD.

    Directory of Open Access Journals (Sweden)

    Xavier Joya

    Full Text Available The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS. In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines.In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification.Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s of ethanol-induced developmental toxicity at very early stages of embryonic development.

  9. Transient Exposure to Ethanol during Zebrafish Embryogenesis Results in Defects in Neuronal Differentiation: An Alternative Model System to Study FASD

    Science.gov (United States)

    Joya, Xavier; Garcia-Algar, Oscar; Vall, Oriol; Pujades, Cristina

    2014-01-01

    Background The exposure of the human embryo to ethanol results in a spectrum of disorders involving multiple organ systems, including the impairment of the development of the central nervous system (CNS). In spite of the importance for human health, the molecular basis of prenatal ethanol exposure remains poorly understood, mainly to the difficulty of sample collection. Zebrafish is now emerging as a powerful organism for the modeling and the study of human diseases. In this work, we have assessed the sensitivity of specific subsets of neurons to ethanol exposure during embryogenesis and we have visualized the sensitive embryonic developmental periods for specific neuronal groups by the use of different transgenic zebrafish lines. Methodology/Principal Findings In order to evaluate the teratogenic effects of acute ethanol exposure, we exposed zebrafish embryos to ethanol in a given time window and analyzed the effects in neurogenesis, neuronal differentiation and brain patterning. Zebrafish larvae exposed to ethanol displayed small eyes and/or a reduction of the body length, phenotypical features similar to the observed in children with prenatal exposure to ethanol. When neuronal populations were analyzed, we observed a clear reduction in the number of differentiated neurons in the spinal cord upon ethanol exposure. There was a decrease in the population of sensory neurons mainly due to a decrease in cell proliferation and subsequent apoptosis during neuronal differentiation, with no effect in motoneuron specification. Conclusion Our investigation highlights that transient exposure to ethanol during early embryonic development affects neuronal differentiation although does not result in defects in early neurogenesis. These results establish the use of zebrafish embryos as an alternative research model to elucidate the molecular mechanism(s) of ethanol-induced developmental toxicity at very early stages of embryonic development. PMID:25383948

  10. Ahr2-dependance of PCB126 effects on the swimbladder in relation to expression of CYP1 and cox-2 genes in developing zebrafish

    Science.gov (United States)

    Jönsson, Maria E.; Kubota, Akira; Timme-Laragy, Alicia; Woodin, Bruce; Stegeman, John J.

    2012-01-01

    The teleost swimbladder is assumed a homolog of the tetrapod lung. Both swimbladder and lung are developmental targets of persistent aryl hydrocarbon receptor (AHR1) agonists; in zebrafish (Danio rerio) the swimbladder fails to inflate with exposure to 3,3’,4,4’,5-pentachlorobiphenyl (PCB126). The mechanism for this effect is unknown, but studies have suggested roles of cytochrome P4501 (CYP1) and cyclooxygenase 2 (Cox-2) in some Ahr-mediated developmental effects in zebrafish. We determined relationships between swimbladder inflation and CYP1 and Cox-2 mRNA expression in PCB126-exposed zebrafish embryos. We also examined effects on β-catenin dependent transcription, histological effects, and Ahr2 dependance of the effect of PCB126 on swimbladder using morpholinos targeting ahr2. One-day-old embryos were exposed to waterborne PCB126 or carrier (DMSO) for 24 h and then held in clean water until day 4, a normal time for swimbladder inflation. The effects of PCB126 were concentration-dependent with EC50 values of 1.4 to 2.0 nM for induction of the CYP1s, 3.7 and 5.1 nM (or higher) for cox-2a and cox-2b induction, and 2.5 nM for inhibition of swimbladder inflation. Histological defects included a compaction of the developing bladder. Ahr2-morpholino treatment rescued the effect of PCB126 (5 nM) on swimbladder inflation and blocked induction of CYP1A, cox-2a, and cox-2b. With 2 nM PCB126 approximately 30% of eleutheroembryos2 failed to inflate the swimbladder, but there was no difference in CYP1 or cox-2 mRNA expression between those embryos and embryos showing inflated swimbladder. Our results indicate that PCB126 blocks swimbladder inflation via an Ahr2-mediated mechanism. This mechanism seems independent of CYP1 or cox-2 mRNA induction but may involve abnormal development of swimbladder cells. PMID:23036320

  11. Examination of a Palatogenic Gene Program in Zebrafish

    Science.gov (United States)

    Swartz, Mary E.; Sheehan-Rooney, Kelly; Dixon, Michael J.; Eberhart, Johann K.

    2011-01-01

    Human palatal clefting is debilitating and difficult to rectify surgically. Animal models enhance our understanding of palatogenesis and are essential in strategies designed to ameliorate palatal malformations in humans. Recent studies have shown that the zebrafish palate, or anterior neurocranium, is under similar genetic control to the amniote palatal skeleton. We extensively analyzed palatogenesis in zebrafish to determine the similarity of gene expression and function across vertebrates. By 36 hpf palatogenic cranial neural crest cells reside in homologous regions of the developing face compared to amniote species. Transcription factors and signaling molecules regulating mouse palatogenesis are expressed in similar domains during palatogenesis in zebrafish. Functional investigation of a subset of these genes, fgf10a, tgfb2, pax9 and smad5 revealed their necessity in zebrafish palatogenesis. Collectively, these results suggest that the gene regulatory networks regulating palatogenesis may be conserved across vertebrate species, demonstrating the utility of zebrafish as a model for palatogenesis. PMID:22016187

  12. Regulation of fertilization and early seed development.

    Science.gov (United States)

    Dresselhaus, Thomas; Doughty, James

    2014-04-01

    Plant reproduction meetings often deal either with pre-fertilization processes such as flowering and pollen biology or post-fertilization processes such as embryogenesis and seed development. The Biochemical Society Focused Meeting entitled 'Regulation of Fertilization and Early Seed Development' was organized to close this gap and to discuss mechanistic similarities and future research directions in the reproductive processes shortly before, during and after fertilization. As an outcome of the workshop, invited speakers and a few selected oral communication presenters contributed focused reviews and technical articles for this issue of Biochemical Society Transactions. We provide here a short overview of the contents and highlights of the various articles.

  13. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    Energy Technology Data Exchange (ETDEWEB)

    Cheng, Xiaoxia [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); University of Chinese Academy of Sciences, Beijing (China); Chen, Xiaowen; Jin, Xia; He, Jiangyan [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Yin, Zhan, E-mail: zyin@ihb.ac.cn [Key Laboratory of Aquatic Biodiversity and Conservation of the Chinese Academy of Sciences, Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei (China); Ningbo Laboratory, State Key Laboratory of Freshwater Ecology and Biotechnology (China)

    2014-07-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO{sub 4}, dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study.

  14. Generation and characterization of gsuα:EGFP transgenic zebrafish for evaluating endocrine-disrupting effects

    International Nuclear Information System (INIS)

    Cheng, Xiaoxia; Chen, Xiaowen; Jin, Xia; He, Jiangyan; Yin, Zhan

    2014-01-01

    The glycoprotein subunit α (gsuα) gene encodes the shared α subunit of the three pituitary heterodimeric glycoprotein hormones: follicle-stimulating hormone β (Fshβ), luteinizing hormone β (Lhβ) and thyroid stimulating hormone β (Tshβ). In our current study, we identified and characterized the promoter region of zebrafish gsuα and generated a stable gsuα:EGFP transgenic line, which recapitulated the endogenous gsuα expression in the early developing pituitary gland. A relatively conserved regulatory element set is presented in the promoter regions of zebrafish and three other known mammalian gsuα promoters. Our results also demonstrated that the expression patterns of the gsuα:EGFP transgene were all identical to those expression patterns of the endogenous gsuα expression in the pituitary tissue when our transgenic fish were treated with various endocrine chemicals, including forskolin (FSK), SP600125, trichostatin A (TSA), KClO 4 , dexamethasone (Dex), β-estradiol and progesterone. Thus, this gsuα:EGFP transgenic fish reporter line provides another valuable tool for investigating the lineage development of gsuα-expressing gonadotrophins and the coordinated regulation of various glycoprotein hormone subunit genes. These reporter fish can serve as a novel platform to perform screenings of endocrine-disrupting chemicals (EDCs) in vivo as well. - Highlights: • Identification of the promoter of zebrafish glycoprotein subunit α (gsuα) gene • Generation of stable transmission gsuα:EGFP transgenic zebrafish reporter • Demonstration of the recapitulation of the gsuα:EGFP and endogenous gsuα expression • Suggestion of the gsuα:EGFP transgenic zebrafish as a novel platform for EDC study

  15. Uncoupling nicotine mediated motoneuron axonal pathfinding errors and muscle degeneration in zebrafish

    International Nuclear Information System (INIS)

    Welsh, Lillian; Tanguay, Robert L.; Svoboda, Kurt R.

    2009-01-01

    Zebrafish embryos offer a unique opportunity to investigate the mechanisms by which nicotine exposure impacts early vertebrate development. Embryos exposed to nicotine become functionally paralyzed by 42 hpf suggesting that the neuromuscular system is compromised in exposed embryos. We previously demonstrated that secondary spinal motoneurons in nicotine-exposed embryos were delayed in development and that their axons made pathfinding errors (Svoboda, K.R., Vijayaraghaven, S., Tanguay, R.L., 2002. Nicotinic receptors mediate changes in spinal motoneuron development and axonal pathfinding in embryonic zebrafish exposed to nicotine. J. Neurosci. 22, 10731-10741). In that study, we did not consider the potential role that altered skeletal muscle development caused by nicotine exposure could play in contributing to the errors in spinal motoneuron axon pathfinding. In this study, we show that an alteration in skeletal muscle development occurs in tandem with alterations in spinal motoneuron development upon exposure to nicotine. The alteration in the muscle involves the binding of nicotine to the muscle-specific AChRs. The nicotine-induced alteration in muscle development does not occur in the zebrafish mutant (sofa potato, [sop]), which lacks muscle-specific AChRs. Even though muscle development is unaffected by nicotine exposure in sop mutants, motoneuron axonal pathfinding errors still occur in these mutants, indicating a direct effect of nicotine exposure on nervous system development.

  16. Development of a convenient in vivo hepatotoxin assay using a transgenic zebrafish line with liver-specific DsRed expression.

    Directory of Open Access Journals (Sweden)

    Xiaoyan Zhang

    Full Text Available Previously we have developed a transgenic zebrafish line (LiPan with liver-specific red fluorescent protein (DsRed expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine] caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics and chlorphenamine (pain killer did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry

  17. Development of a Convenient In Vivo Hepatotoxin Assay Using a Transgenic Zebrafish Line with Liver-Specific DsRed Expression

    Science.gov (United States)

    Zhang, Xiaoyan; Li, Caixia; Gong, Zhiyuan

    2014-01-01

    Previously we have developed a transgenic zebrafish line (LiPan) with liver-specific red fluorescent protein (DsRed) expression under the fabp10a promoter. Since red fluorescence in the liver greatly facilitates the observation of liver in live LiPan fry, we envision that the LiPan zebrafish may provide a useful tool in analyses of hepatotoxicity based on changes of liver red fluorescence intensity and size. In this study, we first tested four well-established hepatotoxins (acetaminophen, aspirin, isoniazid and phenylbutazone) in LiPan fry and demonstrated that these hepatotoxins could significantly reduce both liver red fluorescence and liver size in a dosage-dependent manner, thus the two measurable parameters could be used as indicators of hepatotoxicity. We then tested the LiPan fry with nine other chemicals including environmental toxicants and human drugs. Three (mefenamic acid, lindane, and arsenate) behave like hepatotoxins in reduction of liver red fluorescence, while three others (17β-estradiol, TCDD [2,3,7,8-tetrachlorodibenzo-p-dioxin] and NDMA [N-nitrosodimethylamine]) caused increase of liver red fluorescence and the liver size. Ethanol and two other chemicals, amoxicillin (antibiotics) and chlorphenamine (pain killer) did not resulted in significant changes of liver red fluorescence and liver size. By quantitative RT-PCR analysis, we found that the changes of red fluorescence intensity caused by different chemicals correlated to the changes of endogenous fabp10a RNA expression, indicating that the measured hepatotoxicity was related to fatty acid transportation and metabolism. Finally we tested a mixture of four hepatotoxins and observed a significant reduction of red fluorescence in the liver at concentrations below the lowest effective concentrations of individual hepatotoxins, suggesting that the transgenic zebrafish assay is capable of reporting compound hepatotoxicity effect from chemical mixtures. Thus, the LiPan transgenic fry provide a rapid

  18. Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

    International Nuclear Information System (INIS)

    Lemaire, Benjamin; Kubota, Akira; O'Meara, Conor M.; Lamb, David C.; Tanguay, Robert L.; Goldstone, Jared V.; Stegeman, John J.

    2016-01-01

    Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease. - Highlights: • The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed. • Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity. • Effects of

  19. Cytochrome P450 20A1 in zebrafish: Cloning, regulation and potential involvement in hyperactivity disorders

    Energy Technology Data Exchange (ETDEWEB)

    Lemaire, Benjamin; Kubota, Akira; O' Meara, Conor M. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States); Lamb, David C. [Institute of Life Science, Medical School, Swansea University, Swansea (United Kingdom); Tanguay, Robert L. [Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, OR (United States); Goldstone, Jared V. [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States); Stegeman, John J., E-mail: jstegeman@whoi.edu [Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA (United States)

    2016-04-01

    Cytochrome P450 (CYP) enzymes for which there is no functional information are considered “orphan” CYPs. Previous studies showed that CYP20A1, an orphan, is expressed in human hippocampus and substantia nigra, and in zebrafish (Danio rerio) CYP20A1 maternal transcript occurs in eggs, suggesting involvement in brain and in early development. Moreover, hyperactivity is reported in humans with chromosome 2 microdeletions including CYP20A1. We examined CYP20A1 in zebrafish, including impacts of chemical exposure on expression. Zebrafish CYP20A1 cDNA was cloned, sequenced, and aligned with cloned human CYP20A1 and predicted vertebrate orthologs. CYP20A1s share a highly conserved N-terminal region and unusual sequences in the I-helix and the heme-binding CYP signature motifs. CYP20A1 mRNA expression was observed in adult zebrafish organs including the liver, heart, gonads, spleen and brain, as well as the eye and optic nerve. Putative binding sites in proximal promoter regions of CYP20A1s, and response of zebrafish CYP20A1 to selected nuclear and xenobiotic receptor agonists, point to up-regulation by agents involved in steroid hormone response, cholesterol and lipid metabolism. There also was a dose-dependent reduction of CYP20A1 expression in embryos exposed to environmentally relevant levels of methylmercury. Morpholino knockdown of CYP20A1 in developing zebrafish resulted in behavioral effects, including hyperactivity and a slowing of the optomotor response in larvae. The results suggest that altered expression of CYP20A1 might be part of a mechanism linking methylmercury exposure to neurobehavioral deficits. The expanded information on CYP20A1 brings us closer to “deorphanization”, that is, identifying CYP20A1 functions and its roles in health and disease. - Highlights: • The “orphan” CYP20A1 was cloned from zebrafish and its sequence analyzed. • Knockdown of CYP20A1 reduced an optomotor response and elicited bursts of activity. • Effects of

  20. The myopathy-causing mutation DNM2-S619L leads to defective tubulation in vitro and in developing zebrafish

    Directory of Open Access Journals (Sweden)

    Elizabeth M. Gibbs

    2014-01-01

    Full Text Available DNM2 is a ubiquitously expressed GTPase that regulates multiple subcellular processes. Mutations in DNM2 are a common cause of centronuclear myopathy, a severe disorder characterized by altered skeletal muscle structure and function. The precise mechanisms underlying disease-associated DNM2 mutations are unresolved. We examined the common DNM2-S619L mutation using both in vitro and in vivo approaches. Expression of DNM2-S619L in zebrafish led to the accumulation of aberrant vesicular structures and to defective excitation-contraction coupling. Expression of DNM2-S619L in COS7 cells resulted in defective BIN1-dependent tubule formation. These data suggest that DNM2-S619L causes disease, in part, by interfering with membrane tubulation.

  1. Discovery, characterization and expression of a novel zebrafish gene, znfr, important for notochord formation.

    Science.gov (United States)

    Xu, Yan; Zou, Peng; Liu, Yao; Deng, Fengjiao

    2010-06-01

    Genes specifically expressed in the notochord may be crucial for proper notochord development. Using the digital differential display program offered by the National Center for Biotechnology Information, we identified a novel EST sequence from a zebrafish ovary library (No. XM_701450). The full-length cDNA of this transcript was cloned by performing 3' and 5'-RACE and was further confirmed by PCR and sequencing. The resulting 614 bp gene was found to encode a novel 94 amino acid protein that did not share significant homology with any other known protein. Characterization of the genomic sequence revealed that the gene spanned 4.9 kb and was composed of four exons and three introns. RT-PCR gene expression analysis revealed that our gene of interest was expressed in ovary, kidney, brain, mature oocytes and during the early stages of embryogenesis. During embryonic development, znfr mRNA was found to be expressed in the embryonic shield, chordamesoderm and the vacuolated notochord cells by in situ hybridization. Based on this information, we hypothesize that this novel gene is an important maternal factor required for zebrafish notochord formation during early embryonic development. We have thus named this gene znfr (zebrafish notochord formation related).

  2. Developmental mechanisms of arsenite toxicity in zebrafish (Danio rerio) embryos

    International Nuclear Information System (INIS)

    Li Dan; Lu Cailing; Wang Ju; Hu Wei; Cao Zongfu; Sun Daguang; Xia Hongfei; Ma Xu

    2009-01-01

    Arsenic usually accumulates in soil, water and airborne particles, from which it is taken up by various organisms. Exposure to arsenic through food and drinking water is a major public health problem affecting some countries. At present there are limited laboratory data on the effects of arsenic exposure on early embryonic development and the mechanisms behind its toxicity. In this study, we used zebrafish as a model system to investigate the effects of arsenite on early development. Zebrafish embryos were exposed to a range of sodium arsenite concentrations (0-10.0 mM) between 4 and 120 h post-fertilization (hpf). Survival and early development of the embryos were not obviously influenced by arsenite concentrations below 0.5 mM. However, embryos exposed to higher concentrations (0.5-10.0 mM) displayed reduced survival and abnormal development including delayed hatching, retarded growth and changed morphology. Alterations in neural development included weak tactile responses to light (2.0-5.0 mM, 30 hpf), malformation of the spinal cord and disordered motor axon projections (2.0 mM, 48 hpf). Abnormal cardiac function was observed as bradycardia (0.5-2.0 mM, 60 hpf) and altered ventricular shape (2.0 mM, 48 hpf). Furthermore, altered cell proliferation (2.0 mM, 24 hpf) and apoptosis status (2.0 mM, 24 and 48 hpf), as well as abnormal genomic DNA methylation patterning (2.0 mM, 24 and 48 hpf) were detected in the arsenite-treated embryos. All of these indicate a possible relationship between arsenic exposure and developmental failure in early embryogenesis. Our studies suggest that the negative effects of arsenic on vertebrate embryogenesis are substantial

  3. Tributyltin and Zebrafish: Swimming in Dangerous Water

    Directory of Open Access Journals (Sweden)

    Clemilson Berto-Júnior

    2018-04-01

    Full Text Available Zebrafish has been established as a reliable biological model with important insertion in academy (morphologic, biochemical, and pathophysiological studies and pharmaceutical industry (toxicology and drug development due to its molecular complexity and similar systems biology that recapitulate those from other organisms. Considering the toxicological aspects, many efforts using zebrafish models are being done in order to elucidate the effects of endocrine disruptors, and some of them are focused on tributyltin (TBT and its mechanism of action. TBT is an antifouling agent applied in ship’s hull that is constantly released into the water and absorbed by marine organisms, leading to bioaccumulation and biomagnification effects. Thus, several findings of malformations and changes in the normal biochemical and physiologic aspects of these marine animals have been related to TBT contamination. In the present review, we have compiled the most significant studies related to TBT effects in zebrafish, also taking into consideration the effects found in other study models.

  4. Tributyltin and Zebrafish: Swimming in Dangerous Water

    Science.gov (United States)

    Berto-Júnior, Clemilson; de Carvalho, Denise Pires; Soares, Paula; Miranda-Alves, Leandro

    2018-01-01

    Zebrafish has been established as a reliable biological model with important insertion in academy (morphologic, biochemical, and pathophysiological studies) and pharmaceutical industry (toxicology and drug development) due to its molecular complexity and similar systems biology that recapitulate those from other organisms. Considering the toxicological aspects, many efforts using zebrafish models are being done in order to elucidate the effects of endocrine disruptors, and some of them are focused on tributyltin (TBT) and its mechanism of action. TBT is an antifouling agent applied in ship’s hull that is constantly released into the water and absorbed by marine organisms, leading to bioaccumulation and biomagnification effects. Thus, several findings of malformations and changes in the normal biochemical and physiologic aspects of these marine animals have been related to TBT contamination. In the present review, we have compiled the most significant studies related to TBT effects in zebrafish, also taking into consideration the effects found in other study models. PMID:29692757

  5. Zebrafish Health Conditions in the China Zebrafish Resource Center and 20 Major Chinese Zebrafish Laboratories.

    Science.gov (United States)

    Liu, Liyue; Pan, Luyuan; Li, Kuoyu; Zhang, Yun; Zhu, Zuoyan; Sun, Yonghua

    2016-07-01

    In China, the use of zebrafish as an experimental animal in the past 15 years has widely expanded. The China Zebrafish Resource Center (CZRC), which was established in 2012, is becoming one of the major resource centers in the global zebrafish community. Large-scale use and regular exchange of zebrafish resources have put forward higher requirements on zebrafish health issues in China. This article reports the current aquatic infrastructure design, animal husbandry, and health-monitoring programs in the CZRC. Meanwhile, through a survey of 20 Chinese zebrafish laboratories, we also describe the current health status of major zebrafish facilities in China. We conclude that it is of great importance to establish a widely accepted health standard and health-monitoring strategy in the Chinese zebrafish research community.

  6. A Zebrafish Heart Failure Model for Assessing Therapeutic Agents.

    Science.gov (United States)

    Zhu, Xiao-Yu; Wu, Si-Qi; Guo, Sheng-Ya; Yang, Hua; Xia, Bo; Li, Ping; Li, Chun-Qi

    2018-03-20

    Heart failure is a leading cause of death and the development of effective and safe therapeutic agents for heart failure has been proven challenging. In this study, taking advantage of larval zebrafish, we developed a zebrafish heart failure model for drug screening and efficacy assessment. Zebrafish at 2 dpf (days postfertilization) were treated with verapamil at a concentration of 200 μM for 30 min, which were determined as optimum conditions for model development. Tested drugs were administered into zebrafish either by direct soaking or circulation microinjection. After treatment, zebrafish were randomly selected and subjected to either visual observation and image acquisition or record videos under a Zebralab Blood Flow System. The therapeutic effects of drugs on zebrafish heart failure were quantified by calculating the efficiency of heart dilatation, venous congestion, cardiac output, and blood flow dynamics. All 8 human heart failure therapeutic drugs (LCZ696, digoxin, irbesartan, metoprolol, qiliqiangxin capsule, enalapril, shenmai injection, and hydrochlorothiazide) showed significant preventive and therapeutic effects on zebrafish heart failure (p failure model developed and validated in this study could be used for in vivo heart failure studies and for rapid screening and efficacy assessment of preventive and therapeutic drugs.

  7. Early androgen exposure and human gender development.

    Science.gov (United States)

    Hines, Melissa; Constantinescu, Mihaela; Spencer, Debra

    2015-01-01

    During early development, testosterone plays an important role in sexual differentiation of the mammalian brain and has enduring influences on behavior. Testosterone exerts these influences at times when the testes are active, as evidenced by higher concentrations of testosterone in developing male than in developing female animals. This article critically reviews the available evidence regarding influences of testosterone on human gender-related development. In humans, testosterone is elevated in males from about weeks 8 to 24 of gestation and then again during early postnatal development. Individuals exposed to atypical concentrations of testosterone or other androgenic hormones prenatally, for example, because of genetic conditions or because their mothers were prescribed hormones during pregnancy, have been consistently found to show increased male-typical juvenile play behavior, alterations in sexual orientation and gender identity (the sense of self as male or female), and increased tendencies to engage in physically aggressive behavior. Studies of other behavioral outcomes following dramatic androgen abnormality prenatally are either too small in their numbers or too inconsistent in their results, to provide similarly conclusive evidence. Studies relating normal variability in testosterone prenatally to subsequent gender-related behavior have produced largely inconsistent results or have yet to be independently replicated. For studies of prenatal exposures in typically developing individuals, testosterone has been measured in single samples of maternal blood or amniotic fluid. These techniques may not be sufficiently powerful to consistently detect influences of testosterone on behavior, particularly in the relatively small samples that have generally been studied. The postnatal surge in testosterone in male infants, sometimes called mini-puberty, may provide a more accessible opportunity for measuring early androgen exposure during typical development. This

  8. The developing hypopharyngeal microbiota in early life

    DEFF Research Database (Denmark)

    Mortensen, Martin Steen; Brejnrod, Asker Daniel; Roggenbuck, Michael

    2016-01-01

    BACKGROUND: The airways of healthy humans harbor a distinct microbial community. Perturbations in the microbial community have been associated with disease, yet little is known about the formation and development of a healthy airway microbiota in early life. Our goal was to understand the establi......BACKGROUND: The airways of healthy humans harbor a distinct microbial community. Perturbations in the microbial community have been associated with disease, yet little is known about the formation and development of a healthy airway microbiota in early life. Our goal was to understand...... the establishment of the airway microbiota within the first 3 months of life. We investigated the hypopharyngeal microbiota in the unselected COPSAC2010 cohort of 700 infants, using 16S rRNA gene sequencing of hypopharyngeal aspirates from 1 week, 1 month, and 3 months of age. RESULTS: Our analysis shows...... that majority of the hypopharyngeal microbiota of healthy infants belong to each individual's core microbiota and we demonstrate five distinct community pneumotypes. Four of these pneumotypes are dominated by the genera Staphylococcus, Streptococcus, Moraxella, and Corynebacterium, respectively. Furthermore, we...

  9. Zebrafish: an animal model for research in veterinary medicine.

    Science.gov (United States)

    Nowik, N; Podlasz, P; Jakimiuk, A; Kasica, N; Sienkiewicz, W; Kaleczyc, J

    2015-01-01

    The zebrafish (Danio rerio) has become known as an excellent model organism for studies of vertebrate biology, vertebrate genetics, embryonal development, diseases and drug screening. Nevertheless, there is still lack of detailed reports about usage of the zebrafish as a model in veterinary medicine. Comparing to other vertebrates, they can lay hundreds of eggs at weekly intervals, externally fertilized zebrafish embryos are accessible to observation and manipulation at all stages of their development, which makes possible to simplify the research techniques such as fate mapping, fluorescent tracer time-lapse lineage analysis and single cell transplantation. Although zebrafish are only 2.5 cm long, they are easy to maintain. Intraperitoneal and intracerebroventricular injections, blood sampling and measurement of food intake are possible to be carry out in adult zebrafish. Danio rerio is a useful animal model for neurobiology, developmental biology, drug research, virology, microbiology and genetics. A lot of diseases, for which the zebrafish is a perfect model organism, affect aquatic animals. For a part of them, like those caused by Mycobacterium marinum or Pseudoloma neutrophila, Danio rerio is a natural host, but the zebrafish is also susceptible to the most of fish diseases including Itch, Spring viraemia of carp and Infectious spleen and kidney necrosis. The zebrafish is commonly used in research of bacterial virulence. The zebrafish embryo allows for rapid, non-invasive and real time analysis of bacterial infections in a vertebrate host. Plenty of common pathogens can be examined using zebrafish model: Streptococcus iniae, Vibrio anguillarum or Listeria monocytogenes. The steps are taken to use the zebrafish also in fungal research, especially that dealing with Candida albicans and Cryptococcus neoformans. Although, the zebrafish is used commonly as an animal model to study diseases caused by external agents, it is also useful in studies of metabolic

  10. Development of Life on Early Mars

    Science.gov (United States)

    Gibson, Everett K.; McKay, David S.; Thomas-Keprta, Kathie L.; Clemett, Simon J.; Wentworth, Susan J.

    2009-01-01

    Exploration of Mars has begun to unveil the history of the planet. Combinations of remote sensing, in situ compositional measurements and photographic observations have shown Mars had a dynamic and active geologic evolution. Mars geologic evolution encompassed conditions that were suitable for supporting life. A habitable planet must have water, carbon and energy sources along with a dynamic geologic past. Mars meets all of these requirements. The first 600 My of Martian history were ripe for life to develop because of the abundance of (i) Water- as shown by carved canyons and oceans or lakes with the early presence of near surface water shown by precipitated carbonates in ALH84001, well-dated at 3.9 Gy, (ii) Energy from the original accretional processes, a molten core which generated a strong magnetic field leaving a permanent record in the early crust, active volcanism continuing throughout Martian history, and continuing impact processes, (iii) Carbon, water and a likely thicker atmosphere from extensive volcanic outgassing (i.e. H20, CO2, CH4, CO, O2, N2, H2S, SO2, etc.) and (iv) crustal tectonics as revealed by faulting and possible plate movement reflected by the magnetic pattern in the crust [1]. The question arises: "Why would life not develop from these favorable conditions on Mars in its first 600 My?" During this period, environmental near-surface conditions on Mars were more favorable to life than at any later time. Standing bodies of water, precipitation and flowing surface water, and possibly abundant hydrothermal energy would favor the formation of early life. (Even if life developed elsewhere on Earth, Venus, or on other bodies-it was transported to Mars where surface conditions were suitable for life to evolve). The commonly stated requirement that life would need hundreds of millions of year to get started is only an assumption; we know of no evidence that requires such a long interval for the development of life, if the proper habitable

  11. Culturable gut microbiota diversity in zebrafish.

    Science.gov (United States)

    Cantas, Leon; Sørby, Jan Roger Torp; Aleström, Peter; Sørum, Henning

    2012-03-01

    The zebrafish (Danio rerio) is an increasingly used laboratory animal model in basic biology and biomedicine, novel drug development, and toxicology. The wide use has increased the demand for optimized husbandry protocols to ensure animal health care and welfare. The knowledge about the correlation between culturable zebrafish intestinal microbiota and health in relation to environmental factors and management procedures is very limited. A semi-quantitative level of growth of individual types of bacteria was determined and associated with sampling points. A total of 72 TAB line zebrafish from four laboratories (Labs A-D) in the Zebrafish Network Norway were used. Diagnostic was based on traditional bacterial culture methods and biochemical characterization using commercial kits, followed by 16S rDNA gene sequencing from pure subcultures. Also selected Gram-negative isolates were analyzed for antibiotic susceptibility to 8 different antibiotics. A total of 13 morphologically different bacterial species were the most prevalent: Aeromonas hydrophila, Aeromonas sobria, Vibrio parahaemolyticus, Photobacterium damselae, Pseudomonas aeruginosa, Pseudomonas fluorescens, Pseudomonas luteola, Comamonas testosteroni, Ochrobactrum anthropi, Staphylococcus cohnii, Staphylococcus epidermidis, Staphylococcus capitis, and Staphylococcus warneri. Only Lab B had significantly higher levels of total bacterial growth (OR=2.03), whereas numbers from Lab C (OR=1.01) and Lab D (OR=1.12) were found to be similar to the baseline Lab A. Sexually immature individuals had a significantly higher level of harvested total bacterial growth than mature fish (OR=0.82), no statistically significant differences were found between male and female fish (OR=1.01), and the posterior intestinal segment demonstrated a higher degree of culturable bacteria than the anterior segment (OR=4.1). Multiple antibiotic (>3) resistance was observed in 17% of the strains. We propose that a rapid conventional

  12. How Early Hormones Shape Gender Development

    Science.gov (United States)

    Berenbaum, Sheri A.; Beltz, Adriene M.

    2015-01-01

    Many important psychological characteristics show sex differences, and are influenced by sex hormones at different developmental periods. We focus on the role of sex hormones in early development, particularly the differential effects of prenatal androgens on aspects of gender development. Increasing evidence confirms that prenatal androgens have facilitative effects on male-typed activity interests and engagement (including child toy preferences and adult careers), and spatial abilities, but relatively minimal effects on gender identity. Recent emphasis has been directed to the psychological mechanisms underlying these effects (including sex differences in propulsive movement, and androgen effects on interest in people versus things), and neural substrates of androgen effects (including regional brain volumes, and neural responses to mental rotation, sexually arousing stimuli, emotion, and reward). Ongoing and planned work is focused on understanding the ways in which hormones act jointly with the social environment across time to produce varying trajectories of gender development, and clarifying mechanisms by which androgens affect behaviors. Such work will be facilitated by applying lessons from other species, and by expanding methodology. Understanding hormonal influences on gender development enhances knowledge of psychological development generally, and has important implications for basic and applied questions, including sex differences in psychopathology, women’s underrepresentation in science and math, and clinical care of individuals with variations in gender expression. PMID:26688827

  13. Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

    International Nuclear Information System (INIS)

    Xu, Ting; Zhao, Jing; Hu, Ping; Dong, Zhangji; Li, Jingyun; Zhang, Hongchang; Yin, Daqiang; Zhao, Qingshun

    2014-01-01

    Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

  14. Pentachlorophenol exposure causes Warburg-like effects in zebrafish embryos at gastrulation stage

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Ting; Zhao, Jing [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Hu, Ping [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); State Key Laboratory of Reproductive Medicine, Department of Prenatal Diagnosis, Nanjing Maternity and Child Health Care Hospital Affiliated to Nanjing Medical University, Nanjing 210029 (China); Dong, Zhangji; Li, Jingyun [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China); Zhang, Hongchang [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Yin, Daqiang, E-mail: yindq@tongji.edu.cn [Key Laboratory of Yangtze River Water Environment, Ministry of Education, College of Environmental Science and Technology, Tongji University, Shanghai 200092 (China); Zhao, Qingshun, E-mail: qingshun@nju.edu.cn [Key Laboratory of Model Animal for Disease Study, Ministry of Education, Model Animal Research Center, Nanjing University, Nanjing 210061 (China)

    2014-06-01

    Pentachlorophenol (PCP) is a prevalent pollutant in the environment and has been demonstrated to be a serious toxicant to humans and animals. However, little is known regarding the molecular mechanism underlying its toxic effects on vertebrate early development. To explore the impacts and underlying mechanisms of PCP on early development, zebrafish (Danio rerio) embryos were exposed to PCP at concentrations of 0, 20 and 50 μg/L, and microscopic observation and cDNA microarray analysis were subsequently conducted at gastrulation stage. The morphological observations revealed that PCP caused a developmental delay of zebrafish embryos in a concentration-dependent manner. Transcriptomic data showed that 50 μg/L PCP treatment resulted in significant changes in gene expression level, and the genes involved in energy metabolism and cell behavior were identified based on gene functional enrichment analysis. The energy production of embryos was influenced by PCP via the activation of glycolysis along with the inhibition of oxidative phosphorylation (OXPHOS). The results suggested that PCP acts as an inhibitor of OXPHOS at 8 hpf (hours postfertilization). Consistent with the activated glycolysis, the cell cycle activity of PCP-treated embryos was higher than the controls. These characteristics are similar to the Warburg effect, which occurs in human tumors. The microinjection of exogenous ATP confirmed that an additional energy supply could rescue PCP-treated embryos from the developmental delay due to the energy deficit. Taken together, our results demonstrated that PCP causes a Warburg-like effect on zebrafish embryos during gastrulation, and the affected embryos had the phenotype of developmental delay. - Highlights: • We treat zebrafish embryos with PCP at gastrula stage. • PCP acts as an oxidative phosphorylation inhibitor, not an uncoupler, in gastrulation. • Exogenous ATP injection will rescue the development of effected embryos. • The transcriptome of PCP

  15. Genetic determinants of hyaloid and retinal vasculature in zebrafish

    Directory of Open Access Journals (Sweden)

    Hyde David R

    2007-10-01

    Full Text Available Abstract Background The retinal vasculature is a capillary network of blood vessels that nourishes the inner retina of most mammals. Developmental abnormalities or microvascular complications in the retinal vasculature result in severe human eye diseases that lead to blindness. To exploit the advantages of zebrafish for genetic, developmental and pharmacological studies of retinal vasculature, we characterised the intraocular vasculature in zebrafish. Results We show a detailed morphological and developmental analysis of the retinal blood supply in zebrafish. Similar to the transient hyaloid vasculature in mammalian embryos, vessels are first found attached to the zebrafish lens at 2.5 days post fertilisation. These vessels progressively lose contact with the lens and by 30 days post fertilisation adhere to the inner limiting membrane of the juvenile retina. Ultrastructure analysis shows these vessels to exhibit distinctive hallmarks of mammalian retinal vasculature. For example, smooth muscle actin-expressing pericytes are ensheathed by the basal lamina of the blood vessel, and vesicle vacuolar organelles (VVO, subcellular mediators of vessel-retinal nourishment, are present. Finally, we identify 9 genes with cell membrane, extracellular matrix and unknown identity that are necessary for zebrafish hyaloid and retinal vasculature development. Conclusion Zebrafish have a retinal blood supply with a characteristic developmental and adult morphology. Abnormalities of these intraocular vessels are easily observed, enabling application of genetic and chemical approaches in zebrafish to identify molecular regulators of hyaloid and retinal vasculature in development and disease.

  16. 2017 Midwest Zebrafish Meeting Report.

    Science.gov (United States)

    Sandquist, Elizabeth; Petersen, Sarah C; Smith, Cody J

    2017-12-01

    The 2017 Midwest Zebrafish meeting was held from June 16 to 18 at the University of Cincinnati, sponsored by the Cincinnati Children's Hospital Divisions of Developmental Biology, Molecular Cardiovascular Biology, and Gastroenterology, Hepatology, and Nutrition. The meeting, organized by Saulius Sumanas, Joshua Waxman, and Chunyue Yin, hosted >130 attendees from 16 different states. Scientific sessions were focused on morphogenesis, neural development, novel technologies, and disease models, with Steve Ekker, Stephen Potter, and Lila Solnica-Krezel presenting keynote talks. In this article, we highlight the results and emerging themes from the meeting.

  17. Transcriptome data on maternal RNA of 24 individual zebrafish eggs from five sibling mothers

    Directory of Open Access Journals (Sweden)

    Johanna F.B. Pagano

    2016-09-01

    Full Text Available Maternal mRNA that is present in the mature oocyte plays an important role in the proper development of the early embryo. To elucidate the role of the maternal transcriptome we recently reported a microarray study on individual zebrafish eggs from five different clutches from sibling mothers and showed differences in maternal RNA abundance between and within clutches, “Mother-specific signature in the maternal transcriptome composition of mature, unfertilized Eggs” [1]. Here we provide in detail the applied preprocessing method as well as the R-code to identify expressed and non-expressed genes in the associated transcriptome dataset. Additionally, we provide a website that allows a researcher to search for the expression of their gene of interest in this experiment. Keywords: Zebrafish, Danio rerio, Egg transcriptome, Single egg

  18. Oral exposure of adult zebrafish (Danio rerio) to 2,4,6-tribromophenol affects reproduction

    DEFF Research Database (Denmark)

    Halden, Anna Norman; Nyholm, Jenny Rattfelt; Andersson, Patrik L

    2010-01-01

    The bromophenol 2,4,6-tribromophenol (TBP) is widely used as an industrial chemical, formed by degradation of tetrabromobisphenol-A, and it occurs naturally in marine organisms. Concentrations of TBP in fish have been related to intake via feed, but little is known about effects on fish health...... after oral exposure. In this study, we exposed adult male and female zebrafish (Danio rerio) to TBP via feed in nominal concentrations of 33, 330, and 3300 mu g/g feed (or control feed) for 6 weeks to assess the effects of TBP on reproductive output, gonad morphology, circulatory vitellogenin levels......, and early embryo development. The aim was also to investigate the extent to which TBP was metabolised to 2,4,6-tribromoanisole (IBA) in dietary exposed zebrafish, and the amounts of TBP and TBA found in offspring. After 6 weeks of exposure, we found about 3% of the daily dose of TBP in adult fish...

  19. Early development of grateloupia turuturu (Halymeniaceae, Rhodophyta)

    Science.gov (United States)

    Wang, Gaoge; Jiang, Chunmei; Wang, Shasha; Wei, Xiaojiao; Zhao, Fengjuan

    2012-03-01

    Grateloupia turuturu is a commercial red alga with potential value in nutraceuticals and pharmaceuticals. To supplement information on its life history and verify whether carpospores can be used for seedling culture, early development of G. turuturu was investigated under culture conditions (27°C, 10-13 μol/(m2·s) in irradiance, photoperiod 10:14 h L:D). Three physiological stages were recognized by continuous microscopic observation: division stage, discoid crust stage, and juvenile seedling stage. At the beginning of the division stage, the carpospores developed germ tubes into which the carpospore protoplasm was evacuated, and then the carpospore protoplasm in the germ tubes began to divide continuously until discoid crusts formed. Finally, upright thalli appeared on the discoid crusts and developed into juvenile seedlings. It took about 60 days for carpospores to develop into juvenile seedlings. The growth parameters, including germination rate for carpospores and discoid crust diameter, were recorded. These results contribute more information on the life cycle, and at the same time are of great significance in the scaling-up of artificial seedling cultures of G. turuturu.

  20. Cardiac Myocyte Diversity and a Fibroblast Network in the Junctional Region of the Zebrafish Heart Revealed by Transmission and Serial Block-Face Scanning Electron Microscopy

    KAUST Repository

    Lafontant, Pascal J.; Behzad, Ali Reza; Brown, Evelyn; Landry, Paul; Hu, Norman; Burns, Alan R.

    2013-01-01

    The zebrafish has emerged as an important model of heart development and regeneration. While the structural characteristics of the developing and adult zebrafish ventricle have been previously studied, little attention has been paid to the nature

  1. Comparative Developmental Toxicity of Flavonoids Using an Integrative Zebrafish System.

    Science.gov (United States)

    Bugel, Sean M; Bonventre, Josephine A; Tanguay, Robert L

    2016-11-01

    Flavonoids are a large, structurally diverse class of bioactive naturally occurring chemicals commonly detected in breast milk, soy based infant formulas, amniotic fluid, and fetal cord blood. The potential for pervasive early life stage exposures raises concerns for perturbation of embryogenesis, though developmental toxicity and bioactivity information is limited for many flavonoids. Therefore, we evaluated a suite of 24 flavonoid and flavonoid-like chemicals using a zebrafish embryo-larval toxicity bioassay-an alternative model for investigating developmental toxicity of environmentally relevant chemicals. Embryos were exposed to 1-50 µM of each chemical from 6 to 120 h postfertilization (hpf), and assessed for 26 adverse developmental endpoints at 24, 72, and 120 hpf. Behavioral changes were evaluated in morphologically normal animals at 24 and 72 hpf, at 120 hpf using a larval photomotor response (LPR) assay. Gene expression was comparatively evaluated for all compounds for effects on biomarker transcripts indicative of AHR (cyp1a) and ER (cyp19a1b, esr1, lhb, vtg) pathway bioactivity. Overall, 15 of 24 flavonoids elicited adverse effects on one or more of the developmental or behavioral endpoints. Hierarchical clustering and principle component analyses compared toxicity profiles and identified 3 distinct groups of bioactive flavonoids. Despite robust induction of multiple estrogen-responsive biomarkers, co-exposure with ER and GPER antagonists did not ameliorate toxicity, suggesting ER-independence and alternative modes of action. Taken together, these studies demonstrate that development is sensitive to perturbation by bioactive flavonoids in zebrafish that are not related to traditional estrogen receptor mode of action pathways. This integrative zebrafish platform provides a useful framework for evaluating flavonoid developmental toxicity and hazard prioritization. © The Author 2016. Published by Oxford University Press on behalf of the Society of

  2. Study of Early Child Care and Youth Development (SECCYD)

    Science.gov (United States)

    ... Facebook Twitter Pinterest Email Print NICHD Study of Early Child Care and Youth Development (SECCYD) Sunsetted/For Reference ... page is not being updated . The Study of Early Child Care and Youth Development (SECCYD) began as the ...

  3. Alcohol exposure leads to unrecoverable cardiovascular defects along with edema and motor function changes in developing zebrafish larvae

    Directory of Open Access Journals (Sweden)

    Xu Li

    2016-08-01

    Full Text Available Maternal alcohol consumption during pregnancy can cause a series of developmental disorders in the fetus called FAS (fetal alcohol syndrome. In the present study we exposed zebrafish embryos to 1% and 2% alcohol and observed the morphology of heart and blood vessels during and after exposure to investigate motor function alterations, and damage and recovery to the cardiovascular system. The results showed that alcohol exposure could induce heart deformation, slower heart rate, and incomplete blood vessels and pericardium. After stopping exposure, larvae exposed to 1% alcohol could recover only in heart morphology, but larvae in 2% alcohol could not recover either morphology or function of cardiovascular system. The edema-like characteristics in the 2% alcohol group became more conspicuous afterwards, with destruction in the dorsal aorta, coarctation in segmental arteries and a decrease in motor function, implying more serious unrecoverable cardiovascular defects in the 2% group. The damaged blood vessels in the 2% alcohol group resulted in an alteration in permeability and a decrease of blood volume, which were the causes of edema in pathology. These findings contribute towards a better understanding of ethanol-induced cardiovascular abnormalities and co-syndrome in patients with FAS, and warns against excessive maternal alcohol consumption during pregnancy.

  4. Development of a Patient-Derived Xenograft (PDX of Breast Cancer Bone Metastasis in a Zebrafish Model

    Directory of Open Access Journals (Sweden)

    Laura Mercatali

    2016-08-01

    Full Text Available Bone metastasis is a complex process that needs to be better understood in order to help clinicians prevent and treat it. Xenografts using patient-derived material (PDX rather than cancer cell lines are a novel approach that guarantees more clinically realistic results. A primary culture of bone metastasis derived from a 67-year-old patient with breast cancer was cultured and then injected into zebrafish (ZF embryos to study its metastatic potential. In vivo behavior and results of gene expression analyses of the primary culture were compared with those of cancer cell lines with different metastatic potential (MCF7 and MDA-MB-231. The MCF7 cell line, which has the same hormonal receptor status as the bone metastasis primary culture, did not survive in the in vivo model. Conversely, MDA-MB-231 disseminated and colonized different parts of the ZF, including caudal hematopoietic tissues (CHT, revealing a migratory phenotype. Primary culture cells disseminated and in later stages extravasated from the vessels, engrafting into ZF tissues and reaching the CHT. Primary cell behavior reflected the clinical course of the patient’s medical history. Our results underline the potential for using PDX models in bone metastasis research and outline new methods for the clinical application of this in vivo model.

  5. Solute Carrier Family 26 Member a2 (slc26a2 Regulates Otic Development and Hair Cell Survival in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Fei Liu

    Full Text Available Hearing loss is one of the most prevalent human birth defects. Genetic factors contribute to the pathogenesis of deafness. It is estimated that one-third of deafness genes have already been identified. The current work is an attempt to find novel genes relevant to hearing loss using guilt-by-profiling and guilt-by-association bioinformatics analyses of approximately 80 known non-syndromic hereditary hearing loss (NSHL genes. Among the 300 newly identified candidate deafness genes, slc26a2 were selected for functional studies in zebrafish. The slc26a2 gene was knocked down using an antisense morpholino (MO, and significant defects were observed in otolith patterns, semicircular canal morphology, and lateral neuromast distributions in morphants. Loss-of-function defects are caused primarily by apoptosis, and morphants are insensitive to sound stimulation and imbalanced swimming behaviours. Morphant defects were found to be partially rescued by co-injection of human SLC26A2 mRNA. All the results suggest that bioinformatics is capable of predicting new deafness genes and this showed slc26a2 is to be a critical otic gene whose dysfunction may induce hearing impairment.

  6. Zebrafish: A Versatile Animal Model for Fertility Research

    Directory of Open Access Journals (Sweden)

    Jing Ying Hoo

    2016-01-01

    Full Text Available The utilization of zebrafish in biomedical research is very common in the research world nowadays. Today, it has emerged as a favored vertebrate organism for the research in science of reproduction. There is a significant growth in amount numbers of scientific literature pertaining to research discoveries in reproductive sciences in zebrafish. It has implied the importance of zebrafish in this particular field of research. In essence, the current available literature has covered from the very specific brain region or neurons of zebrafish, which are responsible for reproductive regulation, until the gonadal level of the animal. The discoveries and findings have proven that this small animal is sharing a very close/similar reproductive system with mammals. More interestingly, the behavioral characteristics and along with the establishment of animal courtship behavior categorization in zebrafish have laid an even stronger foundation and firmer reason on the suitability of zebrafish utilization in research of reproductive sciences. In view of the immense importance of this small animal for the development of reproductive sciences, this review aimed at compiling and describing the proximate close similarity of reproductive regulation on zebrafish and human along with factors contributing to the infertility, showing its versatility and its potential usage for fertility research.

  7. The mechanism for primordial germ-cell migration is conserved between Japanese eel and zebrafish.

    Directory of Open Access Journals (Sweden)

    Taiju Saito

    Full Text Available Primordial germ cells (PGCs are segregated and specified from somatic cells during early development. These cells arise elsewhere and have to migrate across the embryo to reach developing gonadal precursors. Several molecules associated with PGC migration (i.e. dead-end, nanos1, and cxcr4 are highly conserved across phylum boundaries. However, since cell migration is a complicated process that is regulated spatially and temporally by multiple adaptors and signal effectors, the process is unlikely to be explained by these known genes only. Indeed, it has been shown that there are variations in PGC migration pattern during development among teleost species. However, it is still unclear whether the actual mechanism of PGC migration is conserved among species. In this study, we studied the migration of PGCs in Japanese eel (Anguilla japonica embryos and tested the migration mechanism between Japanese eel and zebrafish (Danio rerio for conservation, by transplanting eel PGCs into zebrafish embryos. The experiments showed that eel PGCs can migrate toward the gonadal region of zebrafish embryos along with endogenous PGCs, even though the migration patterns, behaviors, and settlements of PGCs are somewhat different between these species. Our results demonstrate that the migration mechanism of PGCs during embryonic development is highly conserved between these two distantly related species (belonging to different teleost orders.

  8. Lactobacillus rhamnosus accelerates zebrafish backbone calcification and gonadal differentiation through effects on the GnRH and IGF systems.

    Directory of Open Access Journals (Sweden)

    Matteo A Avella

    Full Text Available Endogenous microbiota play essential roles in the host's immune system, physiology, reproduction and nutrient metabolism. We hypothesized that a continuous administration of an exogenous probiotic might also influence the host's development. Thus, we treated zebrafish from birth to sexual maturation (2-months treatment with Lactobacillus rhamnosus, a probiotic species intended for human use. We monitored for the presence of L. rhamnosus during the entire treatment. Zebrafish at 6 days post fertilization (dpf exhibited elevated gene expression levels for Insulin-like growth factors -I and -II, Peroxisome proliferator activated receptors -α and -β, VDR-α and RAR-γ when compared to untreated-10 days old zebrafish. Using a gonadotropin-releasing hormone 3 GFP transgenic zebrafish (GnRH3-GFP, higher GnRH3 expression was found at 6, 8 and 10 dpf upon L. rhamnosus treatment. The same larvae exhibited earlier backbone calcification and gonad maturation. Noteworthy in the gonad development was the presence of first testes differentiation at 3 weeks post fertilization in the treated zebrafish population -which normally occurs at 8 weeks- and a dramatic sex ratio modulation (93% females, 7% males in control vs. 55% females, 45% males in the treated group. We infer that administration of L. rhamnosus stimulated the IGF system, leading to a faster backbone calcification. Moreover we hypothesize a role for administration of L. rhamnosus on GnRH3 modulation during early larval development, which in turn affects gonadal development and sex differentiation. These findings suggest a significant role of the microbiota composition on the host organism development profile and open new perspectives in the study of probiotics usage and application.

  9. Early Vascular Ageing - A Concept in Development.

    Science.gov (United States)

    M Nilsson, Peter

    2015-04-01

    Cardiovascular disease (CVD) is a prevalent condition in the elderly, often associated with metabolic disturbance and type 2 diabetes. For a number of years, research dedicated to understand atherosclerosis dominated, and for many good reasons, this pathophysiological process being proximal to the CVD events. In recent years, research has been devoted to an earlier stage of vascular pathology named arteriosclerosis (arterial stiffness) and the new concept of early vascular ageing (EVA), developed by a group of mostly European researchers. This overview describes recent developments in research dedicated to EVA and new emerging aspects found in studies of families at high cardiovascular risk. There are new aspects related to genetics, telomere biology and the role of gut microbiota. However, there is still no unifying definition available of EVA and no direct treatment, but rather only recommendations for conventional cardiovascular risk factor control. New interventions are being developed - not only new antihypertensive drugs, but also new drugs for vascular protection - the selective angiotensin-II (AT2) agonist Compound 21 (C21). Human studies are eagerly awaited. Even new functional food products could have the potential to positively influence cardiometabolic regulation, to be confirmed.

  10. Towards a comprehensive catalog of zebrafish behavior 1.0 and beyond.

    Science.gov (United States)

    Kalueff, Allan V; Gebhardt, Michael; Stewart, Adam Michael; Cachat, Jonathan M; Brimmer, Mallorie; Chawla, Jonathan S; Craddock, Cassandra; Kyzar, Evan J; Roth, Andrew; Landsman, Samuel; Gaikwad, Siddharth; Robinson, Kyle; Baatrup, Erik; Tierney, Keith; Shamchuk, Angela; Norton, William; Miller, Noam; Nicolson, Teresa; Braubach, Oliver; Gilman, Charles P; Pittman, Julian; Rosemberg, Denis B; Gerlai, Robert; Echevarria, David; Lamb, Elisabeth; Neuhauss, Stephan C F; Weng, Wei; Bally-Cuif, Laure; Schneider, Henning

    2013-03-01

    Zebrafish (Danio rerio) are rapidly gaining popularity in translational neuroscience and behavioral research. Physiological similarity to mammals, ease of genetic manipulations, sensitivity to pharmacological and genetic factors, robust behavior, low cost, and potential for high-throughput screening contribute to the growing utility of zebrafish models in this field. Understanding zebrafish behavioral phenotypes provides important insights into neural pathways, physiological biomarkers, and genetic underpinnings of normal and pathological brain function. Novel zebrafish paradigms continue to appear with an encouraging pace, thus necessitating a consistent terminology and improved understanding of the behavioral repertoire. What can zebrafish 'do', and how does their altered brain function translate into behavioral actions? To help address these questions, we have developed a detailed catalog of zebrafish behaviors (Zebrafish Behavior Catalog, ZBC) that covers both larval and adult models. Representing a beginning of creating a more comprehensive ethogram of zebrafish behavior, this effort will improve interpretation of published findings, foster cross-species behavioral modeling, and encourage new groups to apply zebrafish neurobehavioral paradigms in their research. In addition, this glossary creates a framework for developing a zebrafish neurobehavioral ontology, ultimately to become part of a unified animal neurobehavioral ontology, which collectively will contribute to better integration of biological data within and across species.

  11. Molecular and Chemical Genetic Approaches to Developmental Origins of Aging and Disease in Zebrafish

    Science.gov (United States)

    Sasaki, Tomoyuki; Kishi, Shuji

    2013-01-01

    The incidence of diseases increases rapidly with age, accompanied by progressive deteriorations of physiological functions in organisms. Aging-associated diseases are sporadic but mostly inevitable complications arising from senescence. Senescence is often considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena over the dynamic process of aging. The association between early development and late-onset disease with advancing age is thought to come from a consequence of developmental plasticity, the phenomenon by which one genotype can give rise to a range of physiologically and/or morphologically adaptive states in response to different environmental or genetic perturbations. On the one hand, we hypothesized that the future aging process can be predictive based on adaptivity during the early developmental period. Modulating the thresholds of adaptive plasticity by chemical genetic approaches, we have been investigating whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We have successfully conducted experiments to isolate zebrafish mutants expressing apparently altered senescence phenotypes during embryogenesis (“embryonic senescence”), subsequently showing shortened lifespan in adulthoods. We anticipate that previously uncharacterized developmental genes may mediate the aging process and play a pivotal role in senescence. On the other hand, unexpected senescence-related genes might also be involved in the early developmental process and regulation. The ease of manipulation using the zebrafish system allows us to conduct an exhaustive exploration of novel genes and small molecular compounds that can be linked to the senescence phenotype, and thereby facilitates searching for the evolutionary and developmental origins

  12. Two-Photon-Based Photoactivation in Live Zebrafish Embryos

    OpenAIRE

    Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

    2010-01-01

    Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a...

  13. Zebrafish in Toxicology and Environmental Health.

    Science.gov (United States)

    Bambino, Kathryn; Chu, Jaime

    2017-01-01

    As manufacturing processes and development of new synthetic compounds increase to keep pace with the expanding global demand, environmental health, and the effects of toxicant exposure are emerging as critical public health concerns. Additionally, chemicals that naturally occur in the environment, such as metals, have profound effects on human and animal health. Many of these compounds are in the news: lead, arsenic, and endocrine disruptors such as bisphenol A have all been widely publicized as causing disease or damage to humans and wildlife in recent years. Despite the widespread appreciation that environmental toxins can be harmful, there is limited understanding of how many toxins cause disease. Zebrafish are at the forefront of toxicology research; this system has been widely used as a tool to detect toxins in water samples and to investigate the mechanisms of action of environmental toxins and their related diseases. The benefits of zebrafish for studying vertebrate development are equally useful for studying teratogens. Here, we review how zebrafish are being used both to detect the presence of some toxins as well as to identify how environmental exposures affect human health and disease. We focus on areas where zebrafish have been most effectively used in ecotoxicology and in environmental health, including investigation of exposures to endocrine disruptors, industrial waste byproducts, and arsenic. © 2017 Elsevier Inc. All rights reserved.

  14. The development of storytelling in early childhood

    Directory of Open Access Journals (Sweden)

    Ljubica Marjanovič Umek

    2011-01-01

    Full Text Available Storytelling is an important aspect of child's language competence, which largely depends on her/his understanding and expression of a decontextualised content and develops rapidly in the period between the second and sixth year of life. The purpose of this study was to examine age differences in children's storytelling in the period between the third and sixth year of age. In addition, we considered the effect of gender on storytelling of children of different ages. The sample included 156 children aged from 3 to 6 years, who were divided into 3 age groups, namely children, aged 3, 4 and 5 years. Child's storytelling competence was assessed with the Little Glove Storytelling Test. Children's stories told by a standard set of illustrations, were analyzed in terms of criteria, designed to assess the developmental level of the stories. The criteria refer to the words, included in the story, the grammatical structure and the content of the story. The obtained results suggested that several important changes in the development of storytelling occur within the period of early childhood. The 5-years-old children told longer stories with a more complex grammatical structure and a coherent content as the 3-years-old children. Children's achievements on the individual criteria for assessing the developmental level of the stories progressed relatively steadily through all three age groups. The results also showed that gender had no significant effect on the storytelling of children of different ages.

  15. Nutrition and brain development in early life.

    Science.gov (United States)

    Prado, Elizabeth L; Dewey, Kathryn G

    2014-04-01

    Presented here is an overview of the pathway from early nutrient deficiency to long-term brain function, cognition, and productivity, focusing on research from low- and middle-income countries. Animal models have demonstrated the importance of adequate nutrition for the neurodevelopmental processes that occur rapidly during pregnancy and infancy, such as neuron proliferation and myelination. However, several factors influence whether nutrient deficiencies during this period cause permanent cognitive deficits in human populations, including the child's interaction with the environment, the timing and degree of nutrient deficiency, and the possibility of recovery. These factors should be taken into account in the design and interpretation of future research. Certain types of nutritional deficiency clearly impair brain development, including severe acute malnutrition, chronic undernutrition, iron deficiency, and iodine deficiency. While strategies such as salt iodization and micronutrient powders have been shown to improve these conditions, direct evidence of their impact on brain development is scarce. Other strategies also require further research, including supplementation with iron and other micronutrients, essential fatty acids, and fortified food supplements during pregnancy and infancy. © 2014 International Life Sciences Institute.

  16. Molecular cloning and expression analysis of a zebrafish novel zinc finger protein gene rnf141

    Directory of Open Access Journals (Sweden)

    Wenqian Deng

    2009-01-01

    Full Text Available ZNF230 is a novel zinc finger gene cloned by our laboratory. In order to understand the potential functions of this gene in vertebrate development, we cloned the zebrafish orthologue of human ZNF230, named rnf141. The cDNA fragment of rnf141 was obtained by rapid amplification of cDNA ends (RACE. The open reading frame (ORF encodes a polypeptide of 222 amino acids which shares 75.65% identity with the human ZNF230. RT-PCR analysis in zebrafish embryo and adult tissues revealed that rnf141 transcripts are maternally derived and that rnf141 mRNA has a broad distribution. Zygotic rnf141 message is strongly localized in the central nervous system, as shown by whole-mount in situ hybridization. Knockdown and over expression of rnf141 can induce abnormal phenotypes, including abnormal development of brain, as well as yolk sac and axis extendsion. Marker gene analysis showed that rnf141 may play a role in normal dorsoventral patterning of zebrafish embryos, suggesting that rnf141 may have a broad function during early development of vertebrates.

  17. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia

    Directory of Open Access Journals (Sweden)

    Chao Liu

    2015-08-01

    Full Text Available Apolipoprotein C-II (APOC2 is an obligatory activator of lipoprotein lipase. Human patients with APOC2 deficiency display severe hypertriglyceridemia while consuming a normal diet, often manifesting xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia is also an important risk factor for development of cardiovascular disease. Animal models to study hypertriglyceridemia are limited, with no Apoc2-knockout mouse reported. To develop a genetic model of hypertriglyceridemia, we generated an apoc2 mutant zebrafish characterized by the loss of Apoc2 function. apoc2 mutants show decreased plasma lipase activity and display chylomicronemia and severe hypertriglyceridemia, which closely resemble the phenotype observed in human patients with APOC2 deficiency. The hypertriglyceridemia in apoc2 mutants is rescued by injection of plasma from wild-type zebrafish or by injection of a human APOC2 mimetic peptide. Consistent with a previous report of a transient apoc2 knockdown, apoc2 mutant larvae have a minor delay in yolk consumption and angiogenesis. Furthermore, apoc2 mutants fed a normal diet accumulate lipid and lipid-laden macrophages in the vasculature, which resemble early events in the development of human atherosclerotic lesions. In addition, apoc2 mutant embryos show ectopic overgrowth of pancreas. Taken together, our data suggest that the apoc2 mutant zebrafish is a robust and versatile animal model to study hypertriglyceridemia and the mechanisms involved in the pathogenesis of associated human diseases.

  18. Dynamic nucleosome organization at hox promoters during zebrafish embryogenesis.

    Directory of Open Access Journals (Sweden)

    Steven E Weicksel

    Full Text Available Nucleosome organization at promoter regions plays an important role in regulating gene activity. Genome-wide studies in yeast, flies, worms, mammalian embryonic stem cells and transformed cell lines have found well-positioned nucleosomes flanking a nucleosome depleted region (NDR at transcription start sites. This nucleosome arrangement depends on DNA sequence (cis-elements as well as DNA binding factors and ATP-dependent chromatin modifiers (trans-factors. However, little is understood about how the nascent embryonic genome positions nucleosomes during development. This is particularly intriguing since the embryonic genome must undergo a broad reprogramming event upon fusion of sperm and oocyte. Using four stages of early embryonic zebrafish development, we map nucleosome positions at the promoter region of 37 zebrafish hox genes. We find that nucleosome arrangement at the hox promoters is a progressive process that takes place over several stages. At stages immediately after fertilization, nucleosomes appear to be largely disordered at hox promoter regions. At stages after activation of the embryonic genome, nucleosomes are detectable at hox promoters, with positions becoming more uniform and more highly occupied. Since the genomic sequence is invariant during embryogenesis, this progressive change in nucleosome arrangement suggests that trans-factors play an important role in organizing nucleosomes during embryogenesis. Separating hox genes into expressed and non-expressed groups shows that expressed promoters have better positioned and occupied nucleosomes, as well as distinct NDRs, than non-expressed promoters. Finally, by blocking the retinoic acid-signaling pathway, we disrupt early hox gene transcription, but observe no effect on nucleosome positions, suggesting that active hox transcription is not a driving force behind the arrangement of nucleosomes at the promoters of hox genes during early development.

  19. The role of Nrf1 and Nrf2 in the regulation of glutathione and redox dynamics in the developing zebrafish embryo

    Directory of Open Access Journals (Sweden)

    Karilyn E. Sant

    2017-10-01

    Full Text Available Redox signaling is important for embryogenesis, guiding pathways that govern processes crucial for embryo patterning, including cell polarization, proliferation, and apoptosis. Exposure to pro-oxidants during this period can be deleterious, resulting in altered physiology, teratogenesis, later-life diseases, or lethality. We previously reported that the glutathione antioxidant defense system becomes increasingly robust, including a doubling of total glutathione and dynamic shifts in the glutathione redox potential at specific stages during embryonic development in the zebrafish, Danio rerio. However, the mechanisms underlying these changes are unclear, as is the effectiveness of the glutathione system in ameliorating oxidative insults to the embryo at different stages. Here, we examine how the glutathione system responds to the model pro-oxidants tert-butylhydroperoxide and tert-butylhydroquinone at different developmental stages, and the role of Nuclear factor erythroid 2-related factor (Nrf proteins in regulating developmental glutathione redox status. Embryos became increasingly sensitive to pro-oxidants after 72 h post-fertilization (hpf, after which the duration of the recovery period for the glutathione redox potential was increased. To determine whether the doubling of glutathione or the dynamic changes in glutathione redox potential are mediated by zebrafish paralogs of Nrf transcription factors, morpholino oligonucleotides were used to knock down translation of Nrf1 and Nrf2 (nrf1a, nrf1b, nrf2a, nrf2b. Knockdown of Nrf1a or Nrf1b perturbed glutathione redox state until 72 hpf. Knockdown of Nrf2 paralogs also perturbed glutathione redox state but did not significantly affect the response of glutathione to pro-oxidants. Nrf1b morphants had decreased gene expression of glutathione synthesis enzymes, while hsp70 increased in Nrf2b morphants. This work demonstrates that despite having a more robust glutathione system, embryos become more

  20. Dynamic assembly of brambleberry mediates nuclear envelope fusion during early development.

    Science.gov (United States)

    Abrams, Elliott W; Zhang, Hong; Marlow, Florence L; Kapp, Lee; Lu, Sumei; Mullins, Mary C

    2012-08-03

    To accommodate the large cells following zygote formation, early blastomeres employ modified cell divisions. Karyomeres are one such modification, mitotic intermediates wherein individual chromatin masses are surrounded by nuclear envelope; the karyomeres then fuse to form a single mononucleus. We identified brambleberry, a maternal-effect zebrafish mutant that disrupts karyomere fusion, resulting in formation of multiple micronuclei. As karyomeres form, Brambleberry protein localizes to the nuclear envelope, with prominent puncta evident near karyomere-karyomere interfaces corresponding to membrane fusion sites. brambleberry corresponds to an unannotated gene with similarity to Kar5p, a protein that participates in nuclear fusion in yeast. We also demonstrate that Brambleberry is required for pronuclear fusion following fertilization in zebrafish. Our studies provide insight into the machinery required for karyomere fusion and suggest that specialized proteins are necessary for proper nuclear division in large dividing blastomeres. Copyright © 2012 Elsevier Inc. All rights reserved.

  1. Zebrafish eda and edar mutants reveal conserved and ancestral roles of ectodysplasin signaling in vertebrates.

    Directory of Open Access Journals (Sweden)

    Matthew P Harris

    2008-10-01

    Full Text Available The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda and ectodysplasin receptor (edar genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100 that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution.

  2. Zebrafish eda and edar Mutants Reveal Conserved and Ancestral Roles of Ectodysplasin Signaling in Vertebrates

    Science.gov (United States)

    Harris, Matthew P.; Rohner, Nicolas; Schwarz, Heinz; Perathoner, Simon; Konstantinidis, Peter; Nüsslein-Volhard, Christiane

    2008-01-01

    The genetic basis of the development and variation of adult form of vertebrates is not well understood. To address this problem, we performed a mutant screen to identify genes essential for the formation of adult skeletal structures of the zebrafish. Here, we describe the phenotypic and molecular characterization of a set of mutants showing loss of adult structures of the dermal skeleton, such as the rays of the fins and the scales, as well as the pharyngeal teeth. The mutations represent adult-viable, loss of function alleles in the ectodysplasin (eda) and ectodysplasin receptor (edar) genes. These genes are frequently mutated in the human hereditary disease hypohidrotic ectodermal dysplasia (HED; OMIM 224900, 305100) that affects the development of integumentary appendages such as hair and teeth. We find mutations in zebrafish edar that affect similar residues as mutated in human cases of HED and show similar phenotypic consequences. eda and edar are not required for early zebrafish development, but are rather specific for the development of adult skeletal and dental structures. We find that the defects of the fins and scales are due to the role of Eda signaling in organizing epidermal cells into discrete signaling centers of the scale epidermal placode and fin fold. Our genetic analysis demonstrates dose-sensitive and organ-specific response to alteration in levels of Eda signaling. In addition, we show substantial buffering of the effect of loss of edar function in different genetic backgrounds, suggesting canalization of this developmental system. We uncover a previously unknown role of Eda signaling in teleosts and show conservation of the developmental mechanisms involved in the formation and variation of both integumentary appendages and limbs. Lastly, our findings point to the utility of adult genetic screens in the zebrafish in identifying essential developmental processes involved in human disease and in morphological evolution. PMID:18833299

  3. 8-Oxoguanine DNA glycosylase 1 (ogg1) maintains the function of cardiac progenitor cells during heart formation in zebrafish

    Energy Technology Data Exchange (ETDEWEB)

    Yan, Lifeng [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Zhou, Yong [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Yu, Shanhe [Shanghai Institute of Hematology, RuiJin Hospital, Shanghai Jiao Tong University, School of Medicine, Shanghai 200025 (China); Ji, Guixiang [Nanjing Institute of Environmental Sciences/Key Laboratory of Pesticide Environmental Assessment and Pollution Control, Ministry of Environmental Protection, Nanjing 210042 (China); Wang, Lei [Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and Shanghai Jiao Tong University School of Medicine, Shanghai 200025 (China); Liu, Wei [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China); Gu, Aihua, E-mail: aihuagu@njmu.edu.cn [State Key Laboratory of Reproductive Medicine, Institute of Toxicology, Nanjing Medical University, Nanjing 210029 (China); Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029 (China)

    2013-11-15

    Genomic damage may devastate the potential of progenitor cells and consequently impair early organogenesis. We found that ogg1, a key enzyme initiating the base-excision repair, was enriched in the embryonic heart in zebrafish. So far, little is known about DNA repair in cardiogenesis. Here, we addressed the critical role of ogg1 in cardiogenesis for the first time. ogg1 mainly expressed in the anterior lateral plate mesoderm (ALPM), the primary heart tube, and subsequently the embryonic myocardium by in situ hybridisation. Loss of ogg1 resulted in severe cardiac morphogenesis and functional abnormalities, including the short heart length, arrhythmia, decreased cardiomyocytes and nkx2.5{sup +} cardiac progenitor cells. Moreover, the increased apoptosis and repressed proliferation of progenitor cells caused by ogg1 deficiency might contribute to the heart phenotype. The microarray analysis showed that the expression of genes involved in embryonic heart tube morphogenesis and heart structure were significantly changed due to the lack of ogg1. Among those, foxh1 is an important partner of ogg1 in the cardiac development in response to DNA damage. Our work demonstrates the requirement of ogg1 in cardiac progenitors and heart development in zebrafish. These findings may be helpful for understanding the aetiology of congenital cardiac deficits. - Highlights: • A key DNA repair enzyme ogg1 is expressed in the embryonic heart in zebrafish. • We found that ogg1 is essential for normal cardiac morphogenesis in zebrafish. • The production of embryonic cardiomyocytes requires appropriate ogg1 expression. • Ogg1 critically regulated proliferation of cardiac progenitor cells in zebrafish. • foxh1 is a partner of ogg1 in the cardiac development in response to DNA damage.

  4. Early Parental Depression and Child Language Development

    Science.gov (United States)

    Paulson, James F.; Keefe, Heather A.; Leiferman, Jenn A.

    2009-01-01

    Objective: To examine the effects of early maternal and paternal depression on child expressive language at age 24 months and the role that parent-to-child reading may play in this pathway. Participants and methods: The 9-month and 24-month waves from a national prospective study of children and their families, the Early Childhood Longitudinal…

  5. Improving early childhood development and well-being in refugee ...

    International Development Research Centre (IDRC) Digital Library (Canada)

    Improving early childhood development and well-being in refugee and other marginalized countries. Early childhood development research has traditionally focused on single-intervention initiatives and non-refugee populations. This project will generate evidence to support effective, integrated and scalable early childhood ...

  6. A stochastic model for early placental development.

    KAUST Repository

    Cotter, Simon L

    2014-08-01

    In the human, placental structure is closely related to placental function and consequent pregnancy outcome. Studies have noted abnormal placental shape in small-for-gestational-age infants which extends to increased lifetime risk of cardiovascular disease. The origins and determinants of placental shape are incompletely understood and are difficult to study in vivo. In this paper, we model the early development of the human placenta, based on the hypothesis that this is driven by a chemoattractant effect emanating from proximal spiral arteries in the decidua. We derive and explore a two-dimensional stochastic model, and investigate the effects of loss of spiral arteries in regions near to the cord insertion on the shape of the placenta. This model demonstrates that disruption of spiral arteries can exert profound effects on placental shape, particularly if this is close to the cord insertion. Thus, placental shape reflects the underlying maternal vascular bed. Abnormal placental shape may reflect an abnormal uterine environment, predisposing to pregnancy complications. Through statistical analysis of model placentas, we are able to characterize the probability that a given placenta grew in a disrupted environment, and even able to distinguish between different disruptions.

  7. The early research and development of ebselen.

    Science.gov (United States)

    Parnham, Michael J; Sies, Helmut

    2013-11-01

    Ebselen (2-phenyl-1,2-benzisoselenazol-3(2H)-one; PZ-51, DR-3305), is an organoselenium compound with glutathione peroxidase (GPx)-like, thiol-dependent, hydroperoxide reducing activity. As an enzyme mimic for activity of the selenoenzyme GPx, this compound has proved to be highly useful in research on mechanisms in redox biology. Furthermore, the reactivity of ebselen with protein thiols has helped to identify novel, selective targets for inhibitory actions on several enzymes of importance in pharmacology and toxicology. Importantly, the selenium in ebselen is not released and thus is not bioavailable, ebselen metabolites being excreted in bile and urine. As a consequence, initial concerns about selenium toxicity, fortunately, were unfounded. Potential applications in medical settings have been explored, notably in brain ischemia and stroke. More recently, there has been a surge in interest as new medical applications have been taken into consideration. The first publication on the biochemical effects of ebselen appeared 30 years ago (Müller et al.), which prompted the authors to retrace the early development from their perspective. It is a fascinating example of fruitful interaction between research-oriented industry and academia. Copyright © 2013 Elsevier Inc. All rights reserved.

  8. Parental Obesity and Early Childhood Development.

    Science.gov (United States)

    Yeung, Edwina H; Sundaram, Rajeshwari; Ghassabian, Akhgar; Xie, Yunlong; Buck Louis, Germaine

    2017-02-01

    Previous studies identified associations between maternal obesity and childhood neurodevelopment, but few examined paternal obesity despite potentially distinct genetic/epigenetic effects related to developmental programming. Upstate KIDS (2008-2010) recruited mothers from New York State (excluding New York City) at ∼4 months postpartum. Parents completed the Ages and Stages Questionnaire (ASQ) when their children were 4, 8, 12, 18, 24, 30, and 36 months of age corrected for gestation. The ASQ is validated to screen for delays in 5 developmental domains (ie, fine motor, gross motor, communication, personal-social functioning, and problem-solving ability). Analyses included 3759 singletons and 1062 nonrelated twins with ≥1 ASQs returned. Adjusted odds ratios (aORs) and 95% confidence intervals were estimated by using generalized linear mixed models accounting for maternal covariates (ie, age, race, education, insurance, marital status, parity, and pregnancy smoking). Compared with normal/underweight mothers (BMI obese mothers (26% with BMI ≥30) had increased odds of failing the fine motor domain (aOR 1.67; confidence interval 1.12-2.47). The association remained after additional adjustment for paternal BMI (1.67; 1.11-2.52). Paternal obesity (29%) was associated with increased risk of failing the personal-social domain (1.75; 1.13-2.71), albeit attenuated after adjustment for maternal obesity (aOR 1.71; 1.08-2.70). Children whose parents both had BMI ≥35 were likely to additionally fail the problem-solving domain (2.93; 1.09-7.85). Findings suggest that maternal and paternal obesity are each associated with specific delays in early childhood development, emphasizing the importance of family information when screening child development. Copyright © 2017 by the American Academy of Pediatrics.

  9. Use of zebrafish and knockdown technology to define proprotein convertase activity.

    Science.gov (United States)

    Chitramuthu, Babykumari P; Bennett, Hugh P J

    2011-01-01

    The Zebrafish (Danio rerio) is a powerful and well-established tool used extensively for the study of early vertebrate development and as a model of human diseases. Zebrafish genes orthologous to their mammalian counterparts generally share conserved biological function. Protein knockdown or overexpression can be effectively achieved by microinjection of morpholino antisense oligonucleotides (MOs) or mRNA, respectively, into developing embryos at the one- to two-cell stage. Correlating gene expression patterns with the characterizing of phenotypes resulting from over- or underexpression can reveal the function of a particular protein. The microinjection technique is simple and results are reproducible. We defined the expression pattern of the proprotein convertase PCSK5 within the lateral line neuromasts and various organs including the liver, gut and otic vesicle by whole-mount in situ hybridization (ISH) and immunofluorescence (IF). MO-mediated knockdown of zebrafish PCSK5 expression generated embryos that display abnormal neuromast deposition within the lateral line system resulting in uncoordinated patterns of swimming.

  10. Study on radiation modifiers with zebrafish as a vertebrate model

    International Nuclear Information System (INIS)

    Lei Jixiao; Ni Jin; Cai Jianming; Shen Jianliang

    2010-01-01

    Zebrafish (Danio rerio) as a vertebrate model system has been used in a series of biomedical experiments by scientists. It offers distinctive benefits as a laboratory model system, especially for embryonic development, gene expression, drug screening and human disease model. In this paper, the typical radiation modifiers, such as Amifostine, DF-1, AG1478, Flavopiridol and DNA repair proteins involved in biomedical process by use of zebrafish have been reviewed. (authors)

  11. An α-smooth muscle actin (acta2/αsma zebrafish transgenic line marking vascular mural cells and visceral smooth muscle cells.

    Directory of Open Access Journals (Sweden)

    Thomas R Whitesell

    Full Text Available Mural cells of the vascular system include vascular smooth muscle cells (SMCs and pericytes whose role is to stabilize and/or provide contractility to blood vessels. One of the earliest markers of mural cell development in vertebrates is α smooth muscle actin (acta2; αsma, which is expressed by pericytes and SMCs. In vivo models of vascular mural cell development in zebrafish are currently lacking, therefore we developed two transgenic zebrafish lines driving expression of GFP or mCherry in acta2-expressing cells. These transgenic fish were used to trace the live development of mural cells in embryonic and larval transgenic zebrafish. acta2:EGFP transgenic animals show expression that largely mirrors native acta2 expression, with early pan-muscle expression starting at 24 hpf in the heart muscle, followed by skeletal and visceral muscle. At 3.5 dpf, expression in the bulbus arteriosus and ventral aorta marks the first expression in vascular smooth muscle. Over the next 10 days of development, the number of acta2:EGFP positive cells and the number of types of blood vessels associated with mural cells increases. Interestingly, the mural cells are not motile and remain in the same position once they express the acta2:EGFP transgene. Taken together, our data suggests that zebrafish mural cells develop relatively late, and have little mobility once they associate with vessels.

  12. Sex reversal in zebrafish fancl mutants is caused by Tp53-mediated germ cell apoptosis.

    Directory of Open Access Journals (Sweden)

    Adriana Rodríguez-Marí

    2010-07-01

    Full Text Available The molecular genetic mechanisms of sex determination are not known for most vertebrates, including zebrafish. We identified a mutation in the zebrafish fancl gene that causes homozygous mutants to develop as fertile males due to female-to-male sex reversal. Fancl is a member of the Fanconi Anemia/BRCA DNA repair pathway. Experiments showed that zebrafish fancl was expressed in developing germ cells in bipotential gonads at the critical time of sexual fate determination. Caspase-3 immunoassays revealed increased germ cell apoptosis in fancl mutants that compromised oocyte survival. In the absence of oocytes surviving through meiosis, somatic cells of mutant gonads did not maintain expression of the ovary gene cyp19a1a and did not down-regulate expression of the early testis gene amh; consequently, gonads masculinized and became testes. Remarkably, results showed that the introduction of a tp53 (p53 mutation into fancl mutants rescued the sex-reversal phenotype by reducing germ cell apoptosis and, thus, allowed fancl mutants to become fertile females. Our results show that Fancl function is not essential for spermatogonia and oogonia to become sperm or mature oocytes, but instead suggest that Fancl function is involved in the survival of developing oocytes through meiosis. This work reveals that Tp53-mediated germ cell apoptosis induces sex reversal after the mutation of a DNA-repair pathway gene by compromising the survival of oocytes and suggests the existence of an oocyte-derived signal that biases gonad fate towards the female developmental pathway and thereby controls zebrafish sex determination.

  13. High-content screening in zebrafish embryos identifies butafenacil as a potent inducer of anemia.

    Directory of Open Access Journals (Sweden)

    Jessica K Leet

    Full Text Available Using transgenic zebrafish (fli1:egfp that stably express enhanced green fluorescent protein (eGFP within vascular endothelial cells, we recently developed and optimized a 384-well high-content screening (HCS assay that enables us to screen and identify chemicals affecting cardiovascular development and function at non-teratogenic concentrations. Within this assay, automated image acquisition procedures and custom image analysis protocols are used to quantify body length, heart rate, circulation, pericardial area, and intersegmental vessel area within individual live embryos exposed from 5 to 72 hours post-fertilization. After ranking developmental toxicity data generated from the U.S. Environmental Protection Agency's (EPA's zebrafish teratogenesis assay, we screened 26 of the most acutely toxic chemicals within EPA's ToxCast Phase-I library in concentration-response format (0.05-50 µM using this HCS assay. Based on this screen, we identified butafenacil as a potent inducer of anemia, as exposure from 0.39 to 3.125 µM butafenacil completely abolished arterial circulation in the absence of effects on all other endpoints evaluated. Butafenacil is an herbicide that inhibits protoporphyrinogen oxidase (PPO--an enzyme necessary for heme production in vertebrates. Using o-dianisidine staining, we then revealed that severe butafenacil-induced anemia in zebrafish was due to a complete loss of hemoglobin following exposure during early development. Therefore, six additional PPO inhibitors within the ToxCast Phase-I library were screened to determine whether anemia represents a common adverse outcome for these herbicides. Embryonic exposure to only one of these PPO inhibitors--flumioxazin--resulted in a similar phenotype as butafenacil, albeit not as severe as butafenacil. Overall, this study highlights the potential utility of this assay for (1 screening chemicals for cardiovascular toxicity and (2 prioritizing chemicals for future hypothesis

  14. Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.

    Science.gov (United States)

    Cheng, Christina N; Li, Yue; Marra, Amanda N; Verdun, Valerie; Wingert, Rebecca A

    2014-07-17

    The zebrafish embryo is now commonly used for basic and biomedical research to investigate the genetic control of developmental processes and to model congenital abnormalities. During the first day of life, the zebrafish embryo progresses through many developmental stages including fertilization, cleavage, gastrulation, segmentation, and the organogenesis of structures such as the kidney, heart, and central nervous system. The anatomy of a young zebrafish embryo presents several challenges for the visualization and analysis of the tissues involved in many of these events because the embryo develops in association with a round yolk mass. Thus, for accurate analysis and imaging of experimental phenotypes in fixed embryonic specimens between the tailbud and 20 somite stage (10 and 19 hours post fertilization (hpf), respectively), such as those stained using whole mount in situ hybridization (WISH), it is often desirable to remove the embryo from the yolk ball and to position it flat on a glass slide. However, performing a flat mount procedure can be tedious. Therefore, successful and efficient flat mount preparation is greatly facilitated through the visual demonstration of the dissection technique, and also helped by using reagents that assist in optimal tissue handling. Here, we provide our WISH protocol for one or two-color detection of gene expression in the zebrafish embryo, and demonstrate how the flat mounting procedure can be performed on this example of a stained fixed specimen. This flat mounting protocol is broadly applicable to the study of many embryonic structures that emerge during early zebrafish development, and can be implemented in conjunction with other staining methods performed on fixed embryo samples.

  15. Microcystin-LR exposure induces developmental neurotoxicity in zebrafish embryo

    International Nuclear Information System (INIS)

    Wu, Qin; Yan, Wei; Liu, Chunsheng; Li, Li; Yu, Liqin; Zhao, Sujuan; Li, Guangyu

    2016-01-01

    Microcystin-LR (MCLR) is a commonly acting potent hepatotoxin and has been pointed out of potentially causing developmental neurotoxicity, but the exact mechanism is little known. In this study, zebrafish embryos were exposed to 0, 0.8, 1.6 or 3.2 mg/L MCLR for 120 h. MCLR exposure through submersion caused serious hatching delay and body length decrease. The content of MCLR in zebrafish larvae was analyzed and the results demonstrated that MCLR can accumulate in zebrafish larvae. The locomotor speed of zebrafish larvae was decreased. Furthermore, the dopamine and acetylcholine (ACh) content were detected to be significantly decreased in MCLR exposure groups. And the acetylcholinesterase (AChE) activity was significantly increased after exposure to 1.6 and 3.2 mg/L MCLR. The transcription pattern of manf, chrnα7 and ache gene was consistent with the change of the dopamine content, ACh content and AChE activity. Gene expression involved in the development of neurons was also measured. α1-tubulin and shha gene expression were down-regulated, whereas mbp and gap43 gene expression were observed to be significantly up-regulated upon exposure to MCLR. The above results indicated that MCLR-induced developmental toxicity might attribute to the disorder of cholinergic system, dopaminergic signaling, and the development of neurons. - Highlights: • MCLR accumulation induces developmental neurotoxicity in zebrafish embryo. • The decrease of dopamine levels might be associated with the MCLR-induced developmental neurotoxicity in zebrafish larvae. • The alternation of cholinergic system might contribute to the change of neurobehavior in zebrafish larvae exposure with MCLR. - MCLR accumulation induces developmental neurotoxicity by affecting cholinergic system, dopaminergic signaling, and the development of neurons in zebrafish embryo.

  16. Phenotype classification of zebrafish embryos by supervised learning.

    Directory of Open Access Journals (Sweden)

    Nathalie Jeanray

    Full Text Available Zebrafish is increasingly used to assess biological properties of chemical substances and thus is becoming a specific tool for toxicological and pharmacological studies. The effects of chemical substances on embryo survival and development are generally evaluated manually through microscopic observation by an expert and documented by several typical photographs. Here, we present a methodology to automatically classify brightfield images of wildtype zebrafish embryos according to their defects by using an image analysis approach based on supervised machine learning. We show that, compared to manual classification, automatic classification results in 90 to 100% agreement with consensus voting of biological experts in nine out of eleven considered defects in 3 days old zebrafish larvae. Automation of the analysis and classification of zebrafish embryo pictures reduces the workload and time required for the biological expert and increases the reproducibility and objectivity of this classification.

  17. Directed Differentiation of Zebrafish Pluripotent Embryonic Cells to Functional Cardiomyocytes

    Directory of Open Access Journals (Sweden)

    Yao Xiao

    2016-09-01

    Full Text Available A cardiomyocyte differentiation in vitro system from zebrafish embryos remains to be established. Here, we have determined pluripotency window of zebrafish embryos by analyzing their gene-expression patterns of pluripotency factors together with markers of three germ layers, and have found that zebrafish undergoes a very narrow period of pluripotency maintenance from zygotic genome activation to a brief moment after oblong stage. Based on the pluripotency and a combination of appropriate conditions, we established a rapid and efficient method for cardiomyocyte generation in vitro from primary embryonic cells. The induced cardiomyocytes differentiated into functional and specific cardiomyocyte subtypes. Notably, these in vitro generated cardiomyocytes exhibited typical contractile kinetics and electrophysiological features. The system provides a new paradigm of cardiomyocyte differentiation from primary embryonic cells in zebrafish. The technology provides a new platform for the study of heart development and regeneration, in addition to drug discovery, disease modeling, and assessment of cardiotoxic agents.

  18. Zebrafish models in neuropsychopharmacology and CNS drug discovery.

    Science.gov (United States)

    Khan, Kanza M; Collier, Adam D; Meshalkina, Darya A; Kysil, Elana V; Khatsko, Sergey L; Kolesnikova, Tatyana; Morzherin, Yury Yu; Warnick, Jason E; Kalueff, Allan V; Echevarria, David J

    2017-07-01

    Despite the high prevalence of neuropsychiatric disorders, their aetiology and molecular mechanisms remain poorly understood. The zebrafish (Danio rerio) is increasingly utilized as a powerful animal model in neuropharmacology research and in vivo drug screening. Collectively, this makes zebrafish a useful tool for drug discovery and the identification of disordered molecular pathways. Here, we discuss zebrafish models of selected human neuropsychiatric disorders and drug-induced phenotypes. As well as covering a broad range of brain disorders (from anxiety and psychoses to neurodegeneration), we also summarize recent developments in zebrafish genetics and small molecule screening, which markedly enhance the disease modelling and the discovery of novel drug targets. © 2017 The British Pharmacological Society.

  19. Stages in the development of a model organism as a platform for mechanistic models in developmental biology: Zebrafish, 1970-2000.

    Science.gov (United States)

    Meunier, Robert

    2012-06-01

    Model organisms became an indispensable part of experimental systems in molecular developmental and cell biology, constructed to investigate physiological and pathological processes. They are thought to play a crucial role for the elucidation of gene function, complementing the sequencing of the genomes of humans and other organisms. Accordingly, historians and philosophers paid considerable attention to various issues concerning this aspect of experimental biology. With respect to the representational features of model organisms, that is, their status as models, the main focus was on generalization of phenomena investigated in such experimental systems. Model organisms have been said to be models for other organisms or a higher taxon. This, however, presupposes a representation of the phenomenon in question. I will argue that prior to generalization, model organisms allow researchers to built generative material models of phenomena - structures, processes or the mechanisms that explain them - through their integration in experimental set-ups that carve out the phenomena from the whole organism and thus represent them. I will use the history of zebrafish biology to show how model organism systems, from around 1970 on, were developed to construct material models of molecular mechanisms explaining developmental or physiological processes. Copyright © 2011 Elsevier Ltd. All rights reserved.

  20. Developing early algebraic reasoning in a mathematical community of inquiry

    OpenAIRE

    Hunter, Jodie Margaret Roberta

    2013-01-01

    This study explores the development of early algebraic reasoning in mathematical communities of inquiry. Under consideration is the different pathways teachers take as they develop their own understanding of early algebra and then enact changes in their classroom to facilitate algebraic reasoning opportunities. Teachers participated in a professional development intervention which focused on understanding of early algebraic concepts, task development, modification, and enactment, and clas...

  1. Pdlim7 is required for maintenance of the mesenchymal/epidermal Fgf signaling feedback loop during zebrafish pectoral fin development

    Directory of Open Access Journals (Sweden)

    Klosowiak Julian

    2010-10-01

    Full Text Available Abstract Background Vertebrate limb development involves a reciprocal feedback loop between limb mesenchyme and the overlying apical ectodermal ridge (AER. Several gene pathways participate in this feedback loop, including Fgf signaling. In the forelimb lateral plate mesenchyme, Tbx5 activates Fgf10 expression, which in turn initiates and maintains the mesenchyme/AER Fgf signaling loop. Recent findings have revealed that Tbx5 transcriptional activity is regulated by dynamic nucleocytoplasmic shuttling and interaction with Pdlim7, a PDZ-LIM protein family member, along actin filaments. This Tbx5 regulation is critical in heart formation, but the coexpression of both proteins in other developing tissues suggests a broader functional role. Results Knock-down of Pdlim7 function leads to decreased pectoral fin cell proliferation resulting in a severely stunted fin phenotype. While early gene induction and patterning in the presumptive fin field appear normal, the pectoral fin precursor cells display compaction and migration defects between 18 and 24 hours post-fertilization (hpf. During fin growth fgf24 is sequentially expressed in the mesenchyme and then in the apical ectodermal ridge (AER. However, in pdlim7 antisense morpholino-treated embryos this switch of expression is prevented and fgf24 remains ectopically active in the mesenchymal cells. Along with the lack of fgf24 in the AER, other critical factors including fgf8 are reduced, suggesting signaling problems to the underlying mesenchyme. As a consequence of perturbed AER function in the absence of Pdlim7, pathway components in the fin mesenchyme are misregulated or absent, indicating a breakdown of the Fgf signaling feedback loop, which is ultimately responsible for the loss of fin outgrowth. Conclusion This work provides the first evidence for the involvement of Pdlim7 in pectoral fin development. Proper fin outgrowth requires fgf24 downregulation in the fin mesenchyme with subsequent

  2. The heartstrings mutation in zebrafish causes heart/fin Tbx5 deficiency syndrome.

    Science.gov (United States)

    Garrity, Deborah M; Childs, Sarah; Fishman, Mark C

    2002-10-01

    Holt-Oram syndrome is one of the autosomal dominant human "heart-hand" disorders, with a combination of upper limb malformations and cardiac defects. Holt-Oram syndrome is caused by mutations in the TBX5 gene, a member of a large family of T-box transcription factors that play important roles in cell-type specification and morphogenesis. In a screen for mutations affecting zebrafish cardiac function, we isolated the recessive lethal mutant heartstrings, which lacks pectoral fins and exhibits severe cardiac dysfunction, beginning with a slow heart rate and progressing to a stretched, non-functional heart. We mapped and cloned the heartstrings mutation and find it to encode the zebrafish ortholog of the TBX5 gene. The heartstrings mutation causes premature termination at amino acid 316. Homozygous mutant embryos never develop pectoral fin buds and do not express several markers of early fin differentiation. The total absence of any fin bud differentiation distinguishes heartstrings from most other mutations that affect zebrafish fin development, suggesting that Tbx5 functions very early in the pectoral fin induction pathway. Moderate reduction of Tbx5 by morpholino causes fin malformations, revealing an additional early requirement for Tbx5 in coordinating the axes of fin outgrowth. The heart of heartstrings mutant embryos appears to form and function normally through the early heart tube stage, manifesting only a slight bradycardia compared with wild-type siblings. However, the heart fails to loop and then progressively deteriorates, a process affecting the ventricle as well as the atrium. Relative to mammals, fish require lower levels of Tbx5 to produce malformed appendages and display whole-heart rather than atrial-predominant cardiac defects. However, the syndromic deficiencies of tbx5 mutation are remarkably well retained between fish and mammals.

  3. Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

    Energy Technology Data Exchange (ETDEWEB)

    Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Brandhof, Evert-Jan van den [Center for Environmental Quality, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Pennings, Jeroen L.A. [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands); Kamstra, Jorke H. [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Jelinek, Jaroslav [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Issa, Jean-Pierre J. [Fels Institute for Cancer Research and Molecular Biology, Temple University School of Medicine, Philadelphia, PA (United States); Department of Leukemia, The University of Texas MD Anderson Cancer Center, Houston, TX (United States); Legler, Juliette [Institute for Environmental Studies (IVM), VU University, De Boelelaan 1085, 1081 HV Amsterdam (Netherlands); Ven, Leo T.M. van der, E-mail: leo.van.der.ven@rivm.nl [Center for Health Protection, National Institute for Public Health and the Environment (RIVM), PO Box 1, 3720 BA Bilthoven (Netherlands)

    2016-01-15

    Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome

  4. Zebrafish embryos as a screen for DNA methylation modifications after compound exposure

    International Nuclear Information System (INIS)

    Bouwmeester, Manon C.; Ruiter, Sander; Lommelaars, Tobias; Sippel, Josefine; Hodemaekers, Hennie M.; Brandhof, Evert-Jan van den; Pennings, Jeroen L.A.; Kamstra, Jorke H.; Jelinek, Jaroslav; Issa, Jean-Pierre J.; Legler, Juliette; Ven, Leo T.M. van der

    2016-01-01

    Modified epigenetic programming early in life is proposed to underlie the development of an adverse adult phenotype, known as the Developmental Origins of Health and Disease (DOHaD) concept. Several environmental contaminants have been implicated as modifying factors of the developing epigenome. This underlines the need to investigate this newly recognized toxicological risk and systematically screen for the epigenome modifying potential of compounds. In this study, we examined the applicability of the zebrafish embryo as a screening model for DNA methylation modifications. Embryos were exposed from 0 to 72 h post fertilization (hpf) to bisphenol-A (BPA), diethylstilbestrol, 17α-ethynylestradiol, nickel, cadmium, tributyltin, arsenite, perfluoroctanoic acid, valproic acid, flusilazole, 5-azacytidine (5AC) in subtoxic concentrations. Both global and site-specific methylation was examined. Global methylation was only affected by 5AC. Genome wide locus-specific analysis was performed for BPA exposed embryos using Digital Restriction Enzyme Analysis of Methylation (DREAM), which showed minimal wide scale effects on the genome, whereas potential informative markers were not confirmed by pyrosequencing. Site-specific methylation was examined in the promoter regions of three selected genes vasa, vtg1 and cyp19a2, of which vasa (ddx4) was the most responsive. This analysis distinguished estrogenic compounds from metals by direction and sensitivity of the effect compared to embryotoxicity. In conclusion, the zebrafish embryo is a potential screening tool to examine DNA methylation modifications after xenobiotic exposure. The next step is to examine the adult phenotype of exposed embryos and to analyze molecular mechanisms that potentially link epigenetic effects and altered phenotypes, to support the DOHaD hypothesis. - Highlights: • Compound induced effects on DNA methylation in zebrafish embryos • Global methylation not an informative biomarker • Minimal genome

  5. Normal anatomy and histology of the adult zebrafish.

    Science.gov (United States)

    Menke, Aswin L; Spitsbergen, Jan M; Wolterbeek, Andre P M; Woutersen, Ruud A

    2011-08-01

    The zebrafish has been shown to be an excellent vertebrate model for studying the roles of specific genes and signaling pathways. The sequencing of its genome and the relative ease with which gene modifications can be performed have led to the creation of numerous human disease models that can be used for testing the potential and the toxicity of new pharmaceutical compounds. Many pharmaceutical companies already use the zebrafish for prescreening purposes. So far, the focus has been on ecotoxicity and the effects on embryonic development, but there is a trend to expand the use of the zebrafish with acute, subchronic, and chronic toxicity studies that are currently still carried out with the more conventional test animals such as rodents. However, before we can fully realize the potential of the zebrafish as an animal model for understanding human development, disease, and toxicology, we must first greatly advance our knowledge of normal zebrafish physiology, anatomy, and histology. To further this knowledge, we describe, in the present article, location and histology of the major zebrafish organ systems with a brief description of their function.

  6. Scrambled eggs: Proteomic portraits and novel biomarkers of egg quality in zebrafish (Danio rerio).

    Science.gov (United States)

    Yilmaz, Ozlem; Patinote, Amélie; Nguyen, Thao Vi; Com, Emmanuelle; Lavigne, Regis; Pineau, Charles; Sullivan, Craig V; Bobe, Julien

    2017-01-01

    Egg quality is a complex biological trait and a major determinant of reproductive fitness in all animals. This study delivered the first proteomic portraits of egg quality in zebrafish, a leading biomedical model for early development. Egg batches of good and poor quality, evidenced by embryo survival for 24 h, were sampled immediately after spawning and used to create pooled or replicated sample sets whose protein extracts were subjected to different levels of fractionation before liquid chromatography and tandem mass spectrometry. Obtained spectra were searched against a zebrafish proteome database and detected proteins were annotated, categorized and quantified based on normalized spectral counts. Manually curated and automated enrichment analyses revealed poor quality eggs to be deficient of proteins involved in protein synthesis and energy and lipid metabolism, and of some vitellogenin products and lectins, and to have a surfeit of proteins involved in endo-lysosomal activities, autophagy, and apoptosis, and of some oncogene products, lectins and egg envelope proteins. Results of pathway and network analyses suggest that this aberrant proteomic profile results from failure of oocytes giving rise to poor quality eggs to properly transit through final maturation, and implicated Wnt signaling in the etiology of this defect. Quantitative comparisons of abundant proteins in good versus poor quality eggs revealed 17 candidate egg quality markers. Thus, the zebrafish egg proteome is clearly linked to embryo developmental potential, a phenomenon that begs further investigation to elucidate the root causes of poor egg quality, presently a serious and intractable problem in livestock and human reproductive medicine.

  7. Clonal origins of cells in the pigmented retina of the zebrafish eye

    International Nuclear Information System (INIS)

    Streisinger, G.; Coale, F.; Taggart, C.; Walker, C.; Grunwald, D.J.

    1989-01-01

    Mosaic analysis has been used to study the clonal basis of the development of the pigmented retina of the zebrafish, Brachydanio rerio. Zebrafish embryos heterozygous for a recessive mutation at the gol-1 locus were exposed to gamma-irradiation at various developmental stages to create mosaic individuals consisting of wild-type pigmented cells and a clone of pigmentless (golden) cells in the eye. The contribution of individual embryonic cells to the pigmented retina was measured and the total number of cells in the embryo that contributed descendants to this tissue was determined. Until the 32-cell stage, almost every blastomere has some descendants that participate in the formation of the pigmented retina of the zebrafish. During subsequent cell divisions, up to the several thousand-cell stage, the number of ancestral cells is constant: approximately 40 cells are present that will give rise to progeny in the pigmented retina. Analysis of the size of clones in the pigmented retina indicates that the cells of this tissue do not arise through a rigid series of cell divisions originating in the early embryo. The findings that each cleavage stage cell contributes to the pigmented retina and yet the contribution of such cells is highly variable are consistent with the interpretation that clonal descendants of different blastomeres normally intermix extensively prior to formation of the pigmented retina

  8. Liver-Enriched Gene 1, a Glycosylated Secretory Protein, Binds to FGFR and Mediates an Anti-stress Pathway to Protect Liver Development in Zebrafish.

    Directory of Open Access Journals (Sweden)

    Minjie Hu

    2016-02-01

    Full Text Available Unlike mammals and birds, teleost fish undergo external embryogenesis, and therefore their embryos are constantly challenged by stresses from their living environment. These stresses, when becoming too harsh, will cause arrest of cell proliferation, abnormal cell death or senescence. Such organisms have to evolve a sophisticated anti-stress mechanism to protect the process of embryogenesis/organogenesis. However, very few signaling molecule(s mediating such activity have been identified. liver-enriched gene 1 (leg1 is an uncharacterized gene that encodes a novel secretory protein containing a single domain DUF781 (domain of unknown function 781 that is well conserved in vertebrates. In the zebrafish genome, there are two copies of leg1, namely leg1a and leg1b. leg1a and leg1b are closely linked on chromosome 20 and share high homology, but are differentially expressed. In this report, we generated two leg1a mutant alleles using the TALEN technique, then characterized liver development in the mutants. We show that a leg1a mutant exhibits a stress-dependent small liver phenotype that can be prevented by chemicals blocking the production of reactive oxygen species. Further studies reveal that Leg1a binds to FGFR3 and mediates a novel anti-stress pathway to protect liver development through enhancing Erk activity. More importantly, we show that the binding of Leg1a to FGFR relies on the glycosylation at the 70th asparagine (Asn(70 or N(70, and mutating the Asn(70 to Ala(70 compromised Leg1's function in liver development. Therefore, Leg1 plays a unique role in protecting liver development under different stress conditions by serving as a secreted signaling molecule/modulator.

  9. Validation of visualized transgenic zebrafish as a high throughput model to assay bradycardia related cardio toxicity risk candidates.

    Science.gov (United States)

    Wen, Dingsheng; Liu, Aiming; Chen, Feng; Yang, Julin; Dai, Renke

    2012-10-01

    Drug-induced QT prolongation usually leads to torsade de pointes (TdP), thus for drugs in the early phase of development this risk should be evaluated. In the present study, we demonstrated a visualized transgenic zebrafish as an in vivo high-throughput model to assay the risk of drug-induced QT prolongation. Zebrafish larvae 48 h post-fertilization expressing green fluorescent protein in myocardium were incubated with compounds reported to induce QT prolongation or block the human ether-a-go-go-related gene (hERG) K⁺ current. The compounds sotalol, indapaminde, erythromycin, ofoxacin, levofloxacin, sparfloxacin and roxithromycin were additionally administrated by microinjection into the larvae yolk sac. The ventricle heart rate was recorded using the automatic monitoring system after incubation or microinjection. As a result, 14 out of 16 compounds inducing dog QT prolongation caused bradycardia in zebrafish. A similar result was observed with 21 out of 26 compounds which block hERG current. Among the 30 compounds which induced human QT prolongation, 25 caused bradycardia in this model. Thus, the risk of compounds causing bradycardia in this transgenic zebrafish correlated with that causing QT prolongation and hERG K⁺ current blockage in established models. The tendency that high logP values lead to high risk of QT prolongation in this model was indicated, and non-sensitivity of this model to antibacterial agents was revealed. These data suggest application of this transgenic zebrafish as a high-throughput model to screen QT prolongation-related cardio toxicity of the drug candidates. Copyright © 2012 John Wiley & Sons, Ltd.

  10. Feature Binding in Zebrafish

    Directory of Open Access Journals (Sweden)

    P Neri

    2012-07-01

    Full Text Available Binding operations are primarily ascribed to cortex or similarly complex avian structures. My experiments show that the zebrafish, a lower vertebrate lacking cortex, supports visual feature binding of form and motion for the purpose of social behavior. These results challenge the notion that feature binding may require highly evolved neural structures and demonstrate that the nervous system of lower vertebrates can afford unexpectedly complex computations.

  11. Expression of prostaglandin synthases (pgds and pges) during zebrafish gonadal differentiation

    DEFF Research Database (Denmark)

    Jørgensen, Anne; Nielsen, John E; Nielsen, Betina Frydenlund

    2010-01-01

    The present study aimed at elucidating whether the expression pattern of the membrane bound form of prostaglandin E2 synthase (pges) and especially the lipocalin-type prostaglandin D2 synthase (pgds) indicates involvement in gonadal sex differentiation in zebrafish as has previously been found....... In this study, a sexually dimorphic expression of pgds was found in gonads of adult zebrafish with expression in testis but not in ovaries. To determine whether the sex-specific expression pattern of pgds was present in gonads of juvenile zebrafish and therefore could be an early marker of sex in zebrafish, we...... microdissected gonads from four randomly selected individual zebrafish for every second day in the period 2-20 days post hatch (dph) and 0-1 dph. The temporal expression of pgds and pges was investigated in the microdissected gonads, however, no differential expression that could indicate sex-specific difference...

  12. Parents' Role in the Early Head Start Children's Language Development

    Science.gov (United States)

    Griswold, Cecelia Smalls

    2014-01-01

    The development of language during a child's early years has been linked to parental involvement. While Early Head Start (EHS) researchers have theorized that parental involvement is an important factor in language development, there has been little research on how parents view their roles in the language development process. The purpose of this…

  13. The Development of Self-Regulation across Early Childhood

    Science.gov (United States)

    Montroy, Janelle J.; Bowles, Ryan P.; Skibbe, Lori E.; McClelland, Megan M.; Morrison, Frederick J.

    2016-01-01

    The development of early childhood self-regulation is often considered an early life marker for later life successes. Yet little longitudinal research has evaluated whether there are different trajectories of self-regulation development across children. This study investigates the development of behavioral self-regulation between the ages of 3 and…

  14. Endocrine-disrupting effect of the ultraviolet filter benzophenone-3 in zebrafish, Danio rerio

    DEFF Research Database (Denmark)

    Kinnberg, Karin Lund; Petersen, Gitte I.; Albrektsen, Mette

    2015-01-01

    of BP-3 in zebrafish (Danio rerio) in the Fish Sexual Development Test (Organisation for Economic Co-operation and Development TG 234) and a 12 day adult male zebrafish study. In TG 234, exposure from 0 to 60 d posthatch caused a monotone dose-dependent skewing of the phenotypic sex ratio towards less...

  15. Whole-body and multispectral photoacoustic imaging of adult zebrafish

    Science.gov (United States)

    Huang, Na; Xi, Lei

    2016-10-01

    Zebrafish is a top vertebrate model to study developmental biology and genetics, and it is becoming increasingly popular for studying human diseases due to its high genome similarity to that of humans and the optical transparency in embryonic stages. However, it becomes difficult for pure optical imaging techniques to volumetric visualize the internal organs and structures of wild-type zebrafish in juvenile and adult stages with excellent resolution and penetration depth. Even with the establishment of mutant lines which remain transparent over the life cycle, it is still a challenge for pure optical imaging modalities to image the whole body of adult zebrafish with micro-scale resolution. However, the method called photoacoustic imaging that combines all the advantages of the optical imaging and ultrasonic imaging provides a new way to image the whole body of the zebrafish. In this work, we developed a non-invasive photoacoustic imaging system with optimized near-infrared illumination and cylindrical scanning to image the zebrafish. The lateral and axial resolution yield to 80 μm and 600 μm, respectively. Multispectral strategy with wavelengths from 690 nm to 930 nm was employed to image various organs inside the zebrafish. From the reconstructed images, most major organs and structures inside the body can be precisely imaged. Quantitative and statistical analysis of absorption for organs under illumination with different wavelengths were carried out.

  16. Thyroid endocrine disruption and external body morphology of Zebrafish

    Science.gov (United States)

    Sharma, Prakash; Grabowski, Timothy B.; Patino, Reynaldo

    2016-01-01

    This study examined the effects thyroid-active compounds during early development on body morphology of Zebrafish (Danio rerio). Three-day postfertilization (dpf) larvae were exposed to goitrogen [methimazole (MZ, 0.15 mM)], combination of MZ (0.15 mM) and thyroxine (T4, 2 nM), T4 (2 nM), or control (reconstituted water) treatments until 33 dpf and subsequently maintained in reconstituted water until 45 dpf. Samples were taken at 33 and 45 dpf for multivariate analysis of geometric distances between selected homologous landmarks placed on digital images of fish, and for histological assessment of thyrocytes. Body mass, standard length, and pectoral fin length were separately measured on remaining fish at 45 dpf. Histological analysis confirmed the hypothyroid effect (increased thyrocyte height) of MZ and rescue effect of T4 co-administration. Geometric distance analysis showed that pectoral and pelvic fins shifted backward along the rostrocaudal axis under hypothyroid conditions at 45 dpf and that T4 co-treatment prevented this shift. Pectoral fin length at 45 dpf was reduced by exposure to MZ and rescued by co-administration of T4, but it was not associated with standard length. Methimazole caused a reduction in body mass and length at 45 dpf that could not be rescued by T4 co-administration, and non-thyroidal effects of MZ on body shape were also recognized at 33 and 45 dpf. Alterations in the length and position of paired fins caused by exposure to thyroid-disrupting chemicals during early development, as shown here for Zebrafish, could affect physical aspects of locomotion and consequently other important organismal functions such as foraging, predator avoidance, and ultimately survival and recruitment into the adult population. Results of this study also suggest the need to include rescue treatments in endocrine disruption studies that rely on goitrogens as reference for thyroid-mediated effects.

  17. Targeted Mutagenesis of the Hypophysiotropic Gnrh3 in Zebrafish (Danio rerio Reveals No Effects on Reproductive Performance.

    Directory of Open Access Journals (Sweden)

    Olivia Smith Spicer

    Full Text Available Gnrh is the major neuropeptide regulator of vertebrate reproduction, triggering a cascade of events in the pituitary-gonadal axis that result in reproductive competence. Previous research in mice and humans has demonstrated that Gnrh/GNRH null mutations result in hypogonadotropic hypogonadism and infertility. The goal of this study was to eliminate gnrh3 (the hypophysiotropic Gnrh form function in zebrafish (Danio rerio to determine how ontogeny and reproductive performance are affected, as well as factors downstream of Gnrh3 along the reproductive axis. Using the TALEN technology, we developed a gnrh3-/- zebrafish line that harbors a 62 bp deletion in the gnrh3 gene. Our gnrh3-/- zebrafish line represents the first targeted and heritable mutation of a Gnrh isoform in any organism. Using immunohistochemistry, we verified that gnrh3-/- fish do not possess Gnrh3 peptide in any regions of the brain. However, other than changes in mRNA levels of pituitary gonadotropin genes (fshb, lhb, and cga during early development, which are corrected by adulthood, there were no changes in ontogeny and reproduction in gnrh3-/- fish. The gnrh3-/- zebrafish are fertile, displaying normal gametogenesis and reproductive performance in males and females. Together with our previous results that Gnrh3 cell ablation causes infertility, these results indicate that a compensatory mechanism is being activated, which is probably primed early on upon Gnrh3 neuron differentiation and possibly confined to Gnrh3 neurons. Potential compensation factors and sensitive windows of time for compensation during development and puberty should be explored.

  18. Biotransformation of ginsenosides F4 and Rg6 in zebrafish.

    Science.gov (United States)

    Shen, Wen-Wen; Zhang, Hai-Xia; Qiu, Shou-Bei; Wei, Ying-Jie; Zhu, Fen-Xia; Wang, Jing; Wang, Dan-Dan; Jia, Xiao-Bin; Tang, Dao-Quan; Chen, Bin

    2017-03-28

    Ginsenosides F 4 and Rg 6 (GF 4 and GRg 6 ), two main active components of steamed notoginseng or red ginseng, are dehydrated disaccharide saponins. In this work, biotransformation of ginsenosides F 4 and Rg 6 in zebrafish was investigated by qualitatively identifying their metabolites and then proposing their possible metabolic pathways. The prediction of possible metabolism of ginsenosides F 4 and Rg 6 using zebrafish model which can effectively simulate existing mammals model was early and quickly performed. Metabolites of ginsenosides F 4 and Rg 6 after exposing to zebrafish for 24 h were identified by Ultraperformance Liquid Chromatography/Quadrupole-Time-of-Flight Mass Spectrometry. A total of 8 and 6 metabolites of ginsenosides F 4 and Rg 6 were identified in zebrafish, respectively. Of these, 7 and 5, including M1, M3-M5, M7-M9 and N1 (N5), N2, N4 (N9), N7-N8 were reported for the first time as far as we know. The mechanisms of their biotransformation involved were further deduced to be desugarization, glucuronidation, sulfation, dehydroxylation, loss of C-17 and/or C-23 residue pathways. It was concluded that loss of rhamnose at position C-6 and glucuronidation at position C-3 in zebrafish were considered as the main physiologic and metabolic processes of ginsenosides F 4 and ginsenosides Rg 6 , respectively.

  19. Constructivist Early Education for Moral Development.

    Science.gov (United States)

    DeVries, Rheta; Hildebrandt, Carolyn; Zan, Betty

    2000-01-01

    Examines role that constructivist teachers play in fostering moral development in young children. Traces development of perspective taking, autonomy, and self- regulation, and examines effects of different teaching and parenting practices on children's character development. Provides suggestions for teachers to promote optimal moral development by…

  20. Ethanol Exposure Causes Muscle Degeneration in Zebrafish

    Directory of Open Access Journals (Sweden)

    Elizabeth C. Coffey

    2018-03-01

    Full Text Available Alcoholic myopathies are characterized by neuromusculoskeletal symptoms such as compromised movement and weakness. Although these symptoms have been attributed to neurological damage, EtOH may also target skeletal muscle. EtOH exposure during zebrafish primary muscle development or adulthood results in smaller muscle fibers. However, the effects of EtOH exposure on skeletal muscle during the growth period that follows primary muscle development are not well understood. We determined the effects of EtOH exposure on muscle during this phase of development. Strikingly, muscle fibers at this stage are acutely sensitive to EtOH treatment: EtOH induces muscle degeneration. The severity of EtOH-induced muscle damage varies but muscle becomes more refractory to EtOH as muscle develops. NF-kB induction in muscle indicates that EtOH triggers a pro-inflammatory response. EtOH-induced muscle damage is p53-independent. Uptake of Evans blue dye shows that EtOH treatment causes sarcolemmal instability before muscle fiber detachment. Dystrophin-null sapje mutant zebrafish also exhibit sarcolemmal instability. We tested whether Trichostatin A (TSA, which reduces muscle degeneration in sapje mutants, would affect EtOH-treated zebrafish. We found that TSA and EtOH are a lethal combination. EtOH does, however, exacerbate muscle degeneration in sapje mutants. EtOH also disrupts adhesion of muscle fibers to their extracellular matrix at the myotendinous junction: some detached muscle fibers retain beta-Dystroglycan indicating failure of muscle end attachments. Overexpression of Paxillin, which reduces muscle degeneration in zebrafish deficient for beta-Dystroglycan, is not sufficient to rescue degeneration. Taken together, our results suggest that EtOH exposure has pleiotropic deleterious effects on skeletal muscle.

  1. Assessing Home Environment for Early Child Development in Pakistan

    Science.gov (United States)

    Nadeem, Sanober; Rafique, Ghazala; Khowaja, Liaquat; Yameen, Anjum

    2014-01-01

    Family environment plays a very important role in early child development and the availability of stimulating material in the early years of a child's life is crucial for optimising development. The Home Observation for Measurement of the Environment (HOME) inventory is one of the most widely used measures to assess the quality and quantity of…

  2. Psychological preconditions of game activity development in the early childhood

    OpenAIRE

    Valeriya Spitsyna; Ekaterina Saraykina

    2013-01-01

    The article is devoted for detection the psychological preconditions of game activity development at early age and interrelation of game formation with the development of subject actions, informative activity and procedural game.

  3. Developments in early intervention for psychosis in Hong Kong.

    Science.gov (United States)

    Wong, G H Y; Hui, C L M; Wong, D Y; Tang, J Y M; Chang, W C; Chan, S K W; Lee, E H M; Xu, J Q; Lin, J J X; Lai, D C; Tam, W; Kok, J; Chung, D W S; Hung, S F; Chen, E Y H

    2012-09-01

    The year 2011 marked the 10-year milestone of early intervention for psychosis in Hong Kong. Since 2001, the landscape of early psychosis services has changed markedly in Hong Kong. Substantial progress has been made in the areas of early intervention service implementation, knowledge generation, and public awareness promotion. Favourable outcomes attributable to the early intervention service are supported by solid evidence from local clinical research studies; early intervention service users showed improved functioning, ameliorated symptoms, and decreased hospitalisation and suicide rates. Continued development of early intervention in Hong Kong over the decade includes the introduction and maturation of several key platforms, such as the Hospital Authority Early Assessment Service for Young People with Psychosis programme, the Psychosis Studies and Intervention Unit by the University of Hong Kong, the Hong Kong Early Psychosis Intervention Society, the Jockey Club Early Psychosis Project, and the postgraduate Psychological Medicine (Psychosis Studies) programme. In this paper, we reviewed some of the major milestones in local service development with reference to features of the Hong Kong mental health system. We describe chronologically the implementation and consolidation of public early intervention services as well as recent progresses in public awareness work that are tied in with knowledge generation and transfer, and outline the prospects for early intervention in the next decade and those that follow.

  4. Sex Role Development in Early Adolescence.

    Science.gov (United States)

    Wittig, Michele Andrisin

    1983-01-01

    Research involving adolescent identification with and development of sex roles is reviewed in the areas of cognitive skills and personality traits, theories of sex role development, and minority group adolescent sex role development. Emerging issues and educational implications in these areas are discussed. (CJ)

  5. Zebrafish embryos as models for embryotoxic and teratological effects of chemicals.

    NARCIS (Netherlands)

    Yang, Lixin; Ho, Nga Yu; Alshut, Rüdiger; Legradi, J.B.; Weiss, Carsten; Reischl, Markus; Mikut, Ralf; Liebel, Urban; Müller, Ferenc; Strähle, Uwe

    2009-01-01

    The experimental virtues of the zebrafish embryo such as small size, development outside of the mother, cheap maintenance of the adult made the zebrafish an excellent model for phenotypic genetic and more recently also chemical screens. The availability of a genome sequence and several thousand

  6. An individual-based model of Zebrafish population dynamics accounting for energy dynamics

    DEFF Research Database (Denmark)

    Beaudouin, Remy; Goussen, Benoit; Piccini, Benjamin

    2015-01-01

    Developing population dynamics models for zebrafish is crucial in order to extrapolate from toxicity data measured at the organism level to biological levels relevant to support and enhance ecological risk assessment. To achieve this, a dynamic energy budget for individual zebrafish (DEB model...

  7. [Application of zebrafish model organism in the research of Chinese materia medica].

    Science.gov (United States)

    Chen, Lei; Liu, Yi; Liang, Sheng-Wang

    2012-04-01

    Zebrafish has become an important model organism in many fields of biomedical studies and been increasingly used in Chinese materia medica studies in recent years. This article summarized the achievements and prospect for zebrafish as a pharmacological and toxicological tool in the study and development of Chinese materia medica.

  8. Potential teratogenicity of methimazole: exposure of zebrafish embryos to methimazole causes similar developmental anomalies to human methimazole embryopathy.

    Science.gov (United States)

    Komoike, Yuta; Matsuoka, Masato; Kosaki, Kenjiro

    2013-06-01

    While methimazole (MMI) is widely used in the therapy for hyperthyroidism, several groups have reported that maternal exposure to MMI results in a variety of congenital anomalies, including choanal and esophageal atresia, iridic and retinal coloboma, and delayed neurodevelopment. Thus, adverse effects of maternal exposure to MMI on fetal development have long been suggested; however, direct evidence for the teratogenicity of MMI has not been presented. Therefore, we studied the effects of MMI on early development by using zebrafish as a model organism. The fertilized eggs of zebrafish were collected immediately after spawning and grown in egg culture water containing MMI at various concentrations. External observation of the embryos revealed that exposure to high concentrations of MMI resulted in loss of pigmentation, hypoplastic hindbrain, turbid tissue in the forebrain, swelling of the notochord, and curly trunk. Furthermore, these effects occurred in a dose-dependent manner. Precise observation of the serial cross-sections of MMI-exposed embryos elucidated delayed development and hypoplasia of the whole brain and spinal cord, narrowing of the pharynx and esophagus, severe disruption of the retina, and aberrant structure of the notochord. These neuronal, pharyngeal, esophageal, and retinal anomalous morphologies have a direct analogy to the congenital anomalies observed in children exposed to MMI in utero. Here, we show the teratogenic effects of MMI on the development of zebrafish and provide the first experimental evidence for the connection between exposure to MMI and human MMI embryopathy. © 2013 Wiley Periodicals, Inc.

  9. Disease modeling in genetic kidney diseases: zebrafish.

    Science.gov (United States)

    Schenk, Heiko; Müller-Deile, Janina; Kinast, Mark; Schiffer, Mario

    2017-07-01

    Growing numbers of translational genomics studies are based on the highly efficient and versatile zebrafish (Danio rerio) vertebrate model. The increasing types of zebrafish models have improved our understanding of inherited kidney diseases, since they not only display pathophysiological changes but also give us the opportunity to develop and test novel treatment options in a high-throughput manner. New paradigms in inherited kidney diseases have been developed on the basis of the distinct genome conservation of approximately 70 % between zebrafish and humans in terms of existing gene orthologs. Several options are available to determine the functional role of a specific gene or gene sets. Permanent genome editing can be induced via complete gene knockout by using the CRISPR/Cas-system, among others, or via transient modification by using various morpholino techniques. Cross-species rescues succeeding knockdown techniques are employed to determine the functional significance of a target gene or a specific mutation. This article summarizes the current techniques and discusses their perspectives.

  10. Effects of prolonged exposure to perchlorate on thyroid and reproductive function in zebrafish

    Science.gov (United States)

    Mukhi, S.; Patino, R.

    2007-01-01

    The objectives of this study were to determine the effects of prolonged exposure to perchlorate on (1) thyroid status and reproductive performance of adult zebrafish (Danio rerio) and (2) F1 embryo survival and early larval development. Using a static-renewal procedure, mixed sex populations of adult zebrafish were exposed to 0, 10, and 100 mg/l nominal concentrations of waterborne perchlorate for 10 weeks. Thyroid histology was qualitatively assessed, and females and males were separated and further exposed to their respective treatments for six additional weeks. Eight females in each tank replicate (n = 3) were paired weekly with four males from the same respective treatment, and packed-egg (spawn) volume (PEV) was measured each of the last five weeks. At least once during weeks 14-16 of exposure, other end points measured included fertilization rate, fertilized egg diameter, hatching rate, standard length, and craniofacial development of 4-day-postfertilization larvae and thyroid hormone content of 3.5-h embryos and of exposed mothers. At 10 weeks of exposure, perchlorate at both concentrations caused thyroidal hypertrophy and colloid depletion. A marked reduction in PEV was observed toward the end of the 6-week spawning period, but fertilization and embryo hatching rates were unaffected. Fertilized egg diameter and larval length were increased by parental exposure to perchlorate. Larval head depth was unaffected but the forward protrusion of the lower jaw-associated cartilage complexes, Meckel's and ceratohyal, was decreased. Exposure to both concentrations of perchlorate inhibited whole-body thyroxine content in mothers and embryos, but triiodothyronine content was unchanged. In conclusion, prolonged exposure of adult zebrafish to perchlorate not only disrupts their thyroid endocrine system but also impairs reproduction and influences early F1 development. ?? 2007 Oxford University Press.

  11. The hazard assessment of nanostructured CeO{sub 2}-based mixed oxides on the zebrafish Danio rerio under environmentally relevant UV-A exposure

    Energy Technology Data Exchange (ETDEWEB)

    Jemec, Anita, E-mail: anita.jemec@bf.uni-lj.si [National Institute of Chemistry, Laboratory for Environmental Sciences and Engineering, Hajdrihova 19, SI-1001 Ljubljana (Slovenia); University of Ljubljana, Biotechnical Faculty, Department of Biology, Večna pot 111, SI-1000 Ljubljana (Slovenia); Djinović, Petar; Črnivec, Ilja Gasan Osojnik; Pintar, Albin [National Institute of Chemistry, Laboratory for Environmental Sciences and Engineering, Hajdrihova 19, SI-1001 Ljubljana (Slovenia)

    2015-02-15

    The effect of nanomaterials on biota under realistic environmental conditions is an important question. However, there is still a lack of knowledge on how different illumination conditions alter the toxicity of some photocatalytic nanomaterials. We have investigated how environmentally relevant UV-A exposure (intensity 8.50 ± 0.61 W/m{sup 2}, exposure dose 9.0 J/cm{sup 2}) affected the toxicity of cerium oxide (CeO{sub 2})-based nanostructured materials to the early-life stages of zebrafish Danio rerio. Pure cerium oxide (CeO{sub 2}), copper–cerium (CuO–CeO{sub 2}) (with a nominal 10, 15 and 20 mol.% CuO content), cerium–zirconium (CeO{sub 2}–ZrO{sub 2}) and nickel and cobalt (Ni–Co) deposited over CeO{sub 2}–ZrO{sub 2} were tested. It was found that under both illumination regimes, none of the tested materials affected the normal development or induced mortality of zebrafish early-life stages up to 100 mg/L. Only in the case of CuO–CeO{sub 2}, the growth of larvae was decreased (96 h LOEC values for CuCe10, CuCe15 and CuCe20 were 50, 50 and 10 mg/L, respectively). To conclude, CeO{sub 2}-based nanostructured materials are not severely toxic to zebrafish and environmentally relevant UV-A exposure does not enhance their toxicity. - Highlights: • CeO{sub 2}–ZrO{sub 2} nanomaterials and pure CeO{sub 2} (up to 100 mg/L) were not harmful to zebrafish. • Only CuO modified CeO{sub 2} affected the growth of zebrafish larvae. • UV-A radiation did not enhance the toxicity of tested nanomaterials.

  12. The ventralizing activity of Radar, a maternally expressed bone morphogenetic protein, reveals complex bone morphogenetic protein interactions controlling dorso-ventral patterning in zebrafish.

    Science.gov (United States)

    Goutel, C; Kishimoto, Y; Schulte-Merker, S; Rosa, F

    2000-12-01

    In Xenopus and zebrafish, BMP2, 4 and 7 have been implicated, after the onset of zygotic expression, in inducing and maintaining ventro-lateral cell fate during early development. We provide evidence here that a maternally expressed bone morphogenetic protein (BMP), Radar, may control early ventral specification in zebrafish. We show that Radar ventralizes zebrafish embryos and induces the early expression of bmp2b and bmp4. The analysis of Radar overexpression in both swirl/bmp2b mutants and embryos expressing truncated BMP receptors shows that Radar-induced ventralization is dependent on functional BMP2/4 pathways, and may initially rely on an Alk6-related signaling pathway. Finally, we show that while radar-injected swirl embryos still exhibit a strongly dorsalized phenotype, the overexpression of Radar into swirl/bmp2b mutant embryos restores ventral marker expression, including bmp4 expression. Our results suggest that a complex regulation of different BMP pathways controls dorso-ventral (DV) patterning from early cleavage stages until somitogenesis.

  13. Early psychosis workforce development: Core competencies for mental health professionals working in the early psychosis field.

    Science.gov (United States)

    Osman, Helen; Jorm, Anthony F; Killackey, Eoin; Francey, Shona; Mulcahy, Dianne

    2017-08-09

    The aim of this study was to identify the core competencies required of mental health professionals working in the early psychosis field, which could function as an evidence-based tool to support the early psychosis workforce and in turn assist early psychosis service implementation and strengthen early psychosis model fidelity. The Delphi method was used to establish expert consensus on the core competencies. In the first stage, a systematic literature search was conducted to generate competency items. In the second stage, a panel consisting of expert early psychosis clinicians from around the world was formed. Panel members then rated each of the competency items on how essential they are to the clinical practice of all early psychosis clinicians. In total, 1023 pieces of literature including textbooks, journal articles and grey literature were reviewed. A final 542 competency items were identified for inclusion in the questionnaire. A total of 63 early psychosis experts participated in 3 rating rounds. Of the 542 competency items, 242 were endorsed as the required core competencies. There were 29 competency items that were endorsed by 62 or more experts, and these may be considered the foundational competencies for early psychosis practice. The study generated a set of core competencies that provide a common language for early psychosis clinicians across professional disciplines and country of practice, and potentially are a useful professional resource to support early psychosis workforce development and service reform. © 2017 John Wiley & Sons Australia, Ltd.

  14. Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio.

    Directory of Open Access Journals (Sweden)

    Ansa W Fiaz

    Full Text Available Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

  15. Early embryonic androgen exposure induces transgenerational epigenetic and metabolic changes.

    Science.gov (United States)

    Xu, Ning; Chua, Angela K; Jiang, Hong; Liu, Ning-Ai; Goodarzi, Mark O

    2014-08-01

    Androgen excess is a central feature of polycystic ovary syndrome (PCOS), which affects 6% to 10% of young women. Mammals exposed to elevated androgens in utero develop PCOS-like phenotypes in adulthood, suggesting fetal origins of PCOS. We hypothesize that excess androgen exposure during early embryonic development may disturb the epigenome and disrupt metabolism in exposed and unexposed subsequent generations. Zebrafish were used to study the underlying mechanism of fetal origins. Embryos were exposed to androgens (testosterone and dihydrotestosterone) early at 26 to 56 hours post fertilization or late at 21 to 28 days post fertilization. Exposed zebrafish (F0) were grown to adults and crossed to generate unexposed offspring (F1). For both generations, global DNA methylation levels were examined in ovaries using a luminometric methylation assay, and fasting and postprandial blood glucose levels were measured. We found that early but not late androgen exposure induced changes in global methylation and glucose homeostasis in both generations. In general, F0 adult zebrafish exhibited altered global methylation levels in the ovary; F1 zebrafish had global hypomethylation. Fasting blood glucose levels were decreased in F0 but increased in F1; postprandial glucose levels were elevated in both F0 and F1. This androgenized zebrafish study suggests that transient excess androgen exposure during early development can result in transgenerational alterations in the ovarian epigenome and glucose homeostasis. Current data cannot establish a causal relationship between epigenetic changes and altered glucose homeostasis. Whether transgenerational epigenetic alteration induced by prenatal androgen exposure plays a role in the development of PCOS in humans deserves study.

  16. Medical students' professional identity development in an early nursing attachment

    NARCIS (Netherlands)

    Helmich, Esther; Derksen, Els; Prevoo, Mathieu; Laan, Roland; Bolhuis, Sanneke; Koopmans, Raymond

    Objectives The importance of early clinical experience for medical training is well documented. However, to our knowledge there are no studies that assess the influence of very early nursing attachments on the professional development and identity construction of medical students. Working as an

  17. Medical students' professional identity development in an early nursing attachment.

    NARCIS (Netherlands)

    Helmich, E.; Derksen, E.; Prevoo, M.; Laan, R.F.J.M.; Bolhuis, S.; Koopmans, R.T.C.M.

    2010-01-01

    OBJECTIVES: The importance of early clinical experience for medical training is well documented. However, to our knowledge there are no studies that assess the influence of very early nursing attachments on the professional development and identity construction of medical students. Working as an

  18. Developing Prosocial Behaviors in Early Adolescence with Reactive Aggression

    Science.gov (United States)

    Fung, Annis L. C.

    2008-01-01

    Despite the alarming rise of early adolescence aggression in Hong Kong, it is the pioneer evidence-based outcome study on Anger Coping Training (ACT) program for early adolescence with reactive aggression to develop their prosocial behaviors. This research program involved experimental and control groups with pre- and post-comparison using a …

  19. Chaos, Poverty, and Parenting: Predictors of Early Language Development

    Science.gov (United States)

    Vernon-Feagans, Lynne; Garrett-Peters, Patricia; Willoughby, Michael; Mills-Koonce, Roger

    2012-01-01

    Studies have shown that distal family risk factors like poverty and maternal education are strongly related to children's early language development. Yet, few studies have examined these risk factors in combination with more proximal day-to-day experiences of children that might be critical to understanding variation in early language. Young…

  20. Alberta Learning: Early Development Instrument Pilot Project Evaluation.

    Science.gov (United States)

    Meaney, Wanda; Harris-Lorenze, Elayne

    The Early Development Instrument (EDI) was designed by McMaster University to measure the outcomes of childrens early years as they influence their readiness to learn at school. The EDI was piloted in several Canadian cities in recent years through two national initiatives. Building on these initiatives, Alberta Learning piloted the EDI as a…

  1. Early Speech Motor Development: Cognitive and Linguistic Considerations

    Science.gov (United States)

    Nip, Ignatius S. B.; Green, Jordan R.; Marx, David B.

    2009-01-01

    This longitudinal investigation examines developmental changes in orofacial movements occurring during the early stages of communication development. The goals were to identify developmental trends in early speech motor performance and to determine how these trends differ across orofacial behaviors thought to vary in cognitive and linguistic…

  2. Cyp1a reporter zebrafish reveals target tissues for dioxin

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Kun-Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Park, Hye-Jeong [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Jin Hee [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Kim, Suhyun [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Williams, Darren R. [New Drug Targets Laboratory, School of Life Sciences, Gwangju Institute of Science and Technology, Gwangju (Korea, Republic of); Kim, Myeong-Kyu [Department of Neurology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Jung, Young Do [Department of Biochemistry, Chonnam National University Medical School, Gwangju (Korea, Republic of); Teraoka, Hiroki [School of Veterinary Medicine, Rakuno Gakuen University, Ebetsu (Japan); Park, Hae-Chul [Graduate School of Medicine, Korea University, Ansan (Korea, Republic of); Choy, Hyon E., E-mail: hyonchoy@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Shin, Boo Ahn, E-mail: bashin@chonnam.ac.kr [Department of Microbiology, Chonnam National University Medical School, Gwangju (Korea, Republic of); Choi, Seok-Yong, E-mail: zebrafish@chonnam.ac.kr [Department of Biomedical Sciences, Chonnam National University Medical School, Gwangju (Korea, Republic of); School of Biological Sciences and Technology, Chonnam National University, Gwangju (Korea, Republic of)

    2013-06-15

    Highlights: •2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the most toxic anthropogenic substance ever identified. •Transgenic cyp1a reporter zebrafish reveals target tissues for TCDD. •The retinal bipolar cells, otic vesicle, lateral line, pancreas, cloaca and pectoral fin bud are novel targets in zebrafish for TCDD. •Our findings will further understanding of human health risks by TCDD. -- Abstract: 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is the unintentional byproduct of various industrial processes, is classified as human carcinogen and could disrupt reproductive, developmental and endocrine systems. Induction of cyp1a1 is used as an indicator of TCDD exposure. We sought to determine tissues that are vulnerable to TCDD toxicity using a transgenic zebrafish (Danio rerio) model. We inserted a nuclear enhanced green fluorescent protein gene (EGFP) into the start codon of a zebrafish cyp1a gene in a fosmid clone using DNA recombineering. The resulting recombineered fosmid was then used to generate cyp1a reporter zebrafish, embryos of which were exposed to TCDD. Expression pattern of EGFP in the reporter zebrafish mirrored that of endogenous cyp1a mRNA. In addition, exposure of the embryos to TCDD at as low as 10 pM for 72 h, which does not elicit morphological abnormalities of embryos, markedly increased GFP expression. Furthermore, the reporter embryos responded to other AhR ligands as well. Exposure of the embryos to TCDD revealed previously reported (the cardiovascular system, liver, pancreas, kidney, swim bladder and skin) and unreported target tissues (retinal bipolar cells, otic vesicle, lateral line, cloaca and pectoral fin bud) for TCDD. Transgenic cyp1a reporter zebrafish we have developed can further understanding of ecotoxicological relevance and human health risks by TCDD. In addition, they could be used to identify agonists of AhR and antidotes to TCDD toxicity.

  3. Evaluation of color preference in zebrafish for learning and memory.

    Science.gov (United States)

    Avdesh, Avdesh; Martin-Iverson, Mathew T; Mondal, Alinda; Chen, Mengqi; Askraba, Sreten; Morgan, Newman; Lardelli, Michael; Groth, David M; Verdile, Giuseppe; Martins, Ralph N

    2012-01-01

    There is growing interest in using zebrafish (Danio rerio) as a model of neurodegenerative disorders such as Alzheimer's disease. A zebrafish model of tauopathies has recently been developed and characterized in terms of presence of the pathological hallmarks (i.e., neurofibrillary tangles and cell death). However, it is also necessary to validate these models for function by assessing learning and memory. The majority of tools to assess memory and learning in animal models involve visual stimuli, including color preference. The color preference of zebrafish has received little attention. To validate zebrafish as a model for color-associated-learning and memory, it is necessary to evaluate its natural preferences or any pre-existing biases towards specific colors. In the present study, we have used four different colors (red, yellow, green, and blue) to test natural color preferences of the zebrafish using two procedures: Place preference and T-maze. Results from both experiments indicate a strong aversion toward blue color relative to all other colors (red, yellow, and green) when tested in combinations. No preferences or biases were found among reds, yellows, and greens in the place preference procedure. However, red and green were equally preferred and both were preferred over yellow by zebrafish in the T-maze procedure. The results from the present study show a strong aversion towards blue color compared to red, green, and yellow, with yellow being less preferred relative to red and green. The findings from this study may underpin any further designing of color-based learning and memory paradigms or experiments involving aversion, anxiety, or fear in the zebrafish.

  4. ECR-MAPK regulation in liver early development.

    Science.gov (United States)

    Zhao, Xiu-Ju; Zhuo, Hexian

    2014-01-01

    Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region)-socs2 (-SH2-containing signals/receptor tyrosine kinases)-ppp2r2a/pik3c3 (MAPK signaling)-hsd3b5/cav2 (metabolism/organization) plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development.

  5. ECR-MAPK Regulation in Liver Early Development

    Directory of Open Access Journals (Sweden)

    Xiu-Ju Zhao

    2014-01-01

    Full Text Available Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region-socs2 (-SH2-containing signals/receptor tyrosine kinases-ppp2r2a/pik3c3 (MAPK signaling-hsd3b5/cav2 (metabolism/organization plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development.

  6. Embryological exposure to valproic acid induces social interaction deficits in zebrafish (Danio rerio): A developmental behavior analysis.

    Science.gov (United States)

    Zimmermann, Fernanda Francine; Gaspary, Karina Vidarte; Leite, Carlos Eduardo; De Paula Cognato, Giana; Bonan, Carla Denise

    2015-01-01

    Changes in social behavior are associated with brain disorders, including mood disorders, stress, schizophrenia, Alzheimer's disease, and autism spectrum disorders (ASD). Autism is a complex neurodevelopmental disorder characterized by deficits in social interaction, impaired communication, anxiety, hyperactivity, and the presence of restricted interests. Zebrafish is one of the most social vertebrates used as a model in biomedical research, contributing to an understanding of the mechanisms that underlie social behavior. Valproic acid (VPA) is used as an anti-epileptic drug and mood stabilizer; however, prenatal VPA exposure in humans has been associated with an increased incidence of autism and it can also affect fetal brain development. Therefore, we conducted a behavioral screening at different periods of zebrafish development at 6, 30, 70, and 120dpf (days postfertilization) after VPA exposure in the early development stage to investigate social behavior, locomotion, aggression, and anxiety. VPA (48μM) exposure during the first 48hpf (hours postfertilization) did not promote changes on survival, morphology, and hatching rate at 24hpf, 48hpf, and 72hpf. The behavioral patterns suggest that VPA exposure induces changes in locomotor activity and anxiety at different developmental periods in zebrafish. Furthermore, a social interaction deficit is present at 70dpf and 120dpf. VPA exposure did not affect aggression in the adult stage at 70dpf and 120dpf. This is the first study that demonstrated zebrafish exposed to VPA during the first 48h of development exhibit deficits in social interaction, anxiety, and hyperactivity at different developmental periods. Copyright © 2015 Elsevier Inc. All rights reserved.

  7. Apoptotic cell elimination during early tooth development

    Czech Academy of Sciences Publication Activity Database

    Matalová, Eva; Míšek, Ivan; Chovancová, Eva

    2003-01-01

    Roč. 72, č. 7 (2003), s. 34 ISSN 0001-7213. [Congress of the European Association of Veterinary Anatomists/24./. 21.07.2002-25.07.2002, Brno] R&D Projects: GA ČR GP204/02/P112 Institutional research plan: CEZ:AV0Z5045916 Keywords : tooth development Subject RIV: FF - HEENT, Dentistry

  8. EARLY POSTNATAL DEVELOPMENT OF THE SOUTH AFRICAN ...

    African Journals Online (AJOL)

    from a 50:50 ratio (53/47 per cent). Minimum ... back became progressively darker and the vibrissae lengthened to about 8-9 mm. On day 6 ... of grey developed around the eyes by day 8, when the belly had a slight white tinge from the short ...

  9. Early developments in solar cooling equipment

    Science.gov (United States)

    Price, J. M.

    1978-01-01

    A brief description of a development program to design, fabricate and field test a series of solar operated or driven cooling devices, undertaken by the Marshall Space Flight Center in the context of the Solar Heating and Cooling Demonstration Act of 1974, is presented. Attention is given to two basic design concepts: the Rankine cycle principle and the use of a dessicant for cooling.

  10. Pharmacometrics in early clinical drug development

    NARCIS (Netherlands)

    Keizer, R.J.

    2010-01-01

    Pharmacometrics, the science of quantitative clinical pharmacology, has been recognized as one of the main research fields able to improve efficiency in drug development, and to reduce attrition rates on the route from drug discovery to approval. This field of drug research, which builds heavily on

  11. Plasticity during Early Brain Development Is Determined by Ontogenetic Potential.

    Science.gov (United States)

    Krägeloh-Mann, Ingeborg; Lidzba, Karen; Pavlova, Marina A; Wilke, Marko; Staudt, Martin

    2017-04-01

    Two competing hypotheses address neuroplasticity during early brain development: the "Kennard principle" describes the compensatory capacities of the immature developing CNS as superior to those of the adult brain, whereas the "Hebb principle" argues that the young brain is especially sensitive to insults. We provide evidence that these principles are not mutually exclusive. Following early brain lesions that are unilateral, the brain can refer to homotopic areas of the healthy hemisphere. This potential for reorganization is unique to the young brain but available only when, during ontogenesis of brain development, these areas have been used for the functions addressed. With respect to motor function, ipsilateral motor tracts can be recruited, which are only available during early brain development. Language can be reorganized to the right after early left hemispheric lesions, as the representation of the language network is initially bilateral. However, even in these situations, compensatory capacities of the developing brain are found to have limitations, probably defined by early determinants. Thus, plasticity and adaptivity are seen only within ontogenetic potential; that is, axonal or cortical structures cannot be recruited beyond early developmental possibilities. The young brain is probably more sensitive and vulnerable to lesions when these are bilateral. This is shown here for bilateral periventricular white matter lesions that clearly have an impact on cortical architecture and function, thus probably interfering with early network building. Georg Thieme Verlag KG Stuttgart · New York.

  12. Gene transcription ontogeny of hypothalamic-pituitary-thyroid-axis development in early-life stage fathead minnow and zebrafish

    Science.gov (United States)

    Disruption of thyroid hormone signaling is a form of endocrine disruption that is of concern to both human health and ecosystems. Research is being conducted to define the biological targets chemicals may interact with to disrupt thyroid hormone signaling and the stages in develo...

  13. Behavior-Based Early Language Development on a Humanoid Robot

    National Research Council Canada - National Science Library

    Varshavskaya, Paulina

    2002-01-01

    We are exploring the idea that early language acquisition could be better modelled on an artificial creature by considering the pragmatic aspect of natural language and of its development in human infants...

  14. Habituation of the C-start response in larval zebrafish exhibits several distinct phases and sensitivity to NMDA receptor blockade.

    Directory of Open Access Journals (Sweden)

    Adam C Roberts

    Full Text Available The zebrafish larva has been a valuable model system for genetic and molecular studies of development. More recently, biologists have begun to exploit the surprisingly rich behavioral repertoire of zebrafish larvae to investigate behavior. One prominent behavior exhibited by zebrafish early in development is a rapid escape reflex (the C-start. This reflex is mediated by a relatively simple neural circuit, and is therefore an attractive model behavior for neurobiological investigations of simple forms of learning and memory. Here, we describe two forms of short-lived habituation of the C-start in response to brief pulses of auditory stimuli. A rapid form, persisting for ≥1 min but <15 min, was induced by 120 pulses delivered at 0.5-2.0 Hz. A more extended form (termed "short-term habituation" here, which persisted for ≥25 min but <1 h, was induced by spaced training. The spaced training consisted of 10 blocks of auditory pulses delivered at 1 Hz (5 min interblock interval, 900 pulses per block. We found that these two temporally distinguishable forms of habituation are mediated by different cellular mechanisms. The short-term form depends on activation of N-methyl-d-aspartate receptors (NMDARs, whereas the rapid form does not.

  15. Early bilingualism, language attainment, and brain development.

    Science.gov (United States)

    Berken, Jonathan A; Gracco, Vincent L; Klein, Denise

    2017-04-01

    The brain demonstrates a remarkable capacity to undergo structural and functional change in response to experience throughout the lifespan. Evidence suggests that, in many domains of skill acquisition, the manifestation of this neuroplasticity depends on the age at which learning begins. The fact that most skills are acquired late in childhood or in adulthood has proven to be a limitation in studies aimed at determining the relationship between age of acquisition and brain plasticity. Bilingualism, however, provides an optimal model for discerning differences in how the brain wires when a skill is acquired from birth, when the brain circuitry for language is being constructed, versus later in life, when the pathways subserving the first language are already well developed. This review examines some of the existing knowledge about optimal periods in language development, with particular attention to the attainment of native-like phonology. It focuses on the differences in brain structure and function between simultaneous and sequential bilinguals and the compensatory mechanisms employed when bilingualism is achieved later in life, based on evidence from studies using a variety of neuroimaging modalities, including positron emission tomography (PET), task-based and resting-state functional magnetic resonance imaging (fMRI), and structural MRI. The discussion concludes with the presentation of recent neuroimaging studies that explore the concept of nested optimal periods in language development and the different neural paths to language proficiency taken by simultaneous and sequential bilinguals, with extrapolation to general notions of the relationship between age of acquisition and ultimate skill performance. Copyright © 2016 Elsevier Ltd. All rights reserved.

  16. Resource Prospector (RP) - Early Prototyping and Development

    Science.gov (United States)

    Andrews, D.; Colaprete, A.; Quinn, J.; Bluethmann, B.; Trimble, J.

    2015-01-01

    The Resource Prospector (RP) is an In-Situ Resource Utilization (ISRU) technology demonstration mission under study by the NASA Human Exploration and Operations Mission Directorate's (HEOMD) Advanced Exploration Systems (AES) Division. The mission, currently planned to launch in 2020, will demonstrate extraction of oxygen from lunar regolith to validate ISRU capability. The mission will address key Strategic Knowledge Gaps (SKGs) for robotic and human exploration to the Moon, Near Earth Asteroids (NEAs), and ultimately Mars, as well as meet the strategic goals of the Global Exploration Roadmap (GER), offered by the International Space Exploration Coordination Group (ISECG). In this roadmap, the use of local resources is specifically addressed relating to human exploration. RP will provide knowledge to inform the selection of future mission destinations, support the development of exploration systems, and reduce the risk associated with human exploration. Expanding human presence beyond low-Earth orbit to asteroids and Mars will require the maximum possible use of local materials, so-called in-situ resources. The moon presents a unique destination to conduct robotic investigations that advance ISRU capabilities, as well as providing significant exploration and science value. Lunar regolith contains useful resources such as oxygen, water, silicon, and light metals, like aluminum and titanium. Oxygen can be separated from the regolith for life support (breathable air), or used to create rocket propellant (oxidizer). Regolith can be used to protect against radiation exposure, be processed into solar cells, or used to manufacture construction materials such as bricks and glass. RP will characterize the constituents and distribution of water and other volatiles at the poles of the Moon, enabling innovative uses of local resources, in addition to validating ISRU capabilities. This capability, as well as a deeper understanding of regolith, will be valuable in the

  17. Early life exposure to environmental levels of the aromatase inhibitor tributyltin causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio).

    Science.gov (United States)

    McAllister, Brian G; Kime, David E

    2003-11-19

    To determine whether early life exposure to tributyltin (TBT), an aromatase inhibitor, impaired reproductive function in fish, Danio rerio were exposed to environmentally realistic levels (0.01-100 ng l(-1)) of TBT from 0 to 30, 30 to 60, and 0 to 70 days post-hatch, and the sex ratio and sperm motility of the adults examined 3-5 months after cessation of exposure. Fish exposed for 70 days to 0.1 ng l(-1) of TBT, a concentration presently below the detection limit in water, showed a male biased population which produced a high incidence of sperm lacking flagella. At 1 ng l(-1), the motility of sperm was significantly lower than that of control fish, while at 10 ng l(-1), all sperm lacked flagella and, at 100 ng l(-1), milt volume had increased. The effect of exposure on sex ratio was similar after exposure from 0 to 70 and 0 to 30 days, but even 100 ng l(-1) gave only 65% males after exposure from 30 to 60 days. Effects on sperm motility and morphology and on milt volume were less pronounced after 30 day than 70 day exposure. Our data suggest that screening for aromatase inhibiting activity and assessment of its risks in early life to human and wildlife fertility needs to be urgently addressed, and that the reproductive toxicity of TBT may presently be underestimated.

  18. Cross-Modal Learning between Visual and Vibration Signals in Zebrafish Danio Rerio

    Directory of Open Access Journals (Sweden)

    Mu-Yun Wang

    2011-10-01

    Full Text Available Animals are always integrating environmental information from multiple sensory modalities, but the mechanisms underneath are highly underexploited. Crossmodal interactions in animal perception have been found in several species including human, mice and flies. Here we subjected zebrafish as model because its genetic effects on brain and sense organ development are well studied, but the attentional processes are mainly unexplored. Zebrafish show impressive behaviour capabilities with relatively small or “simple” brains which make their nervous system relatively more accessible to experimentation than large-brained mammals. When conditioned with both vision and vibration signals, zebrafish were able to make higher correct choices than only one sensation. After multimodal training, zebrafish were also able to transfer the memory to unimodal conditioning when only given vision or vibration signals. This study provided basic findings for how animals process multimodal sensory from the environment, and showed crossmodal interactions in zebrafish for the first time.

  19. Zebrafish models of cardiovascular diseases and their applications in herbal medicine research.

    Science.gov (United States)

    Seto, Sai-Wang; Kiat, Hosen; Lee, Simon M Y; Bensoussan, Alan; Sun, Yu-Ting; Hoi, Maggie P M; Chang, Dennis

    2015-12-05

    The zebrafish (Danio rerio) has recently become a powerful animal model for cardiovascular research and drug discovery due to its ease of maintenance, genetic manipulability and ability for high-throughput screening. Recent advances in imaging techniques and generation of transgenic zebrafish have greatly facilitated in vivo analysis of cellular events of cardiovascular development and pathogenesis. More importantly, recent studies have demonstrated the functional similarity of drug metabolism systems between zebrafish and humans, highlighting the clinical relevance of employing zebrafish in identifying lead compounds in Chinese herbal medicine with potential beneficial cardiovascular effects. This paper seeks to summarise the scope of zebrafish models employed in cardiovascular studies and the application of these research models in Chinese herbal medicine to date. Crown Copyright © 2015. Published by Elsevier B.V. All rights reserved.

  20. Advancing Early Childhood Development: from Science to Scale 1

    Science.gov (United States)

    Black, Maureen M; Walker, Susan P; Fernald, Lia C H; Andersen, Christopher T; DiGirolamo, Ann M; Lu, Chunling; McCoy, Dana C; Fink, Günther; Shawar, Yusra R; Shiffman, Prof Jeremy; Devercelli, Amanda E; Wodon, Quentin T; Vargas-Barón, Emily; Grantham-McGregor, Sally

    2018-01-01

    Early childhood development programmes vary in coordination and quality, with inadequate and inequitable access, especially for children younger than 3 years. New estimates, based on proxy measures of stunting and poverty, indicate that 250 million children (43%) younger than 5 years in low-income and middle-income countries are at risk of not reaching their developmental potential. There is therefore an urgent need to increase multisectoral coverage of quality programming that incorporates health, nutrition, security and safety, responsive caregiving, and early learning. Equitable early childhood policies and programmes are crucial for meeting Sustainable Development Goals, and for children to develop the intellectual skills, creativity, and wellbeing required to become healthy and productive adults. In this paper, the first in a three part Series on early childhood development, we examine recent scientific progress and global commitments to early childhood development. Research, programmes, and policies have advanced substantially since 2000, with new neuroscientific evidence linking early adversity and nurturing care with brain development and function throughout the life course. PMID:27717614

  1. Two-photon-based photoactivation in live zebrafish embryos.

    Science.gov (United States)

    Russek-Blum, Niva; Nabel-Rosen, Helit; Levkowitz, Gil

    2010-12-24

    Photoactivation of target compounds in a living organism has proven a valuable approach to investigate various biological processes such as embryonic development, cellular signaling and adult physiology. In this respect, the use of multi-photon microscopy enables quantitative photoactivation of a given light responsive agent in deep tissues at a single cell resolution. As zebrafish embryos are optically transparent, their development can be monitored in vivo. These traits make the zebrafish a perfect model organism for controlling the activity of a variety of chemical agents and proteins by focused light. Here we describe the use of two-photon microscopy to induce the activation of chemically caged fluorescein, which in turn allows us to follow cell's destiny in live zebrafish embryos. We use embryos expressing a live genetic landmark (GFP) to locate and precisely target any cells of interest. This procedure can be similarly used for precise light induced activation of proteins, hormones, small molecules and other caged compounds.

  2. Hypoxia-induced retinopathy model in adult zebrafish

    DEFF Research Database (Denmark)

    Cao, Ziquan; Jensen, Lasse D.; Rouhi, Pegah

    2010-01-01

    Hypoxia-induced vascular responses, including angiogenesis, vascular remodeling and vascular leakage, significantly contribute to the onset, development and progression of retinopathy. However, until recently there were no appropriate animal disease models recapitulating adult retinopathy available....... In this article, we describe protocols that create hypoxia-induced retinopathy in adult zebrafish. Adult fli1: EGFP zebrafish are placed in hypoxic water for 3-10 d and retinal neovascularization is analyzed using confocal microscopy. It usually takes 11 d to obtain conclusive results using the hypoxia......-induced retinopathy model in adult zebrafish. This model provides a unique opportunity to study kinetically the development of retinopathy in adult animals using noninvasive protocols and to assess therapeutic efficacy of orally active antiangiogenic drugs....

  3. Molecular cloning and developmental expression of Tlx (Hox11) genes in zebrafish (Danio rerio).

    Science.gov (United States)

    Langenau, D M; Palomero, T; Kanki, J P; Ferrando, A A; Zhou, Y; Zon, L I; Look, A T

    2002-09-01

    Tlx (Hox11) genes are orphan homeobox genes that play critical roles in the regulation of early developmental processes in vertebrates. Here, we report the identification and expression patterns of three members of the zebrafish Tlx family. These genes share similar, but not identical, expression patterns with other vertebrate Tlx-1 and Tlx-3 genes. Tlx-1 is expressed early in the developing hindbrain and pharyngeal arches, and later in the putative splenic primordium. However, unlike its orthologues, zebrafish Tlx-1 is not expressed in the cranial sensory ganglia or spinal cord. Two homologues of Tlx-3 were identified: Tlx-3a and Tlx-3b, which are both expressed in discrete regions of the developing nervous system, including the cranial sensory ganglia and Rohon-Beard neurons. However, only Tlx-3a is expressed in the statoacoustic cranial ganglia, enteric neurons and non-neural tissues such as the fin bud and pharyngeal arches and Tlx-3b is only expressed in the dorsal root ganglia. Copyright 2002 Elsevier Science Ireland Ltd.

  4. Sinup, a novel Siaz-interacting nuclear protein, modulates neural plate formation in the zebrafish embryos

    International Nuclear Information System (INIS)

    Ro, Hyunju; Won, Minho; Lee, Su-Ui; Kim, Kyoon E.; Huh, Tae-Lin; Kim, Cheol-Hee; Rhee, Myungchull

    2005-01-01

    Siah, the vertebrate homologue of the Drosophila seven in absentia (sina) gene, is well conserved from Drosophila to mammal and involved in ubiquitination and proteasome-dependent degradation of various target proteins. To identify cellular proteins interacting with Siah, we screened a zebrafish cDNA library with zebrafish Siah (Siaz) as bait in a yeast two-hybrid assay. We identified a cDNA encoding a novel protein composed of 145 amino acids and termed it as Sinup (Siaz-interacting-nuclear-protein). Sinup is a novel nuclear protein that binds to the highly conserved C-terminal protein-interacting domain of Siaz both in vivo and in vitro. During development, sinup transcripts are abundant from the one-cell stage to the early blastula and then markedly diminished, suggesting sinup largely exists as maternal transcripts. sinup overexpression induced lateral expansion of the neural plate and in consequence caused ectopic expression of otx-2 and hoxb1b during the late gastrula stage. In addition, the lateral/paraxial expression of wnt8 at the onset of gastrulation is suppressed by the forced expression of sinup while the expression levels of various dorso-ventral markers are unaffected. In contrast, interfering with sinup functions using sinup morpholino oligonucleotides gradually diminished the anterior neuroectoderm from the posterior region, and resulted in compete loss of hindbrain at the 3-somites stage. Our report suggests that sinup expression should be tightly regulated during early embryonic development for the proper neural plate formation

  5. Clofibrate and gemfibrozil induce an embryonic malabsorption syndrome in zebrafish

    International Nuclear Information System (INIS)

    Raldua, Demetrio; Andre, Michele; Babin, Patrick J.

    2008-01-01

    Nutrient availability is one of the major non-genetic factors determining embryonic growth and larval or fetal size. Due to the high human consumption of blood lipid regulators, fibrates have recently been reported as pollutants in rivers. Our study investigated the developmental toxicity of fibrates in zebrafish. Treatment with micromolar concentrations of clofibrate or gemfibrozil induced an embryonic malabsorption syndrome (EMS) with very little yolk consumption, resulting in small-sized larvae. This effect was reversible on removing the drug from the water. Clofibrate delayed hatching time and decreased the amount of oil red O lipid staining in the vasculature. It also induced higher density, round-shaped neuromuscular junctions associated with disorganization and less striation of muscular fibers, and pericardial edema, as well as impairing thyroid gland morphogenesis. acox1, apoa1 and mtp hybridization transcript signals were not affected in the yolk syncytial layer (YSL) after clofibrate exposure. Di-(2-ethylhexyl)-phthalate did not slow down yolk resorption, whereas brefeldin A induced EMS. These findings suggest that the inhibition of yolk sac resorption on exposure to fibrate is not at a pre-translational level or peroxisome proliferator-activated receptor alpha dependent and may be due to an inhibition of the YSL constitutive cell secretion. The effects of fibrates and the potential bioconcentration in eggs as well as the additive action of structurally related toxicants warrant an evaluation of the developmental impact of these compounds after long-term exposure at environmentally relevant concentrations. Fibrate-induced EMS in zebrafish seems useful for studying the morphogenetic consequences of impaired nutrient availability during the early stages of vertebrate development

  6. Parametric Cost and Schedule Modeling for Early Technology Development

    Science.gov (United States)

    2018-04-02

    Research NoteNational Security Rep rt PARAMETRIC MODELING FOR EARLY TECHNOLOGY DEVELOPMENT COST AND SCHEDULE Chuck...Alexander NSR_11x17_Cover_CostModeling_v8.indd 1 11/20/17 3:15 PM PARAMETRIC COST AND SCHEDULE MODELING FOR EARLY  TECHNOLOGY DEVELOPMENT Chuck...COST AND SCHEDULE MODELING FOR EARLY  TECHNOLOGY DEVELOPMENT iii Contents Figures

  7. Nppa and Nppb act redundantly during zebrafish cardiac development to confine AVC marker expression and reduce cardiac jelly volume.

    Science.gov (United States)

    Grassini, Daniela R; Lagendijk, Anne K; De Angelis, Jessica E; Da Silva, Jason; Jeanes, Angela; Zettler, Nicole; Bower, Neil I; Hogan, Benjamin M; Smith, Kelly A

    2018-05-11

    Atrial natriuretic peptide ( nppa/anf ) and brain natriuretic peptide ( nppb/bnp ) form a gene cluster with expression in the chambers of the developing heart. Despite restricted expression, a function in cardiac development has not been demonstrated by mutant analysis. This is attributed to functional redundancy however their genomic location in cis has impeded formal analysis. Using genome-editing, we generated mutants for nppa and nppb and found single mutants indistinguishable from wildtype whereas nppa / nppb double mutants display heart morphogenesis defects and pericardial oedema. Analysis of atrioventricular canal (AVC) markers show expansion of bmp4 , tbx2b, has2 and versican expression into the atrium of double mutants. This expanded expression correlates with increased extracellular matrix in the atrium. Using a biosensor for Hyaluronic acid to measure the cardiac jelly (cardiac extracellular matrix), we confirm cardiac jelly expansion in nppa / nppb double mutants. Finally, bmp4 knockdown rescues the expansion of has2 expression and cardiac jelly in double mutants. This definitively shows that nppa and nppb function redundantly during cardiac development to restrict gene expression to the AVC, preventing excessive cardiac jelly synthesis in the atrial chamber. © 2018. Published by The Company of Biologists Ltd.

  8. Myomaker mediates fusion of fast myocytes in zebrafish embryos

    Energy Technology Data Exchange (ETDEWEB)

    Landemaine, Aurélie; Rescan, Pierre-Yves; Gabillard, Jean-Charles, E-mail: Jean-charles.gabillard@rennes.inra.fr

    2014-09-05

    Highlights: • Myomaker is transiently expressed in fast myocytes during embryonic myogenesis. • Myomaker is essential for fast myocyte fusion in zebrafish. • The function of myomaker is conserved among Teleostomi. - Abstract: Myomaker (also called Tmem8c), a new membrane activator of myocyte fusion was recently discovered in mice. Using whole mount in situ hybridization on zebrafish embryos at different stages of embryonic development, we show that myomaker is transiently expressed in fast myocytes forming the bulk of zebrafish myotome. Zebrafish embryos injected with morpholino targeted against myomaker were alive after yolk resorption and appeared morphologically normal, but they were unable to swim, even under effect of a tactile stimulation. Confocal observations showed a marked phenotype characterized by the persistence of mononucleated muscle cells in the fast myotome at developmental stages where these cells normally fuse to form multinucleated myotubes. This indicates that myomaker is essential for myocyte fusion in zebrafish. Thus, there is an evolutionary conservation of myomaker expression and function among Teleostomi.

  9. Neurotransmitter-Regulated Regeneration in the Zebrafish Retina

    Directory of Open Access Journals (Sweden)

    Mahesh B. Rao

    2017-04-01

    Full Text Available Summary: Current efforts to repair damaged or diseased mammalian retinas are inefficient and largely incapable of fully restoring vision. Conversely, the zebrafish retina is capable of spontaneous regeneration upon damage using Müller glia (MG-derived progenitors. Understanding how zebrafish MG initiate regeneration may help develop new treatments that prompt mammalian retinas to regenerate. We show that inhibition of γ-aminobutyric acid (GABA signaling facilitates initiation of MG proliferation. GABA levels decrease following damage, and MG are positioned to detect decreased ambient levels and undergo dedifferentiation. Using pharmacological and genetic approaches, we demonstrate that GABAA receptor inhibition stimulates regeneration in undamaged retinas while activation inhibits regeneration in damaged retinas. : Unlike mammals, zebrafish regenerate following retina damage from a resident adult stem cell (Müller glia. Dissecting the mechanisms that zebrafish use could lead to new therapeutic targets to treat retinal diseases. Patton and colleagues have discovered a mechanism by which decreased GABA levels are sensed by Müller glia to initiate a regenerative response. Keywords: zebrafish, retina, regeneration, Müller glia, GABA

  10. Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

    Science.gov (United States)

    Bailey, Jordan M; Oliveri, Anthony N; Levin, Edward D

    2015-12-01

    Over the last decade, zebrafish (Danio rerio) have become valuable as a complementary model in behavioral pharmacology, opening a new avenue for understanding the relationships between drug action and behavior. This species offers a useful intermediate approach bridging the gap between in vitro studies and traditional mammalian models. Zebrafish offer great advantages of economy compared to their rodent counterparts, their complex brains and behavioral repertoire offer great translational potential relative to in vitro models. The development and validation of a variety of tests to measure behavior, including cognition, in zebrafish have set the stage for the use of this animal for behavioral pharmacology studies. This has led to research into the basic mechanisms of cognitive function as well as screening for potential cognition-improving drug therapies, among other lines of research. As with all models, zebrafish have limitations, which span pharmacokinetic challenges to difficulties quantifying behavior. The use, efficacy and limitations associated with a zebrafish model of cognitive function are discussed in this review, within the context of behavioral pharmacology. Copyright © 2015 Elsevier Inc. All rights reserved.

  11. Comprehensive analysis of coding-lncRNA gene co-expression network uncovers conserved functional lncRNAs in zebrafish.

    Science.gov (United States)

    Chen, Wen; Zhang, Xuan; Li, Jing; Huang, Shulan; Xiang, Shuanglin; Hu, Xiang; Liu, Changning

    2018-05-09

    Zebrafish is a full-developed model system for studying development processes and human disease. Recent studies of deep sequencing had discovered a large number of long non-coding RNAs (lncRNAs) in zebrafish. However, only few of them had been functionally characterized. Therefore, how to take advantage of the mature zebrafish system to deeply investigate the lncRNAs' function and conservation is really intriguing. We systematically collected and analyzed a series of zebrafish RNA-seq data, then combined them with resources from known database and literatures. As a result, we obtained by far the most complete dataset of zebrafish lncRNAs, containing 13,604 lncRNA genes (21,128 transcripts) in total. Based on that, a co-expression network upon zebrafish coding and lncRNA genes was constructed and analyzed, and used to predict the Gene Ontology (GO) and the KEGG annotation of lncRNA. Meanwhile, we made a conservation analysis on zebrafish lncRNA, identifying 1828 conserved zebrafish lncRNA genes (1890 transcripts) that have their putative mammalian orthologs. We also found that zebrafish lncRNAs play important roles in regulation of the development and function of nervous system; these conserved lncRNAs present a significant sequential and functional conservation, with their mammalian counterparts. By integrative data analysis and construction of coding-lncRNA gene co-expression network, we gained the most comprehensive dataset of zebrafish lncRNAs up to present, as well as their systematic annotations and comprehensive analyses on function and conservation. Our study provides a reliable zebrafish-based platform to deeply explore lncRNA function and mechanism, as well as the lncRNA commonality between zebrafish and human.

  12. Excessive nitrite affects zebrafish valvulogenesis through yielding too much NO signaling.

    Directory of Open Access Journals (Sweden)

    Junbo Li

    Full Text Available Sodium nitrite, a common food additive, exists widely not only in the environment but also in our body. Excessive nitrite causes toxicological effects on human health; however, whether it affects vertebrate heart valve development remains unknown. In vertebrates, developmental defects of cardiac valves usually lead to congenital heart disease. To understand the toxic effects of nitrite on valvulogenesis, we exposed zebrafish embryos with different concentrations of sodium nitrite. Our results showed that sodium nitrite caused developmental defects of zebrafish heart dose dependently. It affected zebrafish heart development starting from 36 hpf (hour post fertilization when heart initiates looping process. Comprehensive analysis on the embryos at 24 hpf and 48 hpf showed that excessive nitrite did not affect blood circulation, vascular network, myocardium and endocardium development. But development of endocardial cells in atrioventricular canal (AVC of the embryos at 48 hpf was disrupted by too much nitrite, leading to defective formation of primitive valve leaflets at 76 hpf. Consistently, excessive nitrite diminished expressions of valve progenitor markers including bmp4, has2, vcana and notch1b at 48 hpf. Furthermore, 3', 5'-cyclic guanosine monophosphate (cGMP, downstream of nitric oxide (NO signaling, was increased its level significantly in the embryos exposed with excessive nitrite and microinjection of soluble guanylate cyclase inhibitor ODQ (1H-[1], [2], [4]Oxadiazolo[4,3-a] quinoxalin-1-one, an antagonist of NO signaling, into nitrite-exposed embryos could partly rescue the cardiac valve malformation. Taken together, our results show that excessive nitrite affects early valve leaflet formation by producing too much NO signaling.

  13. Premature aging in telomerase-deficient zebrafish

    Directory of Open Access Journals (Sweden)

    Monique Anchelin

    2013-09-01

    The study of telomere biology is crucial to the understanding of aging and cancer. In the pursuit of greater knowledge in the field of human telomere biology, the mouse has been used extensively as a model. However, there are fundamental differences between mouse and human cells. Therefore, additional models are required. In light of this, we have characterized telomerase-deficient zebrafish (Danio rerio as the second vertebrate model for human telomerase-driven diseases. We found that telomerase-deficient zebrafish show p53-dependent premature aging and reduced lifespan in the first generation, as occurs in humans but not in mice, probably reflecting the similar telomere length in fish and humans. Among these aging symptoms, spinal curvature, liver and retina degeneration, and infertility were the most remarkable. Although the second-generation embryos died in early developmental stages, restoration of telomerase activity rescued telomere length and survival, indicating that telomerase dosage is crucial. Importantly, this model also reproduces the disease anticipation observed in humans with dyskeratosis congenita (DC. Thus, telomerase haploinsufficiency leads to anticipation phenomenon in longevity, which is related to telomere shortening and, specifically, with the proportion of short telomeres. Furthermore, p53 was induced by telomere attrition, leading to growth arrest and apoptosis. Importantly, genetic inhibition of p53 rescued the adverse effects of telomere loss, indicating that the molecular mechanisms induced by telomere shortening are conserved from fish to mammals. The partial rescue of telomere length and longevity by restoration of telomerase activity, together with the feasibility of the zebrafish for high-throughput chemical screening, both point to the usefulness of this model for the discovery of new drugs able to reactivate telomerase in individuals with DC.

  14. Function of JARID2 in bovines during early embryonic development

    Directory of Open Access Journals (Sweden)

    Yao Fu

    2017-12-01

    Full Text Available Histone lysine modifications are important epigenetic modifications in early embryonic development. JARID2, which is a member of the jumonji demethylase protein family, is a regulator of early embryonic development and can regulate mouse development and embryonic stem cell (ESC differentiation by modifying histone lysines. JARID2 can affect early embryonic development by regulating the methylation level of H3K27me3, which is closely related to normal early embryonic development. To investigate the expression pattern of JARID2 and the effect of JARID2-induced H3K27 methylation in bovine oocytes and early embryonic stages, JARID2 mRNA expression and localization were detected in bovine oocytes and early embryos via qRT-PCR and immunofluorescence in the present study. The results showed that JARID2 is highly expressed in the germinal vesicle (GV, MII, 2-cell, 4-cell, 8-cell, 16-cell and blastocyst stages, but the relative expression level of JARID2 in bovine GV oocytes is significantly lower than that at other oocyte/embryonic stages (p < 0.05, and JARID2 is expressed primarily in the nucleus. We next detected the mRNA expression levels of embryonic development-related genes (OCT4, SOX2 and c-myc after JARID2 knockdown through JARID2-2830-siRNA microinjection to investigate the molecularpathwayunderlying the regulation of H3K27me3 by JARID2 during early embryonic development. The results showed that the relative expression levels of these genes in 2-cell embryos weresignificantly higher than those in the blastocyst stage, and expression levels were significantly increased after JARID2 knockdown. In summary, the present study identified the expression pattern of JARID2 in bovine oocytes and at each early embryonic stage, and the results suggest that JARID2 plays a key role in early embryonic development by regulating the expression of OCT4, SOX2 and c-myc via modification of H3K27me3 expression. This work provides new data for improvements in the

  15. Targeted knock-in of CreER T2 in zebrafish using CRISPR/Cas9.

    Science.gov (United States)

    Kesavan, Gokul; Hammer, Juliane; Hans, Stefan; Brand, Michael

    2018-04-01

    New genome-editing approaches, such as the CRISPR/Cas system, have opened up great opportunities to insert or delete genes at targeted loci and have revolutionized genetics in model organisms like the zebrafish. The Cre-loxp recombination system is widely used to activate or inactivate genes with high spatial and temporal specificity. Using a CRISPR/Cas9-mediated knock-in strategy, we inserted a zebrafish codon-optimized CreER T2 transgene at the otx2 gene locus to generate a conditional Cre-driver line. We chose otx2 as it is a patterning gene of the anterior neural plate that is expressed during early development. By knocking in CreER T2 upstream of the endogenous ATG of otx2, we utilized this gene's native promoter and enhancer elements to perfectly match CreER T2 and endogenous otx2 expression patterns. Next, by combining this novel driver line with a Cre-dependent reporter line, we show that only in the presence of tamoxifen can efficient Cre-loxp-mediated recombination be achieved in the anterior neural plate-derived tissues like the telencephalon, the eye and the optic tectum. Our results imply that the otx2:CreER T2 transgenic fish will be a valuable tool for lineage tracing and conditional mutant studies in larval and adult zebrafish.

  16. Determination of monoamine neurotransmitters in zebrafish (Danio rerio) by gas chromatography coupled to mass spectrometry with a two-step derivatization.

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    Aragon, Alvaro; Legradi, Jessica; Ballesteros-Gómez, Ana; Legler, Juliette; van Velzen, Martin; de Boer, Jacob; Leonards, Pim

    2017-04-01

    A sensitive analytical method for the determination of monoamine neurotransmitters (MNTs) in zebrafish larvae was developed using gas chromatography coupled to mass spectrometry. Six MNTs were selected as target compounds for neurotoxicity testing. MNTs underwent a two-step derivatization with hexamethyldisilazane (HDMS) for O-silylation followed by N-methyl-bis-heptafluorobutyramide (MBHFBA) for N-perfluoroacylation. Derivatization conditions were optimized by an experimental design approach. Method validation showed linear calibration curves (r 2  > 0.9976) in the range of 1-100 ng for all the compounds. The recovery rates were between 92 and 119%. The method was repeatable and reproducible with relative standard deviations (RSD) in the range of 2.5-9.3% for intra-day and 4.8-12% for inter-day variation. The limits of detection and the limits of quantitation were 0.4-0.8 and 1.2-2.7 ng/mL, respectively. The method was successfully applied to detect and quantify trace levels of MNTs in 5-day-old zebrafish larvae that were exposed to low concentrations of neurotoxic chemicals such as pesticides and methylmercury. Although visual malformations were not detected, the MNT levels varied significantly during early zebrafish development. These results show that exposure to neurotoxic chemicals can alter neurotransmitter levels and thereby may influence early brain development. Graphical abstract ᅟ.

  17. Integrated and Early Childhood Education: Preparation for Social Development. Theme A: Relevant Provision for Early Childhood.

    Science.gov (United States)

    Axton, J. H. M.

    Factors which influence child development are listed and briefly discussed. These factors are (1) mother's childhood, (2) mother's age, (3) care during pregnancy and delivery, (4) early neonatal factors, (5) birth interval, (6) effect of repeated infection and malnutrition on brain growth and intellectual development, and (7) home environment. The…

  18. Early Brain and Child Development: Connections to Early Education and Child Care

    Science.gov (United States)

    Romano, Judith T.

    2013-01-01

    The vast majority of young children spend time in settings outside of the home, and the nature of those settings directly impacts the child's health and development. The ecobiodevelopmental framework of early brain and child development serve as the backdrop for establishing quality. This article describes the use of quality rating systems,…

  19. The neurogenetic frontier—lessons from misbehaving zebrafish

    Science.gov (United States)

    Granato, Michael

    2008-01-01

    One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish. PMID:18836206

  20. The neurogenetic frontier--lessons from misbehaving zebrafish.

    Science.gov (United States)

    Burgess, Harold A; Granato, Michael

    2008-11-01

    One of the central questions in neuroscience is how refined patterns of connectivity in the brain generate and monitor behavior. Genetic mutations can influence neural circuits by disrupting differentiation or maintenance of component neuronal cells or by altering functional patterns of nervous system connectivity. Mutagenesis screens therefore have the potential to reveal not only the molecular underpinnings of brain development and function, but to illuminate the cellular basis of behavior. Practical considerations make the zebrafish an organism of choice for undertaking forward genetic analysis of behavior. The powerful array of experimental tools at the disposal of the zebrafish researcher makes it possible to link molecular function to neuronal properties that underlie behavior. This review focuses on specific challenges to isolating and analyzing behavioral mutants in zebrafish.

  1. Automated visual tracking for studying the ontogeny of zebrafish swimming

    NARCIS (Netherlands)

    Fontaine, E.; Lentink, D.; Kranenbarg, S.; Müller, U.K.; Leeuwen, van J.L.; Barr, A.H.; Burdick, J.W.

    2008-01-01

    The zebrafish Danio rerio is a widely used model organism in studies of genetics, developmental biology, and recently, biomechanics. In order to quantify changes in swimming during all stages of development, we have developed a visual tracking system that estimates the posture of fish. Our current

  2. Ontogeny of Classical and Operant Learning Behaviors in Zebrafish

    Science.gov (United States)

    Valente, Andre; Huang, Kuo-Hua; Portugues, Ruben; Engert, Florian

    2012-01-01

    The performance of developing zebrafish in both classical and operant conditioning assays was tested with a particular focus on the emergence of these learning behaviors during development. Strategically positioned visual cues paired with electroshocks were used in two fully automated assays to investigate both learning paradigms. These allow the…

  3. Investigating the Flow and Biomechanics of the Embryonic Zebrafish Heart

    Science.gov (United States)

    Johnson, Brennan; Garrity, Deborah; Dasi, Lakshmi

    2010-11-01

    Understanding flow and kinematic characteristics of the embryonic heart is a prerequisite to devise early intervention or detection methods in the context of congenital heart defects. In this study, the kinematics and fluid dynamics of the embryonic zebrafish heart were analyzed through the early stages of cardiac development (24-48 hours post-fertilization) in vivo using optical microscopy and high-speed video. Endocardial walls and individual blood cells were segmented from raw images and were tracked through the cardiac cycle. Particle tracking velocimetry analysis yielded quantitative blood cell velocity field, chamber volume, and flow rate information. It was seen that the pumping mechanism starts as a combined peristaltic and suction pump while the heart is in the tube configuration and transforms into a positive displacement pump after cardiac looping. Strong two-phase nature of the fluid is evident. This work provides us new understanding of the spatio-temporal characteristics of kinematics and blood cell velocity field inside the developing heart.

  4. The search for evolutionary developmental origins of aging in zebrafish: a novel intersection of developmental and senescence biology in the zebrafish model system.

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    Kishi, Shuji

    2011-09-01

    Senescence may be considered the antithesis of early development, but yet there may be factors and mechanisms in common between these two phenomena during the process of aging. We investigated whether any relationship exists between the regulatory mechanisms that function in early development and in senescence using the zebrafish (Danio rerio), a small freshwater fish and a useful model animal for genetic studies. We conducted experiments to isolate zebrafish mutants expressing an apparent senescence phenotype during embryogenesis (embryonic senescence). Some of the genes we thereby identified had already been associated with cellular senescence and chronological aging in other organisms, but many had not yet been linked to these processes. Complete loss-of-function of developmentally essential genes induce embryonic (or larval) lethality, whereas it seems like their partial loss-of-function (i.e., decrease-of-function by heterozygote or hypomorphic mutations) still remains sufficient to go through the early developmental process because of its adaptive plasticity or rather heterozygote advantage. However, in some cases, such partial loss-of-function of genes compromise normal homeostasis due to haploinsufficiency later in adult life having many environmental stress challenges. By contrast, any heterozygote-advantageous genes might gain a certain benefit(s) (much more fitness) by such partial loss-of-function later in life. Physiological senescence may evolutionarily arise from both genetic and epigenetic drifts as well as from losing adaptive developmental plasticity in face of stress signals from the external environment that interacts with functions of multiple genes rather than effects of only a single gene mutation or defect. Previously uncharacterized developmental genes may thus mediate the aging process and play a pivotal role in senescence. Moreover, unexpected senescence-related genes might also be involved in the early developmental process and

  5. Somatic mutagenesis with a Sleeping Beauty transposon system leads to solid tumor formation in zebrafish.

    Directory of Open Access Journals (Sweden)

    Maura McGrail

    2011-04-01

    Full Text Available Large-scale sequencing of human cancer genomes and mouse transposon-induced tumors has identified a vast number of genes mutated in different cancers. One of the outstanding challenges in this field is to determine which genes, when mutated, contribute to cellular transformation and tumor progression. To identify new and conserved genes that drive tumorigenesis we have developed a novel cancer model in a distantly related vertebrate species, the zebrafish, Danio rerio. The Sleeping Beauty (SB T2/Onc transposon system was adapted for somatic mutagenesis in zebrafish. The carp ß-actin promoter was cloned into T2/Onc to create T2/OncZ. Two transgenic zebrafish lines that contain large concatemers of T2/OncZ were isolated by injection of linear DNA into the zebrafish embryo. The T2/OncZ transposons were mobilized throughout the zebrafish genome from the transgene array by injecting SB11 transposase RNA at the 1-cell stage. Alternatively, the T2/OncZ zebrafish were crossed to a transgenic line that constitutively expresses SB11 transposase. T2/OncZ transposon integration sites were cloned by ligation-mediated PCR and sequenced on a Genome Analyzer II. Between 700-6800 unique integration events in individual fish were mapped to the zebrafish genome. The data show that introduction of transposase by transgene expression or RNA injection results in an even distribution of transposon re-integration events across the zebrafish genome. SB11 mRNA injection resulted in neoplasms in 10% of adult fish at ∼10 months of age. T2/OncZ-induced zebrafish tumors contain many mutated genes in common with human and mouse cancer genes. These analyses validate our mutagenesis approach and provide additional support for the involvement of these genes in human cancers. The zebrafish T2/OncZ cancer model will be useful for identifying novel and conserved genetic drivers of human cancers.

  6. Promoting equity through integrated early child development and nutrition interventions.

    Science.gov (United States)

    Black, Maureen M; Dewey, Kathryn G

    2014-01-01

    Sustainable