Characterization and classification of zebrafish brain morphology mutants

PubMed Central

Lowery, Laura Anne; De Rienzo, Gianluca; Gutzman, Jennifer H.; Sive, Hazel

2010-01-01

The mechanisms by which the vertebrate brain achieves its three-dimensional structure are clearly complex, requiring the functions of many genes. Using the zebrafish as a model, we have begun to define genes required for brain morphogenesis, including brain ventricle formation, by studying 16 mutants previously identified as having embryonic brain morphology defects. We report the phenotypic characterization of these mutants at several time-points, using brain ventricle dye injection, imaging, and immunohistochemistry with neuronal markers. Most of these mutants display early phenotypes, affecting initial brain shaping, while others show later phenotypes, affecting brain ventricle expansion. In the early phenotype group, we further define four phenotypic classes and corresponding functions required for brain morphogenesis. Although we did not use known genotypes for this classification, basing it solely on phenotypes, many mutants with defects in functionally related genes clustered in a single class. In particular, class 1 mutants show midline separation defects, corresponding to epithelial junction defects; class 2 mutants show reduced brain ventricle size; class 3 mutants show midbrain-hindbrain abnormalities, corresponding to basement membrane defects; and class 4 mutants show absence of ventricle lumen inflation, corresponding to defective ion pumping. Later brain ventricle expansion requires the extracellular matrix, cardiovascular circulation, and transcription/splicing-dependent events. We suggest that these mutants define processes likely to be used during brain morphogenesis throughout the vertebrates. PMID:19051268

Smoc2 modulates embryonic myelopoiesis during zebrafish development.

PubMed

Mommaerts, Hendrik; Esguerra, Camila V; Hartmann, Ursula; Luyten, Frank P; Tylzanowski, Przemko

2014-11-01

SMOC2 is a member of the BM-40 (SPARC) family of matricellular proteins, reported to influence signaling in the extracellular compartment. In mice, Smoc2 is expressed in many different tissues and was shown to enhance the response to angiogenic growth factors, mediate cell adhesion, keratinocyte migration, and metastasis. Additionally, SMOC2 is associated with vitiligo and craniofacial and dental defects. The function of Smoc2 during early zebrafish development has not been determined to date. In pregastrula zebrafish embryos, smoc2 is expressed ubiquitously. As development progresses, the expression pattern becomes more anteriorly restricted. At the onset of blood cell circulation, smoc2 morphants presented a mild ventralization of posterior structures. Molecular analysis of the smoc2 morphants indicated myelopoietic defects in the rostral blood islands during segmentation stages. Hemangioblast development and further specification of the myeloid progenitor cells were shown to be impaired. Additional experiments indicated that Bmp target genes were down-regulated in smoc2 morphants. Our findings reveal that Smoc2 is an essential player in the development of myeloid cells of the anterior lateral plate mesoderm during embryonic zebrafish development. Furthermore, our data show that Smoc2 affects the transcription of Bmp target genes without affecting initial dorsoventral patterning or mesoderm development. Copyright © 2014 Wiley Periodicals, Inc.

What is the Thalamus in Zebrafish?

PubMed Central

Mueller, Thomas

2012-01-01

Current research on the thalamus and related structures in the zebrafish diencephalon identifies an increasing number of both neurological structures and ontogenetic processes as evolutionary conserved between teleosts and mammals. The patterning processes, for example, which during the embryonic development of zebrafish form the thalamus proper appear largely conserved. Yet also striking differences between zebrafish and other vertebrates have been observed, particularly when we look at mature and histologically differentiated brains. A case in point is the migrated preglomerular complex of zebrafish which evolved only within the lineage of ray-finned fish and has no counterpart in mammals or tetrapod vertebrates. Based on its function as a sensory relay station with projections to pallial zones, the preglomerular complex has been compared to specific thalamic nuclei in mammals. However, no thalamic projections to the zebrafish dorsal pallium, which corresponds topologically to the mammalian isocortex, have been identified. Merely one teleostean thalamic nucleus proper, the auditory nucleus, projects to a part of the dorsal telencephalon, the pallial amygdala. Studies on patterning mechanisms identify a rostral and caudal domain in the embryonic thalamus proper. In both, teleosts and mammals, the rostral domain gives rise to GABAergic neurons, whereas glutamatergic neurons originate in the caudal domain of the zebrafish thalamus. The distribution of GABAergic derivatives in the adult zebrafish brain, furthermore, revealed previously overlooked thalamic nuclei and redefined already established ones. These findings require some reconsideration regarding the topological origin of these adult structures. In what follows, I discuss how evolutionary conserved and newly acquired features of the developing and adult zebrafish thalamus can be compared to the mammalian situation. PMID:22586363

Two knockdown models of the autism genes SYNGAP1 and SHANK3 in zebrafish produce similar behavioral phenotypes associated with embryonic disruptions of brain morphogenesis

PubMed Central

Kozol, Robert A.; Cukier, Holly N.; Zou, Bing; Mayo, Vera; De Rubeis, Silvia; Cai, Guiqing; Griswold, Anthony J.; Whitehead, Patrice L.; Haines, Jonathan L.; Gilbert, John R.; Cuccaro, Michael L.; Martin, Eden R.; Baker, James D.; Buxbaum, Joseph D.; Pericak-Vance, Margaret A.; Dallman, Julia E.

2015-01-01

Despite significant progress in the genetics of autism spectrum disorder (ASD), how genetic mutations translate to the behavioral changes characteristic of ASD remains largely unknown. ASD affects 1–2% of children and adults, and is characterized by deficits in verbal and non-verbal communication, and social interactions, as well as the presence of repetitive behaviors and/or stereotyped interests. ASD is clinically and etiologically heterogeneous, with a strong genetic component. Here, we present functional data from syngap1 and shank3 zebrafish loss-of-function models of ASD. SYNGAP1, a synaptic Ras GTPase activating protein, and SHANK3, a synaptic scaffolding protein, were chosen because of mounting evidence that haploinsufficiency in these genes is highly penetrant for ASD and intellectual disability (ID). Orthologs of both SYNGAP1 and SHANK3 are duplicated in the zebrafish genome and we find that all four transcripts (syngap1a, syngap1b, shank3a and shank3b) are expressed at the earliest stages of nervous system development with pronounced expression in the larval brain. Consistent with early expression of these genes, knockdown of syngap1b or shank3a cause common embryonic phenotypes including delayed mid- and hindbrain development, disruptions in motor behaviors that manifest as unproductive swim attempts, and spontaneous, seizure-like behaviors. Our findings indicate that both syngap1b and shank3a play novel roles in morphogenesis resulting in common brain and behavioral phenotypes. PMID:25882707

Zebrafish Caudal Haematopoietic Embryonic Stromal Tissue (CHEST) Cells Support Haematopoiesis.

PubMed

Wolf, Anja; Aggio, Julian; Campbell, Clyde; Wright, Francis; Marquez, Gabriel; Traver, David; Stachura, David L

2017-03-16

Haematopoiesis is an essential process in early vertebrate development that occurs in different distinct spatial locations in the embryo that shift over time. These different sites have distinct functions: in some anatomical locations specific hematopoietic stem and progenitor cells (HSPCs) are generated de novo. In others, HSPCs expand. HSPCs differentiate and renew in other locations, ensuring homeostatic maintenance. These niches primarily control haematopoiesis through a combination of cell-to-cell signalling and cytokine secretion that elicit unique biological effects in progenitors. To understand the molecular signals generated by these niches, we report the generation of caudal hematopoietic embryonic stromal tissue (CHEST) cells from 72-hours post fertilization (hpf) caudal hematopoietic tissue (CHT), the site of embryonic HSPC expansion in fish. CHEST cells are a primary cell line with perivascular endothelial properties that expand hematopoietic cells in vitro. Morphological and transcript analysis of these cultures indicates lymphoid, myeloid, and erythroid differentiation, indicating that CHEST cells are a useful tool for identifying molecular signals critical for HSPC proliferation and differentiation in the zebrafish. These findings permit comparison with other temporally and spatially distinct haematopoietic-supportive zebrafish niches, as well as with mammalian haematopoietic-supportive cells to further the understanding of the evolution of the vertebrate hematopoietic system.

The Fanconi anemia/BRCA gene network in zebrafish: embryonic expression and comparative genomics.

PubMed

Titus, Tom A; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M; Frohnmayer, Jonathan D; Bremiller, Ruth A; Cañestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H

2009-07-31

Fanconi anemia (FA) is a genetic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn), and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only

The Fanconi anemia/BRCA gene network in zebrafish: Embryonic expression and comparative genomics

PubMed Central

Titus, Tom A.; Yan, Yi-Lin; Wilson, Catherine; Starks, Amber M.; Frohnmayer, Jonathan D.; Canestro, Cristian; Rodriguez-Mari, Adriana; He, Xinjun; Postlethwait, John H.

2008-01-01

Fanconi anemia (FA) is a genic disease resulting in bone marrow failure, high cancer risks, and infertility, and developmental anomalies including microphthalmia, microcephaly, hypoplastic radius and thumb. Here we present cDNA sequences, genetic mapping, and genomic analyses for the four previously undescribed zebrafish FA genes (fanci, fancj, fancm, and fancn, and show that they reverted to single copy after the teleost genome duplication. We tested the hypothesis that FA genes are expressed during embryonic development in tissues that are disrupted in human patients by investigating fanc gene expression patterns. We found fanc gene maternal message, which can provide Fanc proteins to repair DNA damage encountered in rapid cleavage divisions. Zygotic expression was broad but especially strong in eyes, central nervous system and hematopoietic tissues. In the pectoral fin bud at hatching, fanc genes were expressed specifically in the apical ectodermal ridge, a signaling center for fin/limb development that may be relevant to the radius/thumb anomaly of FA patients. Hatching embryos expressed fanc genes strongly in the oral epithelium, a site of squamous cell carcinomas in FA patients. Larval and adult zebrafish expressed fanc genes in proliferative regions of the brain, which may be related to microcephaly in FA. Mature ovaries and testes expressed fanc genes in specific stages of oocyte and spermatocyte development, which may be related to DNA repair during homologous recombination in meiosis and to infertility in human patients. The intestine strongly expressed some fanc genes specifically in proliferative zones. Our results show that zebrafish has a complete complement of fanc genes in single copy and that these genes are expressed in zebrafish embryos and adults in proliferative tissues that are often affected in FA patients. These results support the notion that zebrafish offers an attractive experimental system to help unravel mechanisms relevant not only to

Immunostaining of dissected zebrafish embryonic heart.

PubMed

Yang, Jingchun; Xu, Xiaolei

2012-01-10

Zebrafish embryo becomes a popular in vivo vertebrate model for studying cardiac development and human heart diseases due to its advantageous embryology and genetics. About 100-200 embryos are readily available every week from a single pair of adult fish. The transparent embryos that develop ex utero make them ideal for assessing cardiac defects. The expression of any gene can be manipulated via morpholino technology or RNA injection. Moreover, forward genetic screens have already generated a list of mutants that affect different perspectives of cardiogenesis. Whole mount immunostaining is an important technique in this animal model to reveal the expression pattern of the targeted protein to a particular tissue. However, high resolution images that can reveal cellular or subcellular structures have been difficult, mainly due to the physical location of the heart and the poor penetration of the antibodies. Here, we present a method to address these bottlenecks by dissecting heart first and then conducting the staining process on the surface of a microscope slide. To prevent the loss of small heart samples and to facilitate solution handling, we restricted the heart samples within a circle on the surface of the microscope slides drawn by an immEdge pen. After the staining, the fluorescence signals can be directly observed by a compound microscope. Our new method significantly improves the penetration for antibodies, since a heart from an embryonic fish only consists of few cell layers. High quality images from intact hearts can be obtained within a much reduced procession time for zebrafish embryos aged from day 2 to day 6. Our method can be potentially extended to stain other organs dissected from either zebrafish or other small animals. Copyright © 2012 Journal of Visualized Experiments

Expression pattern of zebrafish rxfp2 homologue genes during embryonic development.

PubMed

Donizetti, Aldo; Fiengo, Marcella; Del Gaudio, Rosanna; Iazzetti, Giovanni; Pariante, Paolo; Minucci, Sergio; Aniello, Francesco

2015-11-01

RXFP2 is one of the 4 receptors for relaxin insulin-like peptides, in particular it binds with high affinity the INSL3 peptide. INSL3/RXFP2 pair is essential for testicular descent during placental mammalian development. The evolutionary history of this ligand/receptor pair has received much attention, since its function in vertebrate species lacking testicular descent, such as the fishes, remains elusive. Herein, we analyzed the expression pattern of three rxfp2 homologue genes in zebrafish embryonic development. For all the three rxfp2 genes (rxfp2a, rxfp2b, and rxfp2-like) we showed the presence of maternally derived transcripts. Later in the development, rxfp2a is only expressed at larval stage, whereas rxfp2b is expressed in all the analyzed stage with highest level in the larvae. The rxfp2-like gene is expressed in all the analyzed stage with a transcript level that increased starting at early pharyngula stage. The spatial localization analysis of rxfp2-like gene showed that it is expressed in many cell clusters in the developing brain. In addition, other rxfp2-like-expressing cells were identified in the retina and oral epithelium. This analysis provides new insights to elucidate the evolution of rxfp2 genes in vertebrate lineage and lays the foundations to study their role in vertebrate embryonic development. © 2015 Wiley Periodicals, Inc.

Longitudinal Effects of Embryonic Exposure to Cocaine on Morphology, Cardiovascular Physiology, and Behavior in Zebrafish.

PubMed

Mersereau, Eric J; Boyle, Cody A; Poitra, Shelby; Espinoza, Ana; Seiler, Joclyn; Longie, Robert; Delvo, Lisa; Szarkowski, Megan; Maliske, Joshua; Chalmers, Sarah; Darland, Diane C; Darland, Tristan

2016-05-31

A sizeable portion of the societal drain from cocaine abuse results from the complications of in utero drug exposure. Because of challenges in using humans and mammalian model organisms as test subjects, much debate remains about the impact of in utero cocaine exposure. Zebrafish offer a number of advantages as a model in longitudinal toxicology studies and are quite sensitive physiologically and behaviorally to cocaine. In this study, we have used zebrafish to model the effects of embryonic pre-exposure to cocaine on development and on subsequent cardiovascular physiology and cocaine-induced conditioned place preference (CPP) in longitudinal adults. Larval fish showed a progressive decrease in telencephalic size with increased doses of cocaine. These treated larvae also showed a dose dependent response in heart rate that persisted 24 h after drug cessation. Embryonic cocaine exposure had little effect on overall health of longitudinal adults, but subtle changes in cardiovascular physiology were seen including decreased sensitivity to isoproterenol and increased sensitivity to cocaine. These longitudinal adult fish also showed an embryonic dose-dependent change in CPP behavior, suggesting an increased sensitivity. These studies clearly show that pre-exposure during embryonic development affects subsequent cocaine sensitivity in longitudinal adults.

Nestin is essential for zebrafish brain and eye development through control of progenitor cell apoptosis.

PubMed

Chen, Hua-Ling; Yuh, Chiou-Hwa; Wu, Kenneth K

2010-02-19

Nestin is expressed in neural progenitor cells (NPC) of developing brain. Despite its wide use as an NPC marker, the function of nestin in embryo development is unclear. As nestin is conserved in zebrafish and its predicted sequence is clustered with the mammalian nestin orthologue, we used zebrafish as a model to investigate its role in embryogenesis. Injection of nestin morpholino (MO) into fertilized eggs induced time- and dose-dependent brain and eye developmental defects. Nestin morphants exhibited characteristic morphological changes including small head, small eyes and hydrocephalus. Histological examinations show reduced hind- and mid-brain size, dilated ventricle, poorly organized retina and underdeveloped lens. Injection of control nestin MO did not induce brain or eye changes. Nestin MO injection reduced expression of ascl1b (achaete-scute complex-like 1b), a marker of NPCs, without affecting its distribution. Nestin MO did not influence Elavl3/4 (Embryonic lethal, abnormal vision, Drosophila-like 3/4) (a neuronal marker), or otx2 (a midbrain neuronal marker), but severely perturbed cranial motor nerve development and axon distribution. To determine whether the developmental defects are due to excessive NPC apoptosis and/or reduced NPC proliferation, we analyzed apoptosis by TUNEL assay and acridine orange staining and proliferation by BrdU incorporation, pcna and mcm5 expressions. Excessive apoptosis was noted in hindbrain and midbrain cells. Apoptotic signals were colocalized with ascl1b. Proliferation markers were not significantly altered by nestin MO. These results suggest that nestin is essential for zebrafish brain and eye development probably through control of progenitor cell apoptosis.

Maternal thyroid hormones are essential for neural development in zebrafish.

PubMed

Campinho, Marco A; Saraiva, João; Florindo, Claudia; Power, Deborah M

2014-07-01

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition.

Ca2+ signalling and early embryonic patterning during zebrafish development.

PubMed

Webb, Sarah E; Miller, Andrew L

2007-09-01

1. It has been proposed that Ca2+ signalling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern-forming events during early vertebrate development. 2. With reference to the embryo of the zebrafish (Danio rerio), herein we review the Ca2+ transients reported from the cleavage to segmentation periods. This time-window includes most of the major pattern-forming events of early development, which transform a single-cell zygote into a complex multicellular embryo with established primary germ layers and body axes. 3. Data are presented to support our proposal that intracellular Ca2+ waves are an essential feature of embryonic cytokinesis and that propagating intercellular Ca2+ waves (both long and short range) may play a crucial role in: (i) the establishment of the embryonic periderm and the coordination of cell movements during epiboly, convergence and extension; (ii) the establishment of the basic embryonic axes and germ layers; and (iii) definition of the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen. 4. The potential downstream targets of these Ca2+ transients are also discussed, as well as how they may integrate with other pattern-forming signalling pathways known to modulate early developmental events.

The effects of triclosan on pluripotency factors and development of mouse embryonic stem cells and zebrafish.

PubMed

Chen, Xiaojiao; Xu, Bo; Han, Xiumei; Mao, Zhilei; Chen, Minjian; Du, Guizhen; Talbot, Prue; Wang, Xinru; Xia, Yankai

2015-04-01

Triclosan (TCS) poses potential risks to reproduction and development due to its endocrine-disrupting properties. However, the mechanism of TCS's effects on early embryonic development is little known. Embryonic stem cells (ESC) and zebrafish embryos provide valuable models for testing the toxic effects of environmental chemicals on early embryogenesis. In this study, mouse embryonic stem cells (mESC) were acutely exposed to TCS for 24 h, and general cytotoxicity and the effect of TCS on pluripotency were then evaluated. In addition, zebrafish embryos were exposed to TCS from 2- to 24-h post-fertilization (hpf), and their morphology was evaluated. In mESC, alkaline phosphatase staining was significantly decreased after treatment with the highest concentration of TCS (50 μM). Although the expression levels of Sox2 mRNA were not changed, the mRNA levels of Oct4 and Nanog in TCS-treated groups were significantly decreased compared to controls. In addition, the protein levels of Oct4, Sox2 and Nanog were significantly reduced in response to TCS treatment. MicroRNA (miR)-134, an expression inhibitor of pluripotency markers, was significantly increased in TCS-treated mESC. In zebrafish experiments, after 24 hpf of treatment, the controls had developed to the late stage of somitogenesis, while embryos exposed to 300 μg/L of TCS were still at the early stage of somitogenesis, and three genes (Oct4, Sox2 and Nanog) were upregulated in treated groups when compared with the controls. The two models demonstrated that TCS may affect early embryonic development by disturbing the expression of the pluripotency markers (Oct4, Sox2 and Nanog).

Identification and expression analysis of zebrafish glypicans during embryonic development.

PubMed

Gupta, Mansi; Brand, Michael

2013-01-01

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.

Maternal Thyroid Hormones Are Essential for Neural Development in Zebrafish

PubMed Central

Saraiva, João; Florindo, Claudia; Power, Deborah M.

2014-01-01

Teleost eggs contain an abundant store of maternal thyroid hormones (THs), and early in zebrafish embryonic development, all the genes necessary for TH signaling are expressed. Nonetheless the function of THs in embryonic development remains elusive. To test the hypothesis that THs are fundamental for zebrafish embryonic development, an monocarboxilic transporter 8 (Mct8) knockdown strategy was deployed to prevent maternal TH uptake. Absence of maternal THs did not affect early specification of the neural epithelia but profoundly modified later dorsal specification of the brain and spinal cord as well as specific neuron differentiation. Maternal THs acted upstream of pax2a, pax7, and pax8 genes but downstream of shha and fgf8a signaling. The lack of inhibitory spinal cord interneurons and increased motoneurons in the mct8 morphants is consistent with their stiff axial body and impaired mobility. The mct8 mutations are associated with X-linked mental retardation in humans, and the cellular and molecular consequences of MCT8 knockdown during embryonic development in zebrafish provides new insight into the potential role of THs in this condition. PMID:24877564

Effects and possible mechanisms of simulated-microgravity on zebrafish embryonic cell

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Cellular level studies are helpful for revealing the underlying mechanisms of microgravity effects on living organisms. Many cell types, ranging from bacteria to mammalian cells, are sensitive to the microgravity environment. In this study, zebrafish embryonic cells (ZF4) were exposed to simulated-microgravity (SMG) for different times to investigate the effects and possible mechanisms of microgravity on fibroblasts. A significant arrest in G2/M phase was detected in ZF4 cells after 24 or 48 hour of SMG exposure, respectively. The mRNA levels of G2/M phase regulators cyclinB1 and cdc2 were significantly decreased, while wee1 was significantly increased. Additionally, CEP135, a core centrosome protein throughout the cell cycle, seems to play a key role in modulating this effect. Quantitative analysis showed that cep135 expression was significantly increased, while CEP135 protein expression level was significantly decreased two times after SMG. Further investigation demonstrated the transfection of dre-miR-22a, a miRNA for targeting cep135, also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells may due to the regulation of dre-miR-22a and its target cep135. Key Words: Simulated-microgravity; zebrafish embryonic cell; G2/M arrest; molecular mechanism

Effect of rearing temperatures during embryonic development on the phenotypic sex in zebrafish (Danio rerio).

PubMed

Abozaid, H; Wessels, S; Hörstgen-Schwark, G

2011-01-01

In zebrafish, Danio rerio, a polygenic pattern of sex determination or a female heterogamety with possible influences of environmental factors is assumed. The present study focuses on the effects of an elevated water temperature (35° C) during the embryonic development on sex determination in zebrafish. Eggs derived from 3 golden females were fertilized by the same mitotic gynogenetic male and exposed to a water temperature of 35° C, applied from 5 to 10 h post fertilization (hpf), from 5 to 24 hpf, and from 5 to 48 hpf, which correspond to the following developmental stages: gastrula, gastrula to segmentation, and gastrula to pharyngula stage, respectively. Hatching and survival rates decreased with increasing exposure to high water temperatures. Reductions in the hatching and survival rates were not responsible for differences in sex ratios. Accordingly, exposition of the fertilized eggs to a high temperature (35° C) leads to an increase of the male proportion from 22.0% in the controls to a balanced sex ratio (48.3, 47.5, and 52.6%) in the gastrula, segmentation, and pharyngula groups, respectively. These results prove the possibility to change the pathway of sexual determination during early embryonic stages in zebrafish by exposure to a high water temperature. Copyright © 2011 S. Karger AG, Basel.

A novel perivascular cell population in the zebrafish brain.

PubMed

Venero Galanternik, Marina; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

2017-04-11

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or 'Mato Cells' in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium.

A novel perivascular cell population in the zebrafish brain

PubMed Central

Galanternik, Marina Venero; Castranova, Daniel; Gore, Aniket V; Blewett, Nathan H; Jung, Hyun Min; Stratman, Amber N; Kirby, Martha R; Iben, James; Miller, Mayumi F; Kawakami, Koichi; Maraia, Richard J; Weinstein, Brant M

2017-01-01

The blood-brain barrier is essential for the proper homeostasis and function of the CNS, but its mechanism of function is poorly understood. Perivascular cells surrounding brain blood vessels are thought to be important for blood-brain barrier establishment, but their roles are not well defined. Here, we describe a novel perivascular cell population closely associated with blood vessels on the zebrafish brain. Based on similarities in their morphology, location, and scavenger behavior, these cells appear to be the zebrafish equivalent of cells variably characterized as Fluorescent Granular Perithelial cells (FGPs), perivascular macrophages, or ‘Mato Cells’ in mammals. Despite their macrophage-like morphology and perivascular location, zebrafish FGPs appear molecularly most similar to lymphatic endothelium, and our imaging studies suggest that these cells emerge by differentiation from endothelium of the optic choroidal vascular plexus. Our findings provide the first report of a perivascular cell population in the brain derived from vascular endothelium. DOI: http://dx.doi.org/10.7554/eLife.24369.001 PMID:28395729

Circadian rhythmicity and light sensitivity of the zebrafish brain.

PubMed

Moore, Helen A; Whitmore, David

2014-01-01

Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

Circadian Rhythmicity and Light Sensitivity of the Zebrafish Brain

PubMed Central

Moore, Helen A.; Whitmore, David

2014-01-01

Traditionally, circadian clocks have been thought of as a neurobiological phenomenon. This view changed somewhat over recent years with the discovery of peripheral tissue circadian oscillators. In mammals, however, the suprachiasmatic nucleus (SCN) in the hypothalamus still retains the critical role of a central synchronizer of biological timing. Zebrafish, in contrast, have always reflected a more highly decentralized level of clock organization, as individual cells and tissues contain directly light responsive circadian pacemakers. As a consequence, clock function in the zebrafish brain has remained largely unexplored, and the precise organization of rhythmic and light-sensitive neurons within the brain is unknown. To address this issue, we used the period3 (per3)-luciferase transgenic zebrafish to confirm that multiple brain regions contain endogenous circadian oscillators that are directly light responsive. In addition, in situ hybridization revealed localised neural expression of several rhythmic and light responsive clock genes, including per3, cryptochrome1a (cry1a) and per2. Adult brain nuclei showing significant clock gene expression include the teleost equivalent of the SCN, as well as numerous hypothalamic nuclei, the periventricular grey zone (PGZ) of the optic tectum, and granular cells of the rhombencephalon. To further investigate the light sensitive properties of neurons, expression of c-fos, a marker for neuronal activity, was examined. c-fos mRNA was upregulated in response to changing light conditions in different nuclei within the zebrafish brain. Furthermore, under constant dark (DD) conditions, c-fos shows a significant circadian oscillation. Taken together, these results show that there are numerous areas of the zebrafish central nervous system, which contain deep brain photoreceptors and directly light-entrainable circadian pacemakers. However, there are also multiple brain nuclei, which possess neither, demonstrating a degree of pacemaker

How the embryonic chick brain twists.

PubMed

Chen, Zi; Guo, Qiaohang; Dai, Eric; Forsch, Nickolas; Taber, Larry A

2016-11-01

During early development, the tubular embryonic chick brain undergoes a combination of progressive ventral bending and rightward torsion, one of the earliest organ-level left-right asymmetry events in development. Existing evidence suggests that bending is caused by differential growth, but the mechanism for the predominantly rightward torsion of the embryonic brain tube remains poorly understood. Here, we show through a combination of in vitro experiments, a physical model of the embryonic morphology and mechanics analysis that the vitelline membrane (VM) exerts an external load on the brain that drives torsion. Our theoretical analysis showed that the force is of the order of 10 micronewtons. We also designed an experiment to use fluid surface tension to replace the mechanical role of the VM, and the estimated magnitude of the force owing to surface tension was shown to be consistent with the above theoretical analysis. We further discovered that the asymmetry of the looping heart determines the chirality of the twisted brain via physical mechanisms, demonstrating the mechanical transfer of left-right asymmetry between organs. Our experiments also implied that brain flexure is a necessary condition for torsion. Our work clarifies the mechanical origin of torsion and the development of left-right asymmetry in the early embryonic brain. © 2016 The Author(s).

Phenotypically anchored transcriptome profiling of developmental exposure to the antimicrobial agent, triclosan, reveals hepatotoxicity in embryonic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Haggard, Derik E.

Triclosan (TCS) is an antimicrobial agent commonly found in a variety of personal care products and cosmetics. TCS readily enters the environment through wastewater and is detected in human plasma, urine, and breast milk due to its widespread use. Studies have implicated TCS as a disruptor of thyroid and estrogen signaling; therefore, research examining the developmental effects of TCS is warranted. In this study, we used embryonic zebrafish to investigate the developmental toxicity and potential mechanism of action of TCS. Embryos were exposed to graded concentrations of TCS from 6 to 120 hours post-fertilization (hpf) and the concentration where 80%more » of the animals had mortality or morbidity at 120 hpf (EC{sub 80}) was calculated. Transcriptomic profiling was conducted on embryos exposed to the EC{sub 80} (7.37 μM). We identified a total of 922 significant differentially expressed transcripts (FDR adjusted P-value ≤ 0.05; fold change ≥ 2). Pathway and gene ontology enrichment analyses identified biological networks and transcriptional hubs involving normal liver functioning, suggesting TCS may be hepatotoxic in zebrafish. Tissue-specific gene enrichment analysis further supported the role of the liver as a target organ for TCS toxicity. We also examined the in vitro bioactivity profile of TCS reported by the ToxCast screening program. TCS had a diverse bioactivity profile and was a hit in 217 of the 385 assay endpoints we identified. We observed similarities in gene expression and hepatic steatosis assays; however, hit data for TCS were more concordant with the hypothesized CAR/PXR activity of TCS from rodent and human in vitro studies. - Highlights: • Triclosan is a common antimicrobial agent with widespread human exposure. • Exposure to the triclosan EC{sub 80} causes robust gene expression changes in zebrafish. • The liver may be a target organ of triclosan toxicity in embryonic zebrafish. • Triclosan disrupts normal liver functioning and

Investigating the Flow and Biomechanics of the Embryonic Zebrafish Heart

NASA Astrophysics Data System (ADS)

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.

Effects of two strobilurins (azoxystrobin and picoxystrobin) on embryonic development and enzyme activities in juveniles and adult fish livers of zebrafish (Danio rerio).

PubMed

Jia, Wei; Mao, Liangang; Zhang, Lan; Zhang, Yanning; Jiang, Hongyun

2018-09-01

Azoxystrobin and picoxystrobin are two primary strobilurin fungicides used worldwide. This study was conducted to test their effects on embryonic development and the activity of several enzyme in the zebrafish (Danio rerio). After fish eggs were separately exposed to azoxystrobin and picoxystrobin from 24 to 144 h post fertilization (hpf), the mortality, hatching, and teratogenetic rates were measured. Additionally, effects of azoxystrobin and picoxystrobin on activities of three important antioxidant enzymes [catalase (CAT), superoxide dismutase (SOD) and peroxidase (POD)] and two primary detoxification enzymes [carboxylesterase (CarE) and glutathione S-transferase (GST)] and malondialdehyde (MDA) content in zebrafish larvae (96 h) and livers of adult zebrafish of both sexes were also assessed for potential toxicity mechanisms. Based on the embryonic development test results, the mortality, hatching, and teratogenetic rates of eggs treated with azoxystrobin and picoxystrobin all showed significant dose- and time-dependent effects, and the 144-h LC 50 values of azoxystrobin and picoxystrobin were 1174.9 and 213.8 μg L -1 , respectively. In the larval zebrafish (96 h) test, activities of CAT, POD, CarE, and GST and MDA content in azoxystrobin and picoxystrobin-treated zebrafish larvae increased significantly with concentrations of the pesticides compared with those in the control. We further revealed that azoxystrobin and picoxystrobin exposure both caused significant oxidative stress in adult fish livers and the changes differed between the sexes. Our results indicated that picoxystrobin led to higher embryonic development toxicity and oxidative stress than azoxystrobin in zebrafish and the male zebrafish liver had stronger ability to detoxify than that of the females. Copyright © 2018 Elsevier Ltd. All rights reserved.

Nasal embryonic LHRH factor plays a role in the developmental migration and projection of gonadotropin-releasing hormone 3 neurons in zebrafish.

PubMed

Palevitch, Ori; Abraham, Eytan; Borodovsky, Natalya; Levkowitz, Gil; Zohar, Yonathan; Gothilf, Yoav

2009-01-01

The initiation of puberty and the functioning of the reproductive system depend on proper development of the hypophysiotropic gonadotropin-releasing hormone (GnRH) system. One critical step in this process is the embryonic migration of GnRH neurons from the olfactory area to the hypothalamus. Using a transgenic zebrafish model, Tg(gnrh3:EGFP), in which GnRH3 neurons and axons are fluorescently labeled, we investigated whether zebrafish NELF is essential for the development of GnRH3 neurons. The zebrafish nelf cDNA was cloned and characterized. During embryonic development, nelf is expressed in GnRH3 neurons and in target sites of GnRH3 projections and perikarya, before the initiation of their migration. Nelf knockdown resulted in a disruption of the GnRH3 system which included absence or misguiding of GnRH3 axonal outgrowth and incorrect or arrested migration of GnRH3 perikarya. These results suggest that Nelf is an important factor in the developmental migration and projection of GnRH3 neurons in zebrafish. Copyright (c) 2008 Wiley-Liss, Inc.

Behavioral, morphometric, and gene expression effects in adult zebrafish (Danio rerio) embryonically exposed to PFOA, PFOS, and PFNA.

PubMed

Jantzen, Carrie E; Annunziato, Kate M; Cooper, Keith R

2016-11-01

Perfluoroalkylated substances (PFAS) are a class of persistent anthropogenic chemicals that have been detected worldwide. PFASs consist of fluorinated carbon chains of varying length, terminal groups, and have a number of industrial uses. A previous zebrafish study from our laboratory showed that acute (3-120h post fertilization, 0.02-2.0μM), waterborne embryonic exposure to these chemicals resulted in chemical specific alterations at 5days post fertilization (dpf), and some effects persisted up to 14 dpf. Using a gene battery consisting of 100 transcripts identified several genes that were up or down regulated. This current study looks at the long-term impacts of PFASs in adult zebrafish using the same exposure regimen. It was hypothesized that sub-lethal exposure of perfluorooctane sulfonate (PFOS), perfluorononanoic acid (PFNA), or perfluorooctane sulfonate (PFOA) in embryonic zebrafish (3-120 hpf) would result in permanent morphometric, gene expression, and behavioral changes in adult fish similar to those observed at 5 and 14 dpf. Zebrafish were exposed to PFOS, PFOA, and PFNA (Control 0μM, 2.0μM) for the first five days post fertilization. At six months post fertilization, no PFAS treatment resulted in a significant change in total body length or weight. In terms of behavior, PFNA males showed a reduction in total distance traveled and time of immobility, and an increase in thigmotaxis behavior, aggressive attacks, and preference for the bright section of the tank. PFOS treated males had a reduced aggression behavior, and PFOA females preferred the dark section of the tank. Gene expression of slco2b1, slco1d1, and tgfb1a were analyzed because these transcripts were previously found to be affected by PFAS exposure in 5dpf and 14 dpf zebrafish and resulted in: significant decrease in expression of slco2b1 for both sexes in PFNA and PFOS treated groups, significant decrease of slco1d1 in all treatment groups for females and PFOS and PFOA exposed males

A conserved role of αA-crystallin in the development of the zebrafish embryonic lens.

PubMed

Zou, Ping; Wu, Shu-Yu; Koteiche, Hanane A; Mishra, Sanjay; Levic, Daniel S; Knapik, Ela; Chen, Wenbiao; Mchaourab, Hassane S

2015-09-01

αA- and αB-crystallins are small heat shock proteins that bind thermodynamically destabilized proteins thereby inhibiting their aggregation. Highly expressed in the mammalian lens, the α-crystallins have been postulated to play a critical role in the maintenance of lens optical properties by sequestering age-damaged proteins prone to aggregation as well as through a multitude of roles in lens epithelial cells. Here, we have examined the role of α-crystallins in the development of the vertebrate zebrafish lens. For this purpose, we have carried out morpholino-mediated knockdown of αA-, αBa- and αBb-crystallin and characterized the gross morphology of the lens. We observed lens abnormalities, including increased reflectance intensity, as a consequence of the interference with expression of these proteins. These abnormalities were less frequent in transgenic zebrafish embryos expressing rat αA-crystallin suggesting a specific role of α-crystallins in embryonic lens development. To extend and confirm these findings, we generated an αA-crystallin knockout zebrafish line. A more consistent and severe lens phenotype was evident in maternal/zygotic αA-crystallin mutants compared to those observed by morpholino knockdown. The penetrance of the lens phenotype was reduced by transgenic expression of rat αA-crystallin and its severity was attenuated by maternal αA-crystallin expression. These findings demonstrate that the role of α-crystallins in lens development is conserved from mammals to zebrafish and set the stage for using the embryonic lens as a model system to test mechanistic aspects of α-crystallin chaperone activity and to develop strategies to fine-tune protein-protein interactions in aging and cataracts. Copyright © 2015 Elsevier Ltd. All rights reserved.

Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

PubMed Central

Chatterjee, Aniruddha; Ozaki, Yuichi; Stockwell, Peter A; Horsfield, Julia A; Morison, Ian M; Nakagawa, Shinichi

2013-01-01

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish. PMID:23975027

Embryonic expression of zebrafish MiT family genes tfe3b, tfeb, and tfec.

PubMed

Lister, James A; Lane, Brandon M; Nguyen, Anhthu; Lunney, Katherine

2011-11-01

The MiT family comprises four genes in mammals: Mitf, Tfe3, Tfeb, and Tfec, which encode transcription factors of the basic-helix-loop-helix/leucine zipper class. Mitf is well-known for its essential role in the development of melanocytes, however the functions of the other members of this family, and of interactions between them, are less well understood. We have now characterized the complete set of MiT genes from zebrafish, which totals six instead of four. The zebrafish genome contain two mitf (mitfa and mitfb), two tfe3 (tfe3a and tfe3b), and single tfeb and tfec genes; this distribution is shared with other teleosts. We present here the sequence and embryonic expression patterns for the zebrafish tfe3b, tfeb, and tfec genes, and identify a new isoform of tfe3a. These findings will assist in elucidating the roles of the MiT gene family over the course of vertebrate evolution. Copyright © 2011 Wiley-Liss, Inc.

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

NASA Astrophysics Data System (ADS)

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.

Clofibrate and gemfibrozil induce an embryonic malabsorption syndrome in zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Raldua, Demetrio; Andre, Michele; Babin, Patrick J.

2008-05-01

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 inducedmore » 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.« less

Egr1 gene knockdown affects embryonic ocular development in zebrafish.

PubMed

Hu, Chao-Yu; Yang, Chang-Hao; Chen, Wei-Yu; Huang, Chiu-Ju; Huang, Hsing-Yen; Chen, Muh-Shy; Tsai, Huai-Jen

2006-10-26

To identify the changes in zebrafish embryonic ocular development after early growth response factor 1 (Egr1) gene knockdown by Egr1-specific translation inhibitor, morpholino oligonucleotides (MO). Two kinds of Egr1-MO were microinjected separately with various dosages into one to four celled zebrafish embryos to find an optimal dose generating an acceptable mortality rate and high frequency of specific phenotype. Chordin-MO served as the positive control; a 5 mismatch MO of Egr1-MO1 and a nonspecific MO served as negative controls. We graded the Egr1 morphants according to their gross abnormalities, and measured their ocular dimensions accordingly. Western blot analysis and synthetic Egr1 mRNA rescue experiments confirmed whether the deformities were caused by Egr1 gene knockdown. Histological examination and three kinds of immunohistochemical staining were applied to identify glutamate receptor one expression in retinal ganglion cells and amacrine cells, to recognize acetylated alpha-tubulin expression which indicated axonogenesis, and to label photoreceptor cells with zpr-1 antibody. After microinjection of 8 ng Egr1-MO1 or 2 ng Egr1-MO2, 81.8% and 97.3% of larvae at 72 h postfertilization had specific defects, respectively. The gross phenotype included string-like heart, flat head, and deformed tail. The more severely deformed larvae had smaller eyes and pupils. Co-injection of 8 ng Egr1-MO1 and supplementary 12 pg synthetic Egr1 mRNA reduced the gross abnormality rate from 84.4% to 29.7%, and decreased the severity of deformities. Egr1 protein appeared in the wildtype and rescued morphants, but was lacking in the Egr1 morphants with specific deformities. Lenses of Egr1 morphants were smaller and had some residual nucleated lens fiber cells. Morphants' retinal cells arranged disorderly and compactly with thin plexiform layers. Immunohistochemical studies showed that morphants had a markedly decreased number of mature retinal ganglion cells, amacrine cells, and

Assessment of diclofenac LC50 reference values in juvenile and embryonic stages of the zebrafish (Danio rerio).

PubMed

Praskova, E; Voslarova, E; Siroka, Z; Plhalova, L; Macova, S; Marsalek, P; Pistekova, V; Svobodova, Z

2011-01-01

The aim of the study was to compare the acute toxicity of diclofenac to juvenile and embryonic stages of the zebrafish (Danio rerio). Acute toxicity tests were performed on the aquarium fish Danio rerio, which is one of the model organisms most commonly used in toxicity testing. The tests were performed using a semi-static method according to OECD guideline No. 203 (Fish, acute toxicity test). Embryo toxicity tests were performed in zebrafish embryos (Danio rerio) in compliance with OECD No. 212 methodology (Fish, short-term toxicity test on embryo and sac-fry stages). The results were subjected to a probit analysis using the EKO-TOX 5.2 programme to determine 96hLC50 and 144hLC50 (median lethal concentration, 50% mortality after a 96 h or 144 h interval, respectively) values of diclofenac. The statistical significance of the difference between LC50 values in juvenile and embryonic stages of Danio rerio was tested using the Mann-Whitney non-parametric test implemented in the Unistat 5.1 programme. The LC50 mean value of diclofenac was 166.6 +/- 9.8 mg/L in juvenile Danio rerio, and 6.11 +/- 2.48 mg/L in embryonic stages of Danio rerio. The study demonstrated a statistically higher sensitivity to diclofenac (P < 0.05) in embryonic stages compared to the juvenile fish.

Role of brain-derived neurotrophic factor during the regenerative response after traumatic brain injury in adult zebrafish.

PubMed

Cacialli, Pietro; Palladino, Antonio; Lucini, Carla

2018-06-01

Several mammalian animal models of traumatic brain injury have been used, mostly rodents. However, reparative mechanisms in mammalian brain are very limited, and newly formed neurons do not survive for long time. The brain of adult zebrafish, a teleost fish widely used as vertebrate model, possesses high regenerative properties after injury due to the presence of numerous stem cells niches. The ventricular lining of the zebrafish dorsal telencephalon is the most studied neuronal stem cell niche because its dorso-lateral zone is considered the equivalent to the hippocampus of mammals which contains one of the two constitutive neurogenic niches of mammals. To mimic TBI, stab wound in the dorso-lateral telencephalon of zebrafish was used in studies devoted to fish regenerative properties. Brain-derived neurotrophic factor, which is known to play key roles in the repair process after traumatic brain lesions, persists around the lesioned area of injured telencephalon of adult zebrafish. These results are extensively compared to reparative processes in rodent brain. Considering the complete repair of the damaged area in fish, it could be tempting to consider brain-derived neurotrophic factor as a factor contributing to create a permissive environment that enables the establishment of new neuronal population in damaged brain.

Monitoring of injury induced brain regeneration of the adult zebrafish by using optical coherence tomography

NASA Astrophysics Data System (ADS)

Yuan, Zhen; Zhang, Jian

2018-02-01

The adult zebrafish has pronounced regenerative capacity of the brain, which makes it an ideal model organism of vertebrate biology for the investigation of recovery of central nervous system injuries. The aim of this study was to employ spectral-domain optical coherence tomography (SD-OCT) system for long-term in vivo monitoring of tissue regeneration using an adult zebrafish model of brain injury. Based on a 1325 nm light source and two high-speed galvo mirrors, the SD-OCT system can offer a large field of view of the three-dimensional (3D) brain structures with high imaging resolution (12 μm axial and 13 μm lateral) at video rate. In vivo experiments based on this system were conducted to monitor the regeneration process of zebrafish brain after injury during a period of 43 days. To monitor and detect the process of tissue regeneration, we performed 3D in vivo imaging in a zebrafish model of adult brain injury during a period of 43 days. The coronal and sagittal views of the injured zebrafish brain at each time point (0 days, 10 days, 20 days and 43 days postlesion) were presented to show the changes of the brain lesion in detail. In addition, the 3D SD-OCT images for an injured zebrafish brain were also reconstructed at days 0 and days 43 post-lesion. We found that SD-OCT is able to effectively and noninvasively monitor the regeneration of the adult zebrafish brain after injury in real time with high 3D spatial resolution and good penetration depth. Our findings also suggested that the adult zebrafish has the extraordinary capability of brain regeneration and is able to repair itself after brain injury.

Notch Receptor Expression in Neurogenic Regions of the Adult Zebrafish Brain

PubMed Central

de Oliveira-Carlos, Vanessa; Ganz, Julia; Hans, Stefan; Kaslin, Jan; Brand, Michael

2013-01-01

The adult zebrash brain has a remarkable constitutive neurogenic capacity. The regulation and maintenance of its adult neurogenic niches are poorly understood. In mammals, Notch signaling is involved in stem cell maintenance both in embryonic and adult CNS. To better understand how Notch signaling is involved in stem cell maintenance during adult neurogenesis in zebrafish we analysed Notch receptor expression in five neurogenic zones of the adult zebrafish brain. Combining proliferation and glial markers we identified several subsets of Notch receptor expressing cells. We found that 90 of proliferating radial glia express notch1a, notch1b and notch3. In contrast, the proliferating non-glial populations of the dorsal telencephalon and hypothalamus rarely express notch3 and about half express notch1a/1b. In the non-proliferating radial glia notch3 is the predominant receptor throughout the brain. In the ventral telencephalon and in the mitotic area of the optic tectum, where cells have neuroepithelial properties, notch1a/1b/3 are expressed in most proliferating cells. However, in the cerebellar niche, although progenitors also have neuroepithelial properties, only notch1a/1b are expressed in a high number of PCNA cells. In this region notch3 expression is mostly in Bergmann glia and at low levels in few PCNA cells. Additionally, we found that in the proliferation zone of the ventral telencephalon, Notch receptors display an apical high to basal low gradient of expression. Notch receptors are also expressed in subpopulations of oligodendrocytes, neurons and endothelial cells. We suggest that the partial regional heterogeneity observed for Notch expression in progenitor cells might be related to the cellular diversity present in each of these neurogenic niches. PMID:24039926

First trimester size charts of embryonic brain structures.

PubMed

Gijtenbeek, M; Bogers, H; Groenenberg, I A L; Exalto, N; Willemsen, S P; Steegers, E A P; Eilers, P H C; Steegers-Theunissen, R P M

2014-02-01

Can reliable size charts of human embryonic brain structures be created from three-dimensional ultrasound (3D-US) visualizations? Reliable size charts of human embryonic brain structures can be created from high-quality images. Previous studies on the visualization of both the cavities and the walls of the brain compartments were performed using 2D-US, 3D-US or invasive intrauterine sonography. However, the walls of the diencephalon, mesencephalon and telencephalon have not been measured non-invasively before. Last-decade improvements in transvaginal ultrasound techniques allow a better visualization and offer the tools to measure these human embryonic brain structures with precision. This study is embedded in a prospective periconceptional cohort study. A total of 141 pregnancies were included before the sixth week of gestation and were monitored until delivery to assess complications and adverse outcomes. For the analysis of embryonic growth, 596 3D-US scans encompassing the entire embryo were obtained from 106 singleton non-malformed live birth pregnancies between 7(+0) and 12(+6) weeks' gestational age (GA). Using 4D View (3D software) the measured embryonic brain structures comprised thickness of the diencephalon, mesencephalon and telencephalon, and the total diameter of the diencephalon and mesencephalon. Of 596 3D scans, 161 (27%) high-quality scans of 79 pregnancies were eligible for analysis. The reliability of all embryonic brain structure measurements, based on the intra-class correlation coefficients (ICCs) (all above 0.98), was excellent. Bland-Altman plots showed moderate agreement for measurements of the telencephalon, but for all other measurements the agreement was good. Size charts were constructed according to crown-rump length (CRL). The percentage of high-quality scans suitable for analysis of these brain structures was low (27%).  The size charts of human embryonic brain structures can be used to study normal and abnormal development of

Ca2+ signaling and early embryonic patterning during the blastula and gastrula periods of zebrafish and Xenopus development.

PubMed

Webb, Sarah E; Miller, Andrew L

2006-11-01

It has been proposed that Ca(2+) signaling, in the form of pulses, waves and steady gradients, may play a crucial role in key pattern forming events during early vertebrate development [L.F. Jaffe, Organization of early development by calcium patterns, BioEssays 21 (1999) 657-667; M.J. Berridge, P. Lipp, M.D. Bootman, The versatility and universality of calcium signaling, Nat. Rev. Mol. Cell Biol. 1 (2000) 11-21; S.E. Webb, A.L. Miller, Calcium signalling during embryonic development, Nat. Rev. Mol. Cell Biol. 4 (2003) 539-551]. With reference to the embryos of zebrafish (Danio rerio) and the frog, Xenopus laevis, we review the Ca(2+) signals reported during the Blastula and Gastrula Periods. This developmental window encompasses the major pattern forming events of epiboly, involution, and convergent extension, which result in the establishment of the basic germ layers and body axes [C.B. Kimmel, W.W. Ballard, S.R. Kimmel, B. Ullmann, T.F. Schilling, Stages of embryonic development of the zebrafish, Dev. Dyn. 203 (1995) 253-310]. Data will be presented to support the suggestion that propagating waves (both long and short range) of Ca(2+) release, followed by sequestration, may play a crucial role in: (1) Coordinating cell movements during these pattern forming events and (2) Contributing to the establishment of the basic embryonic axes, as well as (3) Helping to define the morphological boundaries of specific tissue domains and embryonic structures, including future organ anlagen [E. Gilland, A.L. Miller, E. Karplus, R. Baker, S.E. Webb, Imaging of multicellular large-scale rhythmic calcium waves during zebrafish gastrulation, Proc. Natl. Acad. Sci. USA 96 (1999) 157-161; J.B. Wallingford, A.J. Ewald, R.M. Harland, S.E. Fraser, Calcium signaling during convergent extension in Xenopus, Curr. Biol. 11 (2001) 652-661]. The various potential targets of these Ca(2+) transients will also be discussed, as well as how they might integrate with other known pattern forming

Maternal Gdf3 is an obligatory cofactor in Nodal signaling for embryonic axis formation in zebrafish

PubMed Central

Bisgrove, Brent W; Su, Yi-Chu

2017-01-01

Zebrafish Gdf3 (Dvr1) is a member of the TGFβ superfamily of cell signaling ligands that includes Xenopus Vg1 and mammalian Gdf1/3. Surprisingly, engineered homozygous mutants in zebrafish have no apparent phenotype. Elimination of Gdf3 in oocytes of maternal-zygotic mutants results in embryonic lethality that can be fully rescued with gdf3 RNA, demonstrating that Gdf3 is required only early in development, beyond which mutants are viable and fertile. Gdf3 mutants are refractory to Nodal ligands and Nodal repressor Lefty1. Signaling driven by TGFβ ligand Activin and constitutively active receptors Alk4 and Alk2 remain intact in gdf3 mutants, indicating that Gdf3 functions at the same pathway step as Nodal. Targeting gdf3 and ndr2 RNA to specific lineages indicates that exogenous gdf3 is able to fully rescue mutants only when co-expressed with endogenous Nodal. Together, these findings demonstrate that Gdf3 is an essential cofactor of Nodal signaling during establishment of the embryonic axis. PMID:29140249

Embryonic Zebrafish Model - A Well-Established Method for Rapidly Assessing the Toxicity of Homeopathic Drugs: - Toxicity Evaluation of Homeopathic Drugs Using Zebrafish Embryo Model.

PubMed

Gupta, Himanshu R; Patil, Yogesh; Singh, Dipty; Thakur, Mansee

2016-12-01

Advancements in nanotechnology have led to nanoparticle (NP) use in various fields of medicine. Although the potential of NPs is promising, the lack of documented evidence on the toxicological effects of NPs is concerning. A few studies have documented that homeopathy uses NPs. Unfortunately, very few sound scientific studies have explored the toxic effects of homeopathic drugs. Citing this lack of high-quality scientific evidence, regulatory agencies have been reluctant to endorse homeopathic treatment as an alternative or adjunct treatment. This study aimed to enhance our insight into the impact of commercially-available homeopathic drugs, to study the presence of NPs in those drugs and any deleterious effects they might have, and to determine the distribution pattern of NPs in zebrafish embryos ( Danio rerio ). Homeopathic dilutions were studied using high-resolution transmission electron microscopy with selected area electron diffraction (SAED). For the toxicity assessment on Zebrafish, embryos were exposed to a test solution from 4 - 6 hours post-fertilization, and embryos/larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. Around 5 dpf was found to be the optimum developmental stage for evaluation. The present study aimed to conclusively prove the presence of NPs in all high dilutions of homeopathic drugs. Embryonic zebrafish were exposed to three homeopathic drugs with two potencies (30CH, 200CH) during early embryogenesis. The resulting morphological and cellular responses were observed. Exposure to these potencies produced no visibly significant malformations, pericardial edema, and mortality and no necrotic and apoptotic cellular death. Our findings clearly demonstrate that no toxic effects were observed for these three homeopathic drugs at the potencies and exposure times used in this study. The embryonic zebrafish

Distinct structure and activity of monoamine oxidase in the brain of zebrafish (Danio rerio).

PubMed

Anichtchik, Oleg; Sallinen, Ville; Peitsaro, Nina; Panula, Pertti

2006-10-10

Monoamine oxidase (MAO) is a mitochondrial flavoprotein involved in the metabolism of, e.g., aminergic neurotransmitters and the parkinsonism-inducing neurotoxin 1-methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP). We have reported earlier MPTP-related alterations of brain catecholaminergic system in zebrafish (Danio rerio) brain. Here we describe the structural and functional properties of zebrafish MAO and the distribution of MAO mRNA and activity in zebrafish brain. The gene is located in chromosome 9 and consists of 15 exons. The amino acid composition of the active center resembles both human MAO-A and MAO-B. The enzyme displayed the highest substrate specificity for tyramine, followed by serotonin, phenylethylamine, MPTP, and dopamine; isoform-specific antagonists blocked the activity of the enzyme with equal potency. Zebrafish MAO mRNA, which was present in several tissues, and enzyme displayed differential distribution in the brain; dopaminergic cell clusters had low to moderate levels of MAO activity, whereas the highest levels of MAO activity were detected in noradrenergic and serotonergic cell groups and the habenulointerpeduncular pathway, including its caudal projection to the medial ventral rhombencephalon. The results of this study confirm the presence of functionally active MAO in zebrafish brain and other tissues and characterize the neural systems that express MAO and areas of intense activity in the brain. They also suggest that MPTP toxicity not related to MAO may affect the zebrafish brain.

Stability of citrate-capped silver nanoparticles in exposure media and their effects on the development of embryonic zebrafish (Danio rerio)

PubMed Central

Park, Kwangsik; Tuttle, George; Sinche, Federico; Harper, Stace L.

2014-01-01

The stability of citrate-capped silver nanoparticles (AgNPs) and the embryonic developmental toxicity were evaluated in the fish test water. Serious aggregation of AgNPs was observed in undiluted fish water (DM-100) in which high concentration of ionic salts exist. However, AgNPs were found to be stable for 7 days in DM-10, prepared by diluting the original fish water (DM-100) with deionized water to 10%. The normal physiology of zebrafish embryos were evaluated in DM-10 to see if DM-10 can be used as a control vehicle for the embryonic fish toxicity test. As results, DM-10 without AgNPs did not induce any significant adverse effects on embryonic development of zebrafish determined by mortality, hatching, malformations and heart rate. When embryonic toxicity of AgNPs was tested in both DM-10 and in DM-100, AgNPs showed higher toxicity in DM-10 than in DM-100. This means that the big-sized aggregates of AgNPs were low toxic compared to the nano-sized AgNPs. AgNPs induced delayed hatching, decreased heart rate, pericardial edema, and embryo death. Accumulation of AgNPs in the embryo bodies was also observed. Based on this study, citrate-capped AgNPs are not aggregated in DM-10 and it can be used as a control vehicle in the toxicity test of fish embryonic development. PMID:23325492

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides

PubMed Central

Sterling, M.E.; Chang, G.-Q.; Karatayev, O.; Chang, S.Y.; Leibowitz, S.F.

2016-01-01

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24 h post-fertilization, zebrafish embryos were exposed for 2 h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol. PMID:26778786

4-dimensional functional profiling in the convulsant-treated larval zebrafish brain.

PubMed

Winter, Matthew J; Windell, Dylan; Metz, Jeremy; Matthews, Peter; Pinion, Joe; Brown, Jonathan T; Hetheridge, Malcolm J; Ball, Jonathan S; Owen, Stewart F; Redfern, Will S; Moger, Julian; Randall, Andrew D; Tyler, Charles R

2017-07-26

Functional neuroimaging, using genetically-encoded Ca 2+ sensors in larval zebrafish, offers a powerful combination of high spatiotemporal resolution and higher vertebrate relevance for quantitative neuropharmacological profiling. Here we use zebrafish larvae with pan-neuronal expression of GCaMP6s, combined with light sheet microscopy and a novel image processing pipeline, for the 4D profiling of chemoconvulsant action in multiple brain regions. In untreated larvae, regions associated with autonomic functionality, sensory processing and stress-responsiveness, consistently exhibited elevated spontaneous activity. The application of drugs targeting different convulsant mechanisms (4-Aminopyridine, Pentylenetetrazole, Pilocarpine and Strychnine) resulted in distinct spatiotemporal patterns of activity. These activity patterns showed some interesting parallels with what is known of the distribution of their respective molecular targets, but crucially also revealed system-wide neural circuit responses to stimulation or suppression. Drug concentration-response curves of neural activity were identified in a number of anatomically-defined zebrafish brain regions, and in vivo larval electrophysiology, also conducted in 4dpf larvae, provided additional measures of neural activity. Our quantification of network-wide chemoconvulsant drug activity in the whole zebrafish brain illustrates the power of this approach for neuropharmacological profiling in applications ranging from accelerating studies of drug safety and efficacy, to identifying pharmacologically-altered networks in zebrafish models of human neurological disorders.

Long-term in vivo study of vertebrate embryonic development using noninvasive harmonics optical microscopy

NASA Astrophysics Data System (ADS)

Chen, Szu-Yu; Hsieh, C.-S.; Chu, S.-W.; Lin, Cheng-Yung; Ko, C.-Y.; Chen, Y.-C.; Tsai, Huai-Jen; Hu, C.-H.; Sun, Chi-Kuang

2005-03-01

Harmonics optical microscopy (HOM) provides a truly "noninvasive" tool for in vivo and long-term study of vertebrate embryonic development. Based on the nonlinear natures, it provides sub-micrometer 3D spatial resolution and high 3D optical-sectioning power (~1μm axial resolution) without using invasive and toxic fluorophores. Since only virtual-level-transition is involved, HOM is known to leave no energy deposition and no photodamages. Combined with second harmonic generation, which is sensitive to specific structure such as nerve and muscle fibers, HOM can be used to do functional studies of early developmental dynamics of many vertebrate physiological systems. Recently, zebrafish has become a standard model for many biological and medical studies of vertebrates, due to the similarity between embryonic development of zebrafish and human being. Zebrafish embryos now have been used to study many vertebrate physiological systems. We have demonstrated an in vivo HOM study of developmental dynamics of several embryonic physiological systems in live zebrafish embryos, with focuses on the developments of brains, eyes, ears, and hearts. Based on a femtosecond Cr:forsterite laser, which provides the deepest penetration (~1.5mm) and least photodamage in the zebrafish embryo, complete developing processes of different physiological systems within a period of time longer than 20 hours can be non-invasively observed inside the same embryo.

Random Walk of Single Gold Nanoparticles in Zebrafish Embryos Leading to Stochastic Toxic Effects on Embryonic Developments

PubMed Central

Browning, Lauren M.; Lee, Kerry J.; Huang, Tao; Nallathamby, Prakash D.; Lowman, Jill E.; Xu, Xiao-Hong Nancy

2010-01-01

We have synthesized and characterized stable (non-aggregation, non-photobleaching and non-blinking), nearly monodisperse and highly-purified Au nanoparticles, and used them to probe transport of cleavage-stage zebrafish embryos and to study their effects on embryonic development in real time. We found that single Au nanoparticles (11.6 ± 0.9 nm in diameter) passively diffused into chorionic space of the embryos via their chorionic-pore-canals and continued their random-walk through chorionic space and into inner mass of embryos. Diffusion coefficients of single nanoparticles vary dramatically (2.8×10-11 to 1.3×10-8 cm2/s) as nanoparticles diffuse through various parts of embryos, suggesting highly diverse transport barriers and viscosity gradients of embryos. The amount of Au nanoparticles accumulated in embryos increase with its concentration. Interestingly, their effects on embryonic development are not proportionally related to the concentration. Majority of embryos (74% on average) incubated chronically with 0.025-1.2 nM Au nanoparticles for 120 h developed to normal zebrafish, with some (24%) being dead and few (2%) deformed. We developed a new approach to image and characterize individual Au nanoparticles embedded in tissues using histology sample preparation methods and LSRP spectra of single nanoparticles. We found that Au nanoparticles in various parts of normally developed and deformed zebrafish, suggesting that random-walk of nanoparticles in embryos during their development might have led to stochastic effects on embryonic development. These results show that Au nanoparticles are much more biocompatible (less toxic) to the embryos than Ag nanoparticles that we reported previously, suggesting that they are better suited as biocompatible probes for imaging embryos in vivo. The results provide powerful evidences that biocompatibility and toxicity of nanoparticles highly depend on their chemical properties, and the embryos can serve as effective in

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

PubMed

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.

Probiotic modulation of the microbiota-gut-brain axis and behaviour in zebrafish.

PubMed

Borrelli, Luca; Aceto, Serena; Agnisola, Claudio; De Paolo, Sofia; Dipineto, Ludovico; Stilling, Roman M; Dinan, Timothy G; Cryan, John F; Menna, Lucia F; Fioretti, Alessandro

2016-07-15

The gut microbiota plays a crucial role in the bi-directional gut-brain axis, a communication that integrates the gut and central nervous system (CNS) activities. Animal studies reveal that gut bacteria influence behaviour, Brain-Derived Neurotrophic Factor (BDNF) levels and serotonin metabolism. In the present study, we report for the first time an analysis of the microbiota-gut-brain axis in zebrafish (Danio rerio). After 28 days of dietary administration with the probiotic Lactobacillus rhamnosus IMC 501, we found differences in shoaling behaviour, brain expression levels of bdnf and of genes involved in serotonin signalling/metabolism between control and treated zebrafish group. In addition, in microbiota we found a significant increase of Firmicutes and a trending reduction of Proteobacteria. This study demonstrates that selected microbes can be used to modulate endogenous neuroactive molecules in zebrafish.

In vivo loss of function study reveals the short stature homeobox-containing (shox) gene plays indispensable roles in early embryonic growth and bone formation in zebrafish.

PubMed

Sawada, Rie; Kamei, Hiroyasu; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Shimizu, Toshiaki

2015-02-01

Congenital loss of the SHOX gene is considered to be a genetic cause of short stature phenotype in Turner syndrome and Leri-Weill dyschondrosteosis patients. Though SHOX expression initiates during early fetal development, little is known about the embryonic roles of SHOX. The evolutionary conservation of the zebrafish shox gene and the convenience of the early developmental stages for analyses make zebrafish a preferred model. Here, we characterized structure, expression, and developmental roles of zebrafish shox through a loss-of-function approach. We found a previously undiscovered Shox protein that has both a homeodomain and an OAR-domain in zebrafish. The shox transcript emerged during the segmentation period and it increased in later stages. The predominant domains of shox expression were mandibular arch, pectoral fin, anterior notochord, rhombencephalon, and mesencephalon, suggesting that Shox is involved in bone and neural development. Translational blockade of Shox mRNA by an antisense morpholino oligo delayed embryonic growth, which was restored by the co-overexpression of morpholino-resistant Shox mRNA. At later stages, impaired Shox expression markedly delayed the calcification process in the anterior vertebral column and craniofacial bones. Our data demonstrate evolutionarily conserved Shox plays roles in early embryonic growth and in later bone formation. © 2014 Wiley Periodicals, Inc.

Cellular responses to recurrent pentylenetetrazole-induced seizures in the adult zebrafish brain

PubMed Central

Duy, Phan Q; Berberoglu, Michael A; Beattie, Christine E; Hall, Charles W

2017-01-01

A seizure is a sustained increase in brain electrical activity that can result in loss of consciousness and injury. Understanding how the brain responds to seizures is important for development of new treatment strategies for epilepsy, a neurological condition characterized by recurrent and unprovoked seizures. Pharmacological induction of seizures in rodent models results in a myriad of cellular alterations, including inflammation, angiogenesis, and adult neurogenesis. The purpose of this study is to investigate the cellular responses to recurrent pentylenetetrazole seizures in the adult zebrafish brain. We subjected zebrafish to five once daily pentylenetetrazole induced seizures and characterized the cellular consequences of these seizures. In response to recurrent seizures, we found histologic evidence of vasodilatation, perivascular leukocyte egress and leukocyte proliferation suggesting seizure-induced acute CNS inflammation. We also found evidence of increased proliferation, neurogenesis, and reactive gliosis. Collectively, our results suggest that the cellular responses to seizures in the adult zebrafish brain are similar to those observed in mammalian brains. PMID:28238851

Embryonic Zebrafish Model - A Well-Established Method for Rapidly Assessing the Toxicity of Homeopathic Drugs

PubMed Central

Gupta, Himanshu R; Patil, Yogesh; Singh, Dipty

2016-01-01

Objectives: Advancements in nanotechnology have led to nanoparticle (NP) use in various fields of medicine. Although the potential of NPs is promising, the lack of documented evidence on the toxicological effects of NPs is concerning. A few studies have documented that homeopathy uses NPs. Unfortunately, very few sound scientific studies have explored the toxic effects of homeopathic drugs. Citing this lack of high-quality scientific evidence, regulatory agencies have been reluctant to endorse homeopathic treatment as an alternative or adjunct treatment. This study aimed to enhance our insight into the impact of commercially-available homeopathic drugs, to study the presence of NPs in those drugs and any deleterious effects they might have, and to determine the distribution pattern of NPs in zebrafish embryos (Danio rerio). Methods: Homeopathic dilutions were studied using high-resolution transmission electron microscopy with selected area electron diffraction (SAED). For the toxicity assessment on Zebrafish, embryos were exposed to a test solution from 4 - 6 hours post-fertilization, and embryos/larvae were assessed up to 5 days post-fertilization (dpf) for viability and morphology. Toxicity was recorded in terms of mortality, hatching delay, phenotypic defects and metal accumulation. Around 5 dpf was found to be the optimum developmental stage for evaluation. Results: The present study aimed to conclusively prove the presence of NPs in all high dilutions of homeopathic drugs. Embryonic zebrafish were exposed to three homeopathic drugs with two potencies (30CH, 200CH) during early embryogenesis. The resulting morphological and cellular responses were observed. Exposure to these potencies produced no visibly significant malformations, pericardial edema, and mortality and no necrotic and apoptotic cellular death. Conclusion: Our findings clearly demonstrate that no toxic effects were observed for these three homeopathic drugs at the potencies and exposure times used

Abnormal cerebellar development and ataxia in CARP VIII morphant zebrafish.

PubMed

Aspatwar, Ashok; Tolvanen, Martti E E; Jokitalo, Eija; Parikka, Mataleena; Ortutay, Csaba; Harjula, Sanna-Kaisa E; Rämet, Mika; Vihinen, Mauno; Parkkila, Seppo

2013-02-01

Congenital ataxia and mental retardation are mainly caused by variations in the genes that affect brain development. Recent reports have shown that mutations in the CA8 gene are associated with mental retardation and ataxia in humans and ataxia in mice. The gene product, carbonic anhydrase-related protein VIII (CARP VIII), is predominantly present in cerebellar Purkinje cells, where it interacts with the inositol 1,4,5-trisphosphate receptor type 1, a calcium channel. In this study, we investigated the effects of the loss of function of CARP VIII during embryonic development in zebrafish using antisense morpholino oligonucleotides against the CA8 gene. Knockdown of CA8 in zebrafish larvae resulted in a curved body axis, pericardial edema and abnormal movement patterns. Histologic examination revealed gross morphologic defects in the cerebellar region and in the muscle. Electron microscopy studies showed increased neuronal cell death in developing larvae injected with CA8 antisense morpholinos. These data suggest a pivotal role for CARP VIII during embryonic development. Furthermore, suppression of CA8 expression leads to defects in motor and coordination functions, mimicking the ataxic human phenotype. This work reveals an evolutionarily conserved function of CARP VIII in brain development and introduces a novel zebrafish model in which to investigate the mechanisms of CARP VIII-related ataxia and mental retardation in humans.

Effects of embryonic ethanol exposure at low doses on neuronal development, voluntary ethanol consumption and related behaviors in larval and adult zebrafish: Role of hypothalamic orexigenic peptides.

PubMed

Sterling, M E; Chang, G-Q; Karatayev, O; Chang, S Y; Leibowitz, S F

2016-05-01

Embryonic exposure to ethanol is known to affect neurochemical systems in rodents and increase alcohol drinking and related behaviors in humans and rodents. With zebrafish emerging as a powerful tool for uncovering neural mechanisms of numerous diseases and exhibiting similarities to rodents, the present report building on our rat studies examined in zebrafish the effects of embryonic ethanol exposure on hypothalamic neurogenesis, expression of orexigenic neuropeptides, and voluntary ethanol consumption and locomotor behaviors in larval and adult zebrafish, and also effects of central neuropeptide injections on these behaviors affected by ethanol. At 24h post-fertilization, zebrafish embryos were exposed for 2h to ethanol, at low concentrations of 0.25% and 0.5%, in the tank water. Embryonic ethanol compared to control dose-dependently increased hypothalamic neurogenesis and the proliferation and expression of the orexigenic peptides, galanin (GAL) and orexin (OX), in the anterior hypothalamus. These changes in hypothalamic peptide neurons were accompanied by an increase in voluntary consumption of 10% ethanol-gelatin and in novelty-induced locomotor and exploratory behavior in adult zebrafish and locomotor activity in larvae. After intracerebroventricular injection, these peptides compared to vehicle had specific effects on these behaviors altered by ethanol, with GAL stimulating consumption of 10% ethanol-gelatin more than plain gelatin food and OX stimulating novelty-induced locomotor behavior while increasing intake of food and ethanol equally. These results, similar to those obtained in rats, suggest that the ethanol-induced increase in genesis and expression of these hypothalamic peptide neurons contribute to the behavioral changes induced by embryonic exposure to ethanol. Copyright © 2016 Elsevier B.V. All rights reserved.

ApoA-II directs morphogenetic movements of zebrafish embryo by preventing chromosome fusion during nuclear division in yolk syncytial layer.

PubMed

Zhang, Ting; Yao, Shaohua; Wang, Ping; Yin, Chaoran; Xiao, Chun; Qian, Meilin; Liu, Donghui; Zheng, Lemin; Meng, Wentong; Zhu, Hongyan; Liu, Jin; Xu, Hong; Mo, Xianming

2011-03-18

The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation.

ApoA-II Directs Morphogenetic Movements of Zebrafish Embryo by Preventing Chromosome Fusion during Nuclear Division in Yolk Syncytial Layer*

PubMed Central

Zhang, Ting; Yao, Shaohua; Wang, Ping; Yin, Chaoran; Xiao, Chun; Qian, Meilin; Liu, Donghui; Zheng, Lemin; Meng, Wentong; Zhu, Hongyan; Liu, Jin; Xu, Hong; Mo, Xianming

2011-01-01

The high density lipoprotein (HDL) represents a class of lipid- and protein-containing particles and consists of two major apolipoproteins apoA-I and apoA-II. ApoA-II has been shown to be involved in the pathogenesis of insulin resistance, adiposity, diabetes, and metabolic syndrome. In embryo, apoa2 mRNAs are abundant in the liver, brain, lung, placenta, and in fish yolk syncytial layer (YSL), suggesting that apoa2 may perform a function during embryonic development. Here we find out that apoa2 modulates zebrafish embryonic development by regulating the organization of YSL. Disruption of apoa2 function in zebrafish caused chromosome fusing, which strongly blocked YSL nuclear division, inducing disorders in YSL organization and finally disturbing the embryonic epiboly. Purified native human apoA-II was able specifically to rescue the defects and induced nuclear division in zebrafish embryos and in human HeLa cells. The C terminus of apoA-II was required for the proper chromosome separation during nuclear division of YSL in zebrafish embryos and in human HeLa cells. Our data indicate that organization of YSL is required for blastoderm patterning and morphogenesis and suggest that apolipoprotein apoA-II is a novel factor of nuclear division in YSL involved in the regulation of early zebrafish embryonic morphogenesis and in mammalian cells for proliferation. PMID:21212265

Venous-derived angioblasts generate organ-specific vessels during zebrafish embryonic development.

PubMed

Hen, Gideon; Nicenboim, Julian; Mayseless, Oded; Asaf, Lihee; Shin, Masahiro; Busolin, Giorgia; Hofi, Roy; Almog, Gabriella; Tiso, Natascia; Lawson, Nathan D; Yaniv, Karina

2015-12-15

Formation and remodeling of vascular beds are complex processes orchestrated by multiple signaling pathways. Although it is well accepted that vessels of a particular organ display specific features that enable them to fulfill distinct functions, the embryonic origins of tissue-specific vessels and the molecular mechanisms regulating their formation are poorly understood. The subintestinal plexus of the zebrafish embryo comprises vessels that vascularize the gut, liver and pancreas and, as such, represents an ideal model in which to investigate the early steps of organ-specific vessel formation. Here, we show that both arterial and venous components of the subintestinal plexus originate from a pool of specialized angioblasts residing in the floor of the posterior cardinal vein (PCV). Using live imaging of zebrafish embryos, in combination with photoconvertable transgenic reporters, we demonstrate that these angioblasts undergo two phases of migration and differentiation. Initially, a subintestinal vein forms and expands ventrally through a Bone Morphogenetic Protein-dependent step of collective migration. Concomitantly, a Vascular Endothelial Growth Factor-dependent shift in the directionality of migration, coupled to the upregulation of arterial markers, is observed, which culminates with the generation of the supraintestinal artery. Together, our results establish the zebrafish subintestinal plexus as an advantageous model for the study of organ-specific vessel development and provide new insights into the molecular mechanisms controlling its formation. More broadly, our findings suggest that PCV-specialized angioblasts contribute not only to the formation of the early trunk vasculature, but also to the establishment of late-forming, tissue-specific vascular beds. © 2015. Published by The Company of Biologists Ltd.

Chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish.

PubMed

Yu, Kaimin; Li, Guochao; Feng, Weimin; Liu, Lili; Zhang, Jiayu; Wu, Wei; Xu, Lei; Yan, Yanchun

2015-09-05

The potential interference of endocrine disrupting chemicals (EDCs) on aquatic animals and humans has drawn wide attention in recent years. Reports have shown that some organophosphorus pesticides were a kind of EDCs, but their effects on fish species are still under research. In present study, flow cytometry data of HEC-1B cell line showed that chlorpyrifos (CPF) could increase cell proliferation index like 17β-estradiol (E2), but the effect of CPF was weaker than of E2 in the same concentration. Moreover, CPF altered the expression pattern of estrogen-responsive gene VTG and ERα in zebrafish embryos. When exposed to CPF at various concentrations (0, 0.10, 0.25, 0.50, 0.75 and 1.00mg/L) for 48h during the embryo stage, compared with controls, the hatching rate of treated groups significantly increased at the same time and the hatching rate of embryos was proportional to CPF concentration. The mRNA expression levels of c-myc, cyclin D1, Bax and Bcl-2, which are closely related to cell proliferation and cell apoptosis, were disturbed by CPF in zebrafish embryos after exposure treated for 48h. In addition, acridine orange (AO) staining of zebrafish embryos showed that cell apoptosis was appeared in the 0.75, 1.00mg/L CPF treated groups. Taken together, the results obtained in the present study indicated that chlorpyrifos is estrogenic and alters embryonic hatching, cell proliferation and apoptosis in zebrafish. Copyright © 2015. Published by Elsevier Ireland Ltd.

BDNF Expression in Larval and Adult Zebrafish Brain: Distribution and Cell Identification

PubMed Central

Cacialli, Pietro; Gueguen, Marie-Madeleine; Coumailleau, Pascal; D’Angelo, Livia; Kah, Olivier; Lucini, Carla; Pellegrini, Elisabeth

2016-01-01

Brain-derived neurotrophic factor (BDNF), a member of the neurotrophin family, has emerged as an active mediator in many essential functions in the central nervous system of mammals. BDNF plays significant roles in neurogenesis, neuronal maturation and/or synaptic plasticity and is involved in cognitive functions such as learning and memory. Despite the vast literature present in mammals, studies devoted to BDNF in the brain of other animal models are scarse. Zebrafish is a teleost fish widely known for developmental genetic studies and is emerging as model for translational neuroscience research. In addition, its brain shows many sites of adult neurogenesis allowing higher regenerative properties after traumatic injuries. To add further knowledge on neurotrophic factors in vertebrate brain models, we decided to determine the distribution of bdnf mRNAs in the larval and adult zebrafish brain and to characterize the phenotype of cells expressing bdnf mRNAs by means of double staining studies. Our results showed that bdnf mRNAs were widely expressed in the brain of 7 days old larvae and throughout the whole brain of mature female and male zebrafish. In adults, bdnf mRNAs were mainly observed in the dorsal telencephalon, preoptic area, dorsal thalamus, posterior tuberculum, hypothalamus, synencephalon, optic tectum and medulla oblongata. By combining immunohistochemistry with in situ hybridization, we showed that bdnf mRNAs were never expressed by radial glial cells or proliferating cells. By contrast, bdnf transcripts were expressed in cells with neuronal phenotype in all brain regions investigated. Our results provide the first demonstration that the brain of zebrafish expresses bdnf mRNAs in neurons and open new fields of research on the role of the BDNF factor in brain mechanisms in normal and brain repairs situations. PMID:27336917

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

PubMed

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).

The effect of silver nanoparticles on zebrafish embryonic development and toxicology.

PubMed

Xia, Guangqing; Liu, Tiantian; Wang, Zhenwei; Hou, Yi; Dong, Lihong; Zhu, Junyi; Qi, Jie

2016-06-01

The unique physical and chemical characteristics of nanomaterials, such as the effects of their small size, surface effects, very high rates of reaction, and quantum tunnel effect, have aroused great interest among scholars. However, improper usage has led to an increasing number of nanomaterials entering the environment through various channels, greatly threatening the security of the ecological environment and human health. The urgent need for a scientific assessment of their biosafety can enable nanomaterials to truly benefit humanity. However, the current research in this field is extremely limited with regard to safety standards and waste disposal. In this study, we used silver nanoparticles (nano-Ag) and zebrafish embryos as experimental subjects, and we have reported the deleterious effect on zebrafish embryos treated with different concentrations of nano-Ag, with respect to morphological features (mortality, deformity rate, and heartbeat) and the analysis of expression of relevant genes (sox17, gsc, ntl, otx2); we found a dose-dependent increase in mortality and hatching delay. The results of in situ hybridization indicated that nano-Ag causes a dose-dependent toxicity in embryonic development, and would affect their development and lead to deformity, delayed development, and even death. The safety limit for the concentration of nano-Ag was found to be less than 5 mg/L.

Cerebroventricular Microinjection (CVMI) into Adult Zebrafish Brain Is an Efficient Misexpression Method for Forebrain Ventricular Cells

PubMed Central

Kizil, Caghan; Brand, Michael

2011-01-01

The teleost fish Danio rerio (zebrafish) has a remarkable ability to generate newborn neurons in its brain at adult stages of its lifespan-a process called adult neurogenesis. This ability relies on proliferating ventricular progenitors and is in striking contrast to mammalian brains that have rather restricted capacity for adult neurogenesis. Therefore, investigating the zebrafish brain can help not only to elucidate the molecular mechanisms of widespread adult neurogenesis in a vertebrate species, but also to design therapies in humans with what we learn from this teleost. Yet, understanding the cellular behavior and molecular programs underlying different biological processes in the adult zebrafish brain requires techniques that allow manipulation of gene function. As a complementary method to the currently used misexpression techniques in zebrafish, such as transgenic approaches or electroporation-based delivery of DNA, we devised a cerebroventricular microinjection (CVMI)-assisted knockdown protocol that relies on vivo morpholino oligonucleotides, which do not require electroporation for cellular uptake. This rapid method allows uniform and efficient knockdown of genes in the ventricular cells of the zebrafish brain, which contain the neurogenic progenitors. We also provide data on the use of CVMI for growth factor administration to the brain – in our case FGF8, which modulates the proliferation rate of the ventricular cells. In this paper, we describe the CVMI method and discuss its potential uses in zebrafish. PMID:22076157

Automatic zebrafish heartbeat detection and analysis for zebrafish embryos.

PubMed

Pylatiuk, Christian; Sanchez, Daniela; Mikut, Ralf; Alshut, Rüdiger; Reischl, Markus; Hirth, Sofia; Rottbauer, Wolfgang; Just, Steffen

2014-08-01

A fully automatic detection and analysis method of heartbeats in videos of nonfixed and nonanesthetized zebrafish embryos is presented. This method reduces the manual workload and time needed for preparation and imaging of the zebrafish embryos, as well as for evaluating heartbeat parameters such as frequency, beat-to-beat intervals, and arrhythmicity. The method is validated by a comparison of the results from automatic and manual detection of the heart rates of wild-type zebrafish embryos 36-120 h postfertilization and of embryonic hearts with bradycardia and pauses in the cardiac contraction.

Methods for studying the zebrafish brain: past, present and future.

PubMed

Wyatt, Cameron; Bartoszek, Ewelina M; Yaksi, Emre

2015-07-01

The zebrafish (Danio rerio) is one of the most promising new model organisms. The increasing popularity of this amazing small vertebrate is evident from the exponentially growing numbers of research articles, funded projects and new discoveries associated with the use of zebrafish for studying development, brain function, human diseases and screening for new drugs. Thanks to the development of novel technologies, the range of zebrafish research is constantly expanding with new tools synergistically enhancing traditional techniques. In this review we will highlight the past and present techniques which have made, and continue to make, zebrafish an attractive model organism for various fields of biology, with a specific focus on neuroscience. © 2015 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.

Estrogenic Effects of Several BPA Analogs in the Developing Zebrafish Brain

PubMed Central

Cano-Nicolau, Joel; Vaillant, Colette; Pellegrini, Elisabeth; Charlier, Thierry D.; Kah, Olivier; Coumailleau, Pascal

2016-01-01

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- 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. PMID:27047331

Exposure to benzidine caused apoptosis and malformation of telencephalon region in zebrafish.

PubMed

Chen, Mark Hung-Chih; Hsu, Li-Chi; Wu, Jia-Lun; Yeh, Chi-Wei; Tsai, Jen-Ning; Hseu, You-Cheng; Hsu, Li-Sung

2014-12-01

Exposure to benzidine has been known to induce human cancers, particularly bladder carcinomas. In this study, the zebrafish model was used to investigate the developmental toxicity of benzidine. Embryos at 6 h postfertilization (hpf) that were exposed to benzidine exhibited embryonic death in a dose- and time-dependent manner. Benzidine induced malformations in zebrafish, such as small brain development, shorter axes, and a slight pericardial edema. High concentrations (50, 100, and 200 µM) of benzidine triggered widespread apoptosis in the brain and dorsal neurons, as evidenced by acridine orange and terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) assays. Real-time polymerase chain reaction analysis also showed that benzidine treatment affected p53, bax, and noxa expression. Decreases in specific brain markers, such as emx1 in the telencephalon, ngn1 in differentiated neurons, and otx2 in the midbrain, were observed in benzidine-treated embryos at 24 hpf. Conversely, no overt changes to pax2.1 expression in the midbrain-hindbrain boundary were found. Moreover, the use of Tg(HuC:GFP) zebrafish showed that benzidine caused a malformation of the telencephalon region. Our findings show that benzidine exposure triggers widespread apoptosis in the zebrafish brain and dorsal neurons, resulting in the development of an abnormal telencephalon. © 2013 Wiley Periodicals, Inc.

Time dependent effect of chronic embryonic exposure to ethanol on zebrafish: Morphology, biochemical and anxiety alterations.

PubMed

Ramlan, Nurul Farhana; Sata, Nurul Syafida Asma Mohd; Hassan, Siti Norhidayah; Bakar, Noraini Abu; Ahmad, Syahida; Zulkifli, Syaizwan Zahmir; Abdullah, Che Azurahanim Che; Ibrahim, Wan Norhamidah Wan

2017-08-14

Exposure to ethanol during critical period of development can cause severe impairments in the central nervous system (CNS). This study was conducted to assess the neurotoxic effects of chronic embryonic exposure to ethanol in the zebrafish, taking into consideration the time dependent effect. Two types of exposure regimen were applied in this study. Withdrawal exposure group received daily exposure starting from gastrulation until hatching, while continuous exposure group received daily exposure from gastrulation until behavioural assessment at 6dpf (days post fertilization). Chronic embryonic exposure to ethanol decreased spontaneous tail coiling at 24hpf (hour post fertilization), heart rate at 48hpf and increased mortality rate at 72hpf. The number of apoptotic cells in the embryos treated with ethanol was significantly increased as compared to the control. We also measured the morphological abnormalities and the most prominent effects can be observed in the treated embryos exposed to 1.50% and 2.00%. The treated embryos showed shorter body length, larger egg yolk, smaller eye diameter and heart edema as compared to the control. Larvae received 0.75% continuous ethanol exposure exhibited decreased swimming activity and increased anxiety related behavior, while withdrawal ethanol exposure showed increased swimming activity and decreased anxiety related behavior as compared to the respective control. Biochemical analysis exhibited that ethanol exposure for both exposure regimens altered proteins, lipids, carbohydrates and nucleic acids of the zebrafish larvae. Our results indicated that time dependent effect of ethanol exposure during development could target the biochemical processes thus leading to induction of apoptosis and neurobehavioral deficits in the zebrafish larvae. Thus it raised our concern about the safe limit of alcohol consumption for pregnant mother especially during critical periods of vulnerability for developing nervous system. Copyright © 2017

Rapid whole brain imaging of neural activity in freely behaving larval zebrafish (Danio rerio)

PubMed Central

Shang, Chunfeng; Yang, Wenbin; Bai, Lu; Du, Jiulin

2017-01-01

The internal brain dynamics that link sensation and action are arguably better studied during natural animal behaviors. Here, we report on a novel volume imaging and 3D tracking technique that monitors whole brain neural activity in freely swimming larval zebrafish (Danio rerio). We demonstrated the capability of our system through functional imaging of neural activity during visually evoked and prey capture behaviors in larval zebrafish. PMID:28930070

GLUT3 gene expression is critical for embryonic growth, brain development and survival.

PubMed

Carayannopoulos, Mary O; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U

2014-04-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. Copyright © 2014 Elsevier Inc. All rights reserved.

GLUT3 Gene Expression is Critical for Embryonic Growth, Brain Development and Survival

PubMed Central

Carayannopoulos, Mary O.; Xiong, Fuxia; Jensen, Penny; Rios-Galdamez, Yesenia; Huang, Haigen; Lin, Shuo; Devaskar, Sherin U.

2015-01-01

Glucose is the primary energy source for eukaryotic cells and the predominant substrate for the brain. GLUT3 is essential for trans-placental glucose transport and highly expressed in the mammalian brain. To further elucidate the role of GLUT3 in embryonic development, we utilized the vertebrate whole animal model system of Danio rerio as a tractable system for defining the cellular and molecular mechanisms altered by impaired glucose transport and metabolism related to perturbed expression of GLUT3. The comparable orthologue of human GLUT3 was identified and the expression of this gene abrogated during early embryonic development. In a dose-dependent manner embryonic brain development was disrupted resulting in a phenotype of aberrant brain organogenesis, associated with embryonic growth restriction and increased cellular apoptosis. Rescue of the morphant phenotype was achieved by providing exogenous GLUT3 mRNA. We conclude that GLUT3 is critically important for brain organogenesis and embryonic growth. Disruption of GLUT3 is responsible for the phenotypic spectrum of embryonic growth restriction to demise and neural apoptosis with microcephaly. PMID:24529979

Benomyl induction of brain aromatase and toxic effects in the zebrafish embryo.

PubMed

Kim, Dong-Jae; Seok, Seung-Hyeok; Baek, Min-Won; Lee, Hui-Young; Na, Yi-Rang; Park, Sung-Hoon; Lee, Hyun-Kyoung; Dutta, Noton Kumar; Kawakami, Koichi; Park, Jae-Hak

2009-05-01

Benomyl is a benzimidazole fungicide that has been widely used on a variety of food crops and ornamental plants. It is known to cause adverse effects on reproductive systems, including decreased testicular and epididymal weights and reduced epididymal sperm counts and fertility. The brain aromatase gene is up-regulated by estrogens and estrogen mimics and considered a target gene to screen estrogen mimics. This study was designed to test the estrogenic potential and toxic effects of benomyl in the zebrafish system, and validated this system as a model that may correspond to the effect of benomyl in rodents. Concentrations of 20 x 10(-6), 40 x 10(-6) and 80 x 10(-6) M of benomyl-treated embryos showed decreased survival, hatching and heart rates, and increased incidence of malformations, such as pericardial edema, spinal lordosis, elongated heart, head edema, eye lens protrusion and caudal fin disappearance. Benomyl induced enhanced green fluorescent protein (EGFP) expression in the mediobasal hypothalamus (MBH) in transient zebrafish embryos with a brain aromatase-based reporter gene. In this study, we determined that benomyl has estrogenic potential based on zebrafish brain aromatase gene induction, and that benomyl is toxic at 20 x 10(-6) M concentration and higher. These results demonstrate the usefulness of zebrafish embryos as an in vivo system to examine the estrogenic and developmental toxic potential of unknown compounds.

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

PubMed

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.

Localization of BDNF expression in the developing brain of zebrafish

PubMed Central

De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

2014-01-01

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. PMID:24588510

Cadmium affects muscle type development and axon growth in zebrafish embryonic somitogenesis.

PubMed

Hen Chow, Elly Suk; Cheng, Shuk Han

2003-05-01

We have previously reported that exposure to cadmium during zebrafish embryonic development caused morphological malformations of organs and ectopic expression of genes involved in regulating developmental process. One of the most common developmental defects observed was altered axial curvature resulting from defects in the myotomes of the somites. In this study, we investigated the mechanisms of cadmium-induced toxicity in zebrafish somitogenesis. We showed that the critical period of exposure was the gastrulation period, which actually preceded the formation of the first morphologically distinct somites. The somites thus formed lost the typical chevron V-shape and are packed disorderly. The myogenic lineage commitment of the axial mesodermal cells was not affected, as the myogenic regulatory transcription factors were expressed normally. There were, however, losses of fast and slow muscle fibers in the myotomes. The innervation of the muscle blocks by spinal motoneurons is an important process of the somitogenesis. Both primary and secondary motoneurons appear to form normally while the axon growth is affected in cadmium-treated embryos. The notochord, which is essential in the patterning of the somites and the central nervous system, showed abnormal morphological features and failed to extend to the tail region. Taken together, it appears that cadmium exposure led to abnormal somite patterning of the muscle fibers and defects in axonogenesis.

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish.

PubMed

Gamse, Joshua T; Gorelick, Daniel A

2016-10-01

For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals.

Localization of BDNF expression in the developing brain of zebrafish.

PubMed

De Felice, E; Porreca, I; Alleva, E; De Girolamo, P; Ambrosino, C; Ciriaco, E; Germanà, A; Sordino, P

2014-05-01

The brain-derived neurotrophic factor (BDNF) gene is expressed in differentiating and post-mitotic neurons of the zebrafish embryo, where it has been implicated in Huntington's disease. Little is known, however, about the full complement of neuronal cell types that express BDNF in this important vertebrate model. Here, we further explored the transcriptional profiles during the first week of development using real-time quantitative polymerase chain reaction (RT-qPCR) and whole-mount in situ hybridization (WISH). RT-qPCR results revealed a high level of maternal contribution followed by a steady increase of zygotic transcription, consistent with the notion of a prominent role of BDNF in neuronal maturation and maintenance. Based on WISH, we demonstrate for the first time that BDNF expression in the developing brain of zebrafish is structure specific. Anatomical criteria and co-staining with genetic markers (shh, pax2a, emx1, krox20, lhx2b and lhx9) visualized major topological domains of BDNF-positive cells in the pallium, hypothalamus, posterior tuberculum and optic tectum. Moreover, the relative timing of BDNF transcription in the eye and tectum may illustrate a mechanism for coordinated development of the retinotectal system. Taken together, our results are compatible with a local delivery and early role of BDNF in the developing brain of zebrafish, adding basic knowledge to the study of neurotrophin functions in neural development and disease. © 2014 Anatomical Society.

BMP signaling modulates hepcidin expression in zebrafish embryos independent of hemojuvelin.

PubMed

Gibert, Yann; Lattanzi, Victoria J; Zhen, Aileen W; Vedder, Lea; Brunet, Frédéric; Faasse, Sarah A; Babitt, Jodie L; Lin, Herbert Y; Hammerschmidt, Matthias; Fraenkel, Paula G

2011-01-21

Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.

Alkbh4 and Atrn Act Maternally to Regulate Zebrafish Epiboly

PubMed Central

Sun, Qingrui; Liu, Xingfeng; Gong, Bo; Wu, Di; Meng, Anming; Jia, Shunji

2017-01-01

During embryonic gastrulation, coordinated cell movements occur to bring cells to their correct position. Among them, epiboly produces the first distinct morphological changes, which is essential for the early development of zebrafish. Despite its fundamental importance, little is known to understand the underlying molecular mechanisms. By generating maternal mutant lines with CRISPR/Cas9 technology and using morpholino knockdown strategy, we showed that maternal Alkbh4 depletion leads to severe epiboly defects in zebrafish. Immunofluorescence assays revealed that Alkbh4 promotes zebrafish embryonic epiboly through regulating actomyosin contractile ring formation, which is composed of Actin and non-muscular myosin II (NMII). To further investigate this process, yeast two hybridization assay was performed and Atrn was identified as a binding partner of Alkbh4. Combining with the functional results of Alkbh4, we found that maternal Atrn plays a similar role in zebrafish embryonic morphogenesis by regulating actomyosin formation. On the molecular level, our data revealed that Atrn prefers to interact with the active form of Alkbh4 and functions together with it to regulate the demethylation of Actin, the actomyosin formation, and subsequently the embryonic epiboly. PMID:28924386

Developmental exposure to acetaminophen does not induce hyperactivity in zebrafish larvae.

PubMed

Reuter, Isabel; Knaup, Sabine; Romanos, Marcel; Lesch, Klaus-Peter; Drepper, Carsten; Lillesaar, Christina

2016-08-01

First line pain relief medication during pregnancy relies nearly entirely on the over-the-counter analgesic acetaminophen, which is generally considered safe to use during gestation. However, recent epidemiological studies suggest a risk of developing attention-deficit/hyperactivity disorder (ADHD)-like symptoms in children if mothers use acetaminophen during pregnancy. Currently, there are no experimental proofs that prenatal acetaminophen exposure causes developmental brain alterations of progeny. Exposure to high acetaminophen concentrations causes liver toxicity, which is well investigated in different model organisms. However, sub-liver-toxic concentrations have not been experimentally investigated with respect to ADHD endophenotypes such as hyperactivity. We used zebrafish to investigate the potential impact of acetaminophen exposure on locomotor activity levels, and compared it to the established zebrafish Latrophilin 3 (Lphn3) ADHD-model. We determined the sub-liver-toxic concentration of acetaminophen in zebrafish larvae and treated wild-type and lphn3.1 knockdown larvae with increasing concentrations of acetaminophen. We were able to confirm that lphn3.1 knockdown alone causes hyperactivity, strengthening the implication of Lphn3 dysfunction as an ADHD risk factor. Neither acute nor chronic exposure to acetaminophen at sub-liver-toxic concentrations in wild-type or lphn3.1 knock-downs increases locomotor activity levels. Together our findings show that embryonic to larval exposure to acetaminophen does not cause hyperactivity in zebrafish larvae. Furthermore, there are no additive and/or synergistic effects of acetaminophen exposure in a susceptible background induced by knock-down of lphn3.1. Our experimental study suggests that there is, at least in zebrafish larvae, no direct link between embryonic acetaminophen exposure and hyperactivity. Further work is necessary to clarify this issue in humans.

Whole-brain activity mapping onto a zebrafish brain atlas.

PubMed

Randlett, Owen; Wee, Caroline L; Naumann, Eva A; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E; Portugues, Ruben; Lacoste, Alix M B; Riegler, Clemens; Engert, Florian; Schier, Alexander F

2015-11-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Whole-brain activity mapping onto a zebrafish brain atlas

PubMed Central

Randlett, Owen; Wee, Caroline L.; Naumann, Eva A.; Nnaemeka, Onyeka; Schoppik, David; Fitzgerald, James E.; Portugues, Ruben; Lacoste, Alix M.B.; Riegler, Clemens; Engert, Florian; Schier, Alexander F.

2015-01-01

In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open source atlas containing molecular labels and anatomical region definitions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated-Extracellular signal-regulated kinase (ERK/MAPK) as a readout of neural activity, we have developed a system to create and contextualize whole brain maps of stimulus- and behavior-dependent neural activity. This MAP-Mapping (Mitogen Activated Protein kinase – Mapping) assay is technically simple, fast, inexpensive, and data analysis is completely automated. Since MAP-Mapping is performed on fish that are freely swimming, it is applicable to nearly any stimulus or behavior. We demonstrate the utility of our high-throughput approach using hunting/feeding, pharmacological, visual and noxious stimuli. The resultant maps outline hundreds of areas associated with behaviors. PMID:26778924

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice.

PubMed

Alonso, Maria I; Lamus, Francisco; Carnicero, Estela; Moro, Jose A; de la Mano, Anibal; Fernández, Jose M F; Desmond, Mary E; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies.

Embryonic Cerebrospinal Fluid Increases Neurogenic Activity in the Brain Ventricular-Subventricular Zone of Adult Mice

PubMed Central

Alonso, Maria I.; Lamus, Francisco; Carnicero, Estela; Moro, Jose A.; de la Mano, Anibal; Fernández, Jose M. F.; Desmond, Mary E.; Gato, Angel

2017-01-01

Neurogenesis is a very intensive process during early embryonic brain development, becoming dramatically restricted in the adult brain in terms of extension and intensity. We have previously demonstrated the key role of embryonic cerebrospinal fluid (CSF) in developing brain neurogenic activity. We also showed that cultured adult brain neural stem cells (NSCs) remain competent when responding to the neurogenic influence of embryonic CSF. However, adult CSF loses its neurogenic inductive properties. Here, by means of an organotypic culture of adult mouse brain sections, we show that local administration of embryonic CSF in the subventricular zone (SVZ) niche is able to trigger a neurogenic program in NSCs. This leads to a significant increase in the number of non-differentiated NSCs, and also in the number of new neurons which show normal migration, differentiation and maturation. These new data reveal that embryonic CSF activates adult brain NSCs, supporting the previous idea that it contains key instructive components which could be useful in adult brain neuroregenerative strategies. PMID:29311854

Mixtures, Metabolites, and Mechanisms: Understanding Toxicology Using Zebrafish

PubMed Central

Gamse, Joshua T.

2016-01-01

Abstract For more than 60 years, zebrafish have been used in toxicological studies. Due to their transparency, genetic tractability, and compatibility with high-throughput screens, zebrafish embryos are uniquely suited to study the effects of pharmaceuticals and environmental insults on embryonic development, organ formation and function, and reproductive success. This special issue of Zebrafish highlights the ways zebrafish are used to investigate the toxic effects of endocrine disruptors, pesticides, and heavy metals. PMID:27618129

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

PubMed

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.

Quantitative assessment of cypermethrin induced behavioural and biochemical anomalies in adult zebrafish.

PubMed

Nema, Shubham; Bhargava, Yogesh

2018-05-23

Cypermethrin is one of the top five pesticides used globally. Although the effect of cypermethrin on the embryonic stages of zebrafish is well characterized, its toxic effect on the behaviour of adult zebrafish is largely unknown. Here we used videogram and automated tracking approach to quantitatively assess behavioural toxicity induced by the short exposure of cypermethrin to adult zebrafish. We observed that cypermethrin at 25 ppb level induced behavioural toxicity in adult zebrafish. Motor activity of the treated group was significantly retarded which affected their overall exploratory behaviour including their visit to the central arena of the open-field test. Furthermore, the treated group showed erratic movements (covered less distance per unit time) without affecting their angle based behavioural endpoints. In contrast to the control group, the cypermethrin exposed group showed frequent freezing behaviour. However, their freezing episodes were characterized by constant drift-like movement caused by the loss of their voluntary control over the motor coordination. These behavioural changes are similar to typical anxiety-like behaviour. Though, cypermethrin exposure at ppb level for just half an hour was sufficient to induce behavioural toxicity, it failed to alter brain superoxide dismutase and acetylcholine esterase enzyme activity. Our data indicates that acute short-term exposure of cypermethrin induces behavioural anomalies in adult zebrafish through a mechanism distinct from alteration of brain superoxide dismutase and the acetylcholine esterase activity. Copyright © 2018 Elsevier Inc. All rights reserved.

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shi, Yanan; Liu, Xiaochun; Zhu, Pei

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.more » 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.« less

Diving into the world of alcohol teratogenesis: a review of zebrafish models of fetal alcohol spectrum disorder.

PubMed

Fernandes, Yohaan; Buckley, Desire M; Eberhart, Johann K

2018-04-01

The term fetal alcohol spectrum disorder (FASD) refers to the entire suite of deleterious outcomes resulting from embryonic exposure to alcohol. Along with other reviews in this special issue, we provide insight into how animal models, specifically the zebrafish, have informed our understanding of FASD. We first provide a brief introduction to FASD. We discuss the zebrafish as a model organism and its strengths for alcohol research. We detail how zebrafish has been used to model some of the major defects present in FASD. These include behavioral defects, such as social behavior as well as learning and memory, and structural defects, disrupting organs such as the brain, sensory organs, heart, and craniofacial skeleton. We provide insights into how zebrafish research has aided in our understanding of the mechanisms of ethanol teratogenesis. We end by providing some relatively recent advances that zebrafish has provided in characterizing gene-ethanol interactions that may underlie FASD.

Quantitative analysis of mechanical force required for cell extrusion in zebrafish embryonic epithelia.

PubMed

Yamada, Sohei; Iino, Takanori; Bessho, Yasumasa; Hosokawa, Yoichiroh; Matsui, Takaaki

2017-10-15

When cells in epithelial sheets are damaged by intrinsic or extrinsic causes, they are eliminated by extrusion from the sheet. Cell extrusion, which is required for maintenance of tissue integrity, is the consequence of contraction of actomyosin rings, as demonstrated by both molecular/cellular biological experimentation and numerical simulation. However, quantitative evaluation of actomyosin contraction has not been performed because of the lack of a suitable direct measurement system. In this study, we developed a new method using a femtosecond laser to quantify the contraction force of the actomyosin ring during cell extrusion in zebrafish embryonic epithelia. In this system, an epithelial cell in zebrafish embryo is first damaged by direct femtosecond laser irradiation. Next, a femtosecond laser-induced impulsive force is loaded onto the actomyosin ring, and the contraction force is quantified to be on the order of kPa as a unit of pressure. We found that cell extrusion was delayed when the contraction force was slightly attenuated, suggesting that a relatively small force is sufficient to drive cell extrusion. Thus, our method is suitable for the relative quantitative evaluation of mechanical dynamics in the process of cell extrusion, and in principle the method is applicable to similar phenomena in different tissues and organs of various species. © 2017. Published by The Company of Biologists Ltd.

Cep55 regulates embryonic growth and development by promoting Akt stability in zebrafish.

PubMed

Jeffery, Jessie; Neyt, Christine; Moore, Wade; Paterson, Scott; Bower, Neil I; Chenevix-Trench, Georgia; Verkade, Heather; Hogan, Benjamin M; Khanna, Kum Kum

2015-05-01

CEP55 was initially described as a centrosome- and midbody-associated protein and a key mediator of cytokinesis. More recently, it has been implicated in PI3K/AKT pathway activation via an interaction with the catalytic subunit of PI3K. However, its role in embryonic development is unknown. Here we describe a cep55 nonsense mutant zebrafish with which we can study the in vivo physiologic role of Cep55. Homozygous mutants underwent extensive apoptosis by 24 hours postfertilization (hpf) concomitant with cell cycle defects, and heterozygous carriers were indistinguishable from their wild-type siblings. A similar phenotype was also observed in zebrafish injected with a cep55 morpholino, suggesting the mutant is a cep55 loss-of-function model. Further analysis revealed that Akt was destabilized in the homozygous mutants, which partially phenocopied Akt1 and Akt2 knockdown. Expression of either constitutively activated PIK3CA or AKT1 could partially rescue the homozygous mutants. Consistent with a role for Cep55 in regulation of Akt stability, treatment with proteasome inhibitor, MG132, partially rescued the homozygous mutants. Taken together, these results provide the first description of Cep55 in development and underline the importance of Cep55 in the regulation of Pi3k/Akt pathway and in particular Akt stability. © FASEB.

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

PubMed

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.

Anesthesia and euthanasia in zebrafish.

PubMed

Matthews, Monte; Varga, Zoltán M

2012-01-01

Because of the relative ease of embryonic manipulation and observation, the ability to produce a great number of genetic mutations, efficient screening methods, and the continued advance of molecular genetic tools, such as the progress in sequencing and mapping of the zebrafish genome, the use of zebrafish (Danio rerio) as a biomedical model organism continues to expand. However, studies involving zebrafish husbandry and veterinary care struggle to keep pace with scientific progress. This article outlines some of the current, acceptable methods for providing anesthesia and euthanasia and provides some examples of how performance-based approaches can be used to advance the relatively limited number of anesthetic and euthanizing techniques available for zebrafish.

Phylostratigraphic Profiles in Zebrafish Uncover Chordate Origins of the Vertebrate Brain

PubMed Central

Šestak, Martin Sebastijan; Domazet-Lošo, Tomislav

2015-01-01

An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan–eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor. PMID:25415965

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish

NASA Astrophysics Data System (ADS)

Lee, Kerry J.; Browning, Lauren M.; Nallathamby, Prakash D.; Osgood, Christopher J.; Xu, Xiao-Hong Nancy

2013-11-01

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 +/- 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c << 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

Characterization and toxicology evaluation of chitosan nanoparticles on the embryonic development of zebrafish, Danio rerio.

PubMed

Wang, Yanbo; Zhou, Jinru; Liu, Lin; Huang, Changjiang; Zhou, Deqing; Fu, Linglin

2016-05-05

In the present study, chitosan nanoparticles were prepared, characterized and used to evaluate the embryonic toxicology on zebrafish (Danio rerio). The average particle size of chitosan nanoparticles was 84.86nm. The increased mortality and decreased hatching rate was found in the zebrafish embryo exposure to normal chitosan particles and chitosan nanoparticles with the increased addition concentration. At 120h post-fertilization (hpf), the rate of mortality were 25.0 and 44.4% in the groups treated with chitosan nanoparticles and normal chitosan particles at 250mg/L, respectively. At 72hpf, the hatching rate in the groups treated with normal chitosan particles were lower (P<0.01) at 300 and 400mg/L than those of the corresponding control groups, respectively. However, there were no significant differences between the groups treated with chitosan nanoparticles and the control groups across all the addition concentrations. More abundant typical malformation of embryos was observed in the groups treated with normal chitosan particles compared with those treated with chitosan nanoparticles. The LC50 (medium lethal concentration) of chitosan nanoparticles was 280mg/L at 96hpf and 270mg/L at 120hpf. As for normal chitosan particles, the LC50 was 257mg/L at both 96hpf and 120hpf. The TC50 (medium teratogenic concentration) of the zebrafish treated with chitosan nanoparticles and normal chitosan particles were 257mg/L and 137mg/L, respectively. It indicated that the chitosan nanoparticles were relatively more secure compared with normal chitosan particles. Copyright © 2016 Elsevier Ltd. All rights reserved.

Non-invasive electrocardiogram detection of in vivo zebrafish embryos using electric potential sensors

NASA Astrophysics Data System (ADS)

Rendon-Morales, E.; Prance, R. J.; Prance, H.; Aviles-Espinosa, R.

2015-11-01

In this letter, we report the continuous detection of the cardiac electrical activity in embryonic zebrafish using a non-invasive approach. We present a portable and cost-effective platform based on the electric potential sensing technology, to monitor in vivo electrocardiogram activity from the zebrafish heart. This proof of principle demonstration shows how electrocardiogram measurements from the embryonic zebrafish may become accessible by using electric field detection. We present preliminary results using the prototype, which enables the acquisition of electrophysiological signals from in vivo 3 and 5 days-post-fertilization zebrafish embryos. The recorded waveforms show electrocardiogram traces including detailed features such as QRS complex, P and T waves.

Kcnh1 Voltage-gated Potassium Channels Are Essential for Early Zebrafish Development*

PubMed Central

Stengel, Rayk; Rivera-Milla, Eric; Sahoo, Nirakar; Ebert, Christina; Bollig, Frank; Heinemann, Stefan H.; Schönherr, Roland; Englert, Christoph

2012-01-01

The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning. PMID:22927438

Embryonic fate map of first pharyngeal arch structures in the sox10: kaede zebrafish transgenic model.

PubMed

Dougherty, Max; Kamel, George; Shubinets, Valeriy; Hickey, Graham; Grimaldi, Michael; Liao, Eric C

2012-09-01

Cranial neural crest cells follow stereotypic patterns of migration to form craniofacial structures. The zebrafish is a powerful vertebrate genetic model where transgenics with reporter proteins under the transcriptional regulation of lineage-specific promoters can be generated. Numerous studies demonstrate that the zebrafish ethmoid plate is embryologically analogous to the mammalian palate. A fate map correlating embryonic cranial neural crest to defined jaw structures would provide a useful context for the morphogenetic analysis of craniofacial development. To that end, the sox10:kaede transgenic was generated, where sox10 provides lineage restriction to the neural crest. Specific regions of neural crest were labeled at the 10-somite stage by photoconversion of the kaede reporter protein. Lineage analysis was carried out during pharyngeal development in wild-type animals, after miR140 injection, and after estradiol treatment. At the 10-somite stage, cranial neural crest cells anterior of the eye contributed to the median ethmoid plate, whereas cells medial to the eye formed the lateral ethmoid plate and trabeculae and a posterior population formed the mandible. miR-140 overexpression and estradiol inhibition of Hedgehog signaling resulted in cleft development, with failed migration of the anterior cell population to form the median ethmoid plate. The sox10:kaede transgenic line provides a useful tool for neural crest lineage analysis. These studies illustrate the advantages of the zebrafish model for application in morphogenetic studies of vertebrate craniofacial development.

Expression and activity profiling of the steroidogenic enzymes of glucocorticoid biosynthesis and the fdx1 co-factors in zebrafish.

PubMed

Weger, M; Diotel, N; Weger, B D; Beil, T; Zaucker, A; Eachus, H L; Oakes, J A; do Rego, J L; Storbeck, K-H; Gut, P; Strähle, U; Rastegar, S; Müller, F; Krone, N

2018-04-01

The spatial and temporal expression of steroidogenic genes in zebrafish has not been fully characterised. Because zebrafish are increasingly employed in endocrine and stress research, a better characterisation of steroidogenic pathways is required to target specific steps in the biosynthetic pathways. In the present study, we have systematically defined the temporal and spatial expression of steroidogenic enzymes involved in glucocorticoid biosynthesis (cyp21a2, cyp11c1, cyp11a1, cyp11a2, cyp17a1, cyp17a2, hsd3b1, hsd3b2), as well as the mitochondrial electron-providing ferredoxin co-factors (fdx1, fdx1b), during zebrafish development. Our studies showed an early expression of all these genes during embryogenesis. In larvae, expression of cyp11a2, cyp11c1, cyp17a2, cyp21a2, hsd3b1 and fdx1b can be detected in the interrenal gland, which is the zebrafish counterpart of the mammalian adrenal gland, whereas the fdx1 transcript is mainly found in the digestive system. Gene expression studies using quantitative reverse transcriptase-PCR and whole-mount in situ hybridisation in the adult zebrafish brain revealed a wide expression of these genes throughout the encephalon, including neurogenic regions. Using ultra-high-performance liquid chromatography tandem mass spectrometry, we were able to demonstrate the presence of the glucocorticoid cortisol in the adult zebrafish brain. Moreover, we demonstrate de novo biosynthesis of cortisol and the neurosteroid tetrahydrodeoxycorticosterone in the adult zebrafish brain from radiolabelled pregnenolone. Taken together, the present study comprises a comprehensive characterisation of the steroidogenic genes and the fdx co-factors facilitating glucocorticoid biosynthesis in zebrafish. Furthermore, we provide additional evidence of de novo neurosteroid biosynthesising in the brain of adult zebrafish facilitated by enzymes involved in glucocorticoid biosynthesis. Our study provides a valuable source for establishing the zebrafish as a

Neurogenesis in the embryonic and adult brain: same regulators, different roles

PubMed Central

Urbán, Noelia; Guillemot, François

2014-01-01

Neurogenesis persists in adult mammals in specific brain areas, known as neurogenic niches. Adult neurogenesis is highly dynamic and is modulated by multiple physiological stimuli and pathological states. There is a strong interest in understanding how this process is regulated, particularly since active neuronal production has been demonstrated in both the hippocampus and the subventricular zone (SVZ) of adult humans. The molecular mechanisms that control neurogenesis have been extensively studied during embryonic development. Therefore, we have a broad knowledge of the intrinsic factors and extracellular signaling pathways driving proliferation and differentiation of embryonic neural precursors. Many of these factors also play important roles during adult neurogenesis, but essential differences exist in the biological responses of neural precursors in the embryonic and adult contexts. Because adult neural stem cells (NSCs) are normally found in a quiescent state, regulatory pathways can affect adult neurogenesis in ways that have no clear counterpart during embryogenesis. BMP signaling, for instance, regulates NSC behavior both during embryonic and adult neurogenesis. However, this pathway maintains stem cell proliferation in the embryo, while it promotes quiescence to prevent stem cell exhaustion in the adult brain. In this review, we will compare and contrast the functions of transcription factors (TFs) and other regulatory molecules in the embryonic brain and in adult neurogenic regions of the adult brain in the mouse, with a special focus on the hippocampal niche and on the regulation of the balance between quiescence and activation of adult NSCs in this region. PMID:25505873

Amigo adhesion protein regulates development of neural circuits in zebrafish brain.

PubMed

Zhao, Xiang; Kuja-Panula, Juha; Sundvik, Maria; Chen, Yu-Chia; Aho, Vilma; Peltola, Marjaana A; Porkka-Heiskanen, Tarja; Panula, Pertti; Rauvala, Heikki

2014-07-18

The Amigo protein family consists of three transmembrane proteins characterized by six leucine-rich repeat domains and one immunoglobulin-like domain in their extracellular moieties. Previous in vitro studies have suggested a role as homophilic adhesion molecules in brain neurons, but the in vivo functions remain unknown. Here we have cloned all three zebrafish amigos and show that amigo1 is the predominant family member expressed during nervous system development in zebrafish. Knockdown of amigo1 expression using morpholino oligonucleotides impairs the formation of fasciculated tracts in early fiber scaffolds of brain. A similar defect in fiber tract development is caused by mRNA-mediated expression of the Amigo1 ectodomain that inhibits adhesion mediated by the full-length protein. Analysis of differentiated neural circuits reveals defects in the catecholaminergic system. At the behavioral level, the disturbed formation of neural circuitry is reflected in enhanced locomotor activity and in the inability of the larvae to perform normal escape responses. We suggest that Amigo1 is essential for the development of neural circuits of zebrafish, where its mechanism involves homophilic interactions within the developing fiber tracts and regulation of the Kv2.1 potassium channel to form functional neural circuitry that controls locomotion. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

Behavioural responses to novelty or to a predator stimulus are not altered in adult zebrafish by early embryonic alcohol exposure

PubMed Central

Seguin, Diane; Shams, Soaleha; Gerlai, Robert

2016-01-01

Background Fetal Alcohol Spectrum Disorders (FASD) may vary in symptoms and severity. In the milder and more prevalent forms of the disease, behavioural abnormalities may include impaired social behaviour, e.g. difficulty interpreting social cues. FASD patients remain often undiagnosed due to lack of biomarkers, and treatment is unavailable because the mechanisms of the disease are not yet understood. Animal models have been proposed to facilitate addressing these problems. More recently, short exposure of the zebrafish embryo to low concentrations of alcohol was shown to lead to significant and lasting impairment of behaviour in response to social stimuli. The impairment may be the result of abnormal social behaviour or altered fear/anxiety. The goal of the current study was to investigate the latter. Methods Here, we employed the alcohol exposure regimen used previously (exposure of 24th hour post-fertilization embryos to 0.00, 0.25, 0.50, 0.75 or 1.00 vol/vol % alcohol for 2 hours), allowed the fish to reach adulthood, and measured the behavioural responses of these adults to a novel tank (anxiety related behaviours) as well as to an animated image of a sympatric predator of zebrafish (fear related behaviours). Results We found behavioural responses of embryonic alcohol exposed adult fish to remain statistically indistinguishable from those of controls, suggesting unaltered anxiety and fear in the embryonic alcohol treated fish. Conclusions Given that motor and perceptual function was previously shown to be also unaltered in the adults after embryonic alcohol exposure, our current results suggest that the impaired response of these fish to social stimuli may be the result of abnormal social behaviour. PMID:27790739

Myomaker mediates fusion of fast myocytes in zebrafish embryos

DOE Office of Scientific and Technical Information (OSTI.GOV)

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 weremore » 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.« less

Phylostratigraphic profiles in zebrafish uncover chordate origins of the vertebrate brain.

PubMed

Šestak, Martin Sebastijan; Domazet-Lošo, Tomislav

2015-02-01

An elaborated tripartite brain is considered one of the important innovations of vertebrates. Other extant chordate groups have a more basic brain organization. For instance, cephalochordates possess a relatively simple brain possibly homologous to the vertebrate forebrain and hindbrain, whereas tunicates display the tripartite organization, but without the specialized brain centers. The difference in anatomical complexity is even more pronounced if one compares chordates with other deuterostomes that have only a diffuse nerve net or alternatively a rather simple central nervous system. To gain a new perspective on the evolutionary roots of the complex vertebrate brain, we made here a phylostratigraphic analysis of gene expression patterns in the developing zebrafish (Danio rerio). The recovered adaptive landscape revealed three important periods in the evolutionary history of the zebrafish brain. The oldest period corresponds to preadaptive events in the first metazoans and the emergence of the nervous system at the metazoan-eumetazoan transition. The origin of chordates marks the next phase, where we found the overall strongest adaptive imprint in almost all analyzed brain regions. This finding supports the idea that the vertebrate brain evolved independently of the brains within the protostome lineage. Finally, at the origin of vertebrates we detected a pronounced signal coming from the dorsal telencephalon, in agreement with classical theories that consider this part of the cerebrum a genuine vertebrate innovation. Taken together, these results reveal a stepwise adaptive history of the vertebrate brain where most of its extant organization was already present in the chordate ancestor. © The Author 2014. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

High resolution fate map of the zebrafish diencephalon.

PubMed

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

2009-07-01

The diencephalon acts as an interactive site between the sensory, central, and endocrine systems and is one of the most elaborate structures in the vertebrate brain. To better understand the embryonic development and morphogenesis of the diencephalon, we developed an improved photoactivation (uncaging)-based lineage tracing strategy. To determine the exact position of a given diencephalic progenitor domain, we used a transgenic line driving green fluorescent protein (GFP) in cells expressing the proneural protein, Neurogenin1 (Neurog1), which was used as a visible neural plate landmark. This approach facilitated precise labeling of defined groups of cells in the prospective diencephalon of the zebrafish neural plate. In this manner, we labeled multiple overlapping areas of the diencephalon, thereby ensuring both accuracy and reproducibility of our lineage tracing regardless of the dynamic changes of the developing neural plate. We present a fate map of the zebrafish diencephalon at a higher spatial resolution than previously described. (c) 2009 Wiley-Liss, Inc.

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

PubMed Central

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

Identification and Characterization of Long Non-Coding RNAs Related to Mouse Embryonic Brain Development from Available Transcriptomic Data

PubMed Central

He, Hongjuan; Xiu, Youcheng; Guo, Jing; Liu, Hui; Liu, Qi; Zeng, Tiebo; Chen, Yan; Zhang, Yan; Wu, Qiong

2013-01-01

Long non-coding RNAs (lncRNAs) as a key group of non-coding RNAs have gained widely attention. Though lncRNAs have been functionally annotated and systematic explored in higher mammals, few are under systematical identification and annotation. Owing to the expression specificity, known lncRNAs expressed in embryonic brain tissues remain still limited. Considering a large number of lncRNAs are only transcribed in brain tissues, studies of lncRNAs in developmental brain are therefore of special interest. Here, publicly available RNA-sequencing (RNA-seq) data in embryonic brain are integrated to identify thousands of embryonic brain lncRNAs by a customized pipeline. A significant proportion of novel transcripts have not been annotated by available genomic resources. The putative embryonic brain lncRNAs are shorter in length, less spliced and show less conservation than known genes. The expression of putative lncRNAs is in one tenth on average of known coding genes, while comparable with known lncRNAs. From chromatin data, putative embryonic brain lncRNAs are associated with active chromatin marks, comparable with known lncRNAs. Embryonic brain expressed lncRNAs are also indicated to have expression though not evident in adult brain. Gene Ontology analysis of putative embryonic brain lncRNAs suggests that they are associated with brain development. The putative lncRNAs are shown to be related to possible cis-regulatory roles in imprinting even themselves are deemed to be imprinted lncRNAs. Re-analysis of one knockdown data suggests that four regulators are associated with lncRNAs. Taken together, the identification and systematic analysis of putative lncRNAs would provide novel insights into uncharacterized mouse non-coding regions and the relationships with mammalian embryonic brain development. PMID:23967161

Morphometric analysis and neuroanatomical mapping of the zebrafish brain.

PubMed

Gupta, Tripti; Marquart, Gregory D; Horstick, Eric J; Tabor, Kathryn M; Pajevic, Sinisa; Burgess, Harold A

2018-06-21

Large-scale genomic studies have recently identified genetic variants causative for major neurodevelopmental disorders, such as intellectual disability and autism. However, determining how underlying developmental processes are affected by these mutations remains a significant challenge in the field. Zebrafish is an established model system in developmental neurogenetics that may be useful in uncovering the mechanisms of these mutations. Here we describe the use of voxel-intensity, deformation field, and volume-based morphometric techniques for the systematic and unbiased analysis of gene knock-down and environmental exposure-induced phenotypes in zebrafish. We first present a computational method for brain segmentation based on transgene expression patterns to create a comprehensive neuroanatomical map. This map allowed us to disclose statistically significant changes in brain microstructure and composition in neurodevelopmental models. We demonstrate the effectiveness of morphometric techniques in measuring changes in the relative size of neuroanatomical subdivisions in atoh7 morphant larvae and in identifying phenotypes in larvae treated with valproic acid, a chemical demonstrated to increase the risk of autism in humans. These tools enable rigorous evaluation of the effects of gene mutations and environmental exposures on neural development, providing an entry point for cellular and molecular analysis of basic developmental processes as well as neurodevelopmental and neurodegenerative disorders. Published by Elsevier Inc.

Novel Insights into the Genetic Controls of Primitive and Definitive Hematopoiesis from Zebrafish Models

PubMed Central

Sood, Raman; Liu, Paul

2012-01-01

Hematopoiesis is a dynamic process where initiation and maintenance of hematopoietic stem cells, as well as their differentiation into erythroid, myeloid and lymphoid lineages, are tightly regulated by a network of transcription factors. Understanding the genetic controls of hematopoiesis is crucial as perturbations in hematopoiesis lead to diseases such as anemia, thrombocytopenia, or cancers, including leukemias and lymphomas. Animal models, particularly conventional and conditional knockout mice, have played major roles in our understanding of the genetic controls of hematopoiesis. However, knockout mice for most of the hematopoietic transcription factors are embryonic lethal, thus precluding the analysis of their roles during the transition from embryonic to adult hematopoiesis. Zebrafish are an ideal model organism to determine the function of a gene during embryonic-to-adult transition of hematopoiesis since bloodless zebrafish embryos can develop normally into early larval stage by obtaining oxygen through diffusion. In this review, we discuss the current status of the ontogeny and regulation of hematopoiesis in zebrafish. By providing specific examples of zebrafish morphants and mutants, we have highlighted the contributions of the zebrafish model to our overall understanding of the roles of transcription factors in regulation of primitive and definitive hematopoiesis. PMID:22888355

Alterations to Juvenile Zebrafish (Danio rerio) Swim Performance after Acute Embryonic Exposure to Sub-lethal Exposures of Hydraulic Fracturing Flowback and Produced Water.

PubMed

Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

2017-12-01

Hydraulic fracturing flowback and produced water (FPW) is a wastewater produced during fracturing activities in an operating well which is hyper saline and chemically heterogeneous in nature, containing both anthropogenic and petrogenic chemicals. Determination of FPW associated toxicity to embryonic fish is limited, while investigation into how embryonic exposures may affect later life stages is not yet studied. Zebrafish embryos (24hrs post fertilization) were acutely exposed to 2.5% and 5% FPW fractions for either 24 or 48hrs and returned to freshwater. After either 24 or 48h exposures, embryos were examined for expression of 3 hypoxia related genes. Erythropoietin (epoa) but not hypoxia inducible factor (hif1aa) nor hemoglobin -ß chain (hbbe1.1) was up-regulated after either 24 or 48h FPW exposure. Surviving embryos were placed in freshwater and grown to a juvenile stage (60days post fertilization). Previously exposed zebrafish were analyzed for both swim performance (U crit and U max ) and aerobic capacity. Fish exposed to both sediment containing (FPW-S) or sediment free (FPW-SF) FPW displayed significantly reduced aerobic scope and U crit /U max values compared to control conditions. Our results collectively suggest that organics present in our FPW sample may be responsible for sub-lethal fitness and metabolic responses. We provide evidence supporting the theory that the cardio-respiratory system is impacted by FPW exposure. This is the first known research associating embryonic FPW exposures to sub-lethal performance related responses in later life fish stages. Copyright © 2017 Elsevier B.V. All rights reserved.

Coordinating cell and tissue behavior during zebrafish neural tube morphogenesis.

PubMed

Araya, Claudio; Ward, Laura C; Girdler, Gemma C; Miranda, Miguel

2016-03-01

The development of a vertebrate neural epithelium with well-organized apico-basal polarity and a central lumen is essential for its proper function. However, how this polarity is established during embryonic development and the potential influence of surrounding signals and tissues on such organization has remained less understood. In recent years the combined superior transparency and genetics of the zebrafish embryo has allowed for in vivo visualization and quantification of the cellular and molecular dynamics that govern neural tube structure. Here, we discuss recent studies revealing how co-ordinated cell-cell interactions coupled with adjacent tissue dynamics are critical to regulate final neural tissue architecture. Furthermore, new findings show how the spatial regulation and timing of orientated cell division is key in defining precise lumen formation at the tissue midline. In addition, we compare zebrafish neurulation with that of amniotes and amphibians in an attempt to understand the conserved cellular mechanisms driving neurulation and resolve the apparent differences among animals. Zebrafish neurulation not only offers fundamental insights into early vertebrate brain development but also the opportunity to explore in vivo cell and tissue dynamics during complex three-dimensional animal morphogenesis. © 2015 Wiley Periodicals, Inc.

Silver nanoparticles induce developmental stage-specific embryonic phenotypes in zebrafish.

PubMed

Lee, Kerry J; Browning, Lauren M; Nallathamby, Prakash D; Osgood, Christopher J; Xu, Xiao-Hong Nancy

2013-12-07

Much is anticipated from the development and deployment of nanomaterials in biological organisms, but concerns remain regarding their biocompatibility and target specificity. Here we report our study of the transport, biocompatibility and toxicity of purified and stable silver nanoparticles (Ag NPs, 13.1 ± 2.5 nm in diameter) upon the specific developmental stages of zebrafish embryos using single NP plasmonic spectroscopy. We find that single Ag NPs passively diffuse into five different developmental stages of embryos (cleavage, early-gastrula, early-segmentation, late-segmentation, and hatching stages), showing stage-independent diffusion modes and diffusion coefficients. Notably, the Ag NPs induce distinctive stage and dose-dependent phenotypes and nanotoxicity, upon their acute exposure to the Ag NPs (0-0.7 nM) for only 2 h. The late-segmentation embryos are most sensitive to the NPs with the lowest critical concentration (CNP,c < 0.02 nM) and highest percentages of cardiac abnormalities, followed by early-segmentation embryos (CNP,c < 0.02 nM), suggesting that disruption of cell differentiation by the NPs causes the most toxic effects on embryonic development. The cleavage-stage embryos treated with the NPs develop into a wide variety of phenotypes (abnormal finfold, tail/spinal cord flexure, cardiac malformation/edema, yolk sac edema, and acephaly). These organ structures are not yet developed in cleavage-stage embryos, suggesting that the earliest determinative events to create these structures are ongoing, and disrupted by NPs, which leads to the downstream effects. In contrast, the hatching embryos are most resistant to the Ag NPs, and majority of embryos (94%) develop normally, and none of them develop abnormally. Interestingly, early-gastrula embryos are less sensitive to the NPs than cleavage and segmentation stage embryos, and do not develop abnormally. These important findings suggest that the Ag NPs are not simple poisons, and they can target

Stab wound injury of the zebrafish adult telencephalon: a method to investigate vertebrate brain neurogenesis and regeneration.

PubMed

Schmidt, Rebecca; Beil, Tanja; Strähle, Uwe; Rastegar, Sepand

2014-08-04

Adult zebrafish have an amazing capacity to regenerate their central nervous system after injury. To investigate the cellular response and the molecular mechanisms involved in zebrafish adult central nervous system (CNS) regeneration and repair, we developed a zebrafish model of adult telencephalic injury. In this approach, we manually generate an injury by pushing an insulin syringe needle into the zebrafish adult telencephalon. At different post injury days, fish are sacrificed, their brains are dissected out and stained by immunohistochemistry and/or in situ hybridization (ISH) with appropriate markers to observe cell proliferation, gliogenesis, and neurogenesis. The contralateral unlesioned hemisphere serves as an internal control. This method combined for example with RNA deep sequencing can help to screen for new genes with a role in zebrafish adult telencephalon neurogenesis, regeneration, and repair.

Characterization of multiciliated ependymal cells that emerge in the neurogenic niche of the aged zebrafish brain.

PubMed

Ogino, Takashi; Sawada, Masato; Takase, Hiroshi; Nakai, Chiemi; Herranz-Pérez, Vicente; Cebrián-Silla, Arantxa; Kaneko, Naoko; García-Verdugo, José Manuel; Sawamoto, Kazunobu

2016-10-15

In mammals, ventricular walls of the developing brain maintain a neurogenic niche, in which radial glial cells act as neural stem cells (NSCs) and generate new neurons in the embryo. In the adult brain, the neurogenic niche is maintained in the ventricular-subventricular zone (V-SVZ) of the lateral wall of lateral ventricles and the hippocampal dentate gyrus. In the neonatal V-SVZ, radial glial cells transform into astrocytic postnatal NSCs and multiciliated ependymal cells. On the other hand, in zebrafish, radial glial cells continue to cover the surface of the adult telencephalic ventricle and maintain a higher neurogenic potential in the adult brain. However, the cell composition of the neurogenic niche of the aged zebrafish brain has not been investigated. Here we show that multiciliated ependymal cells emerge in the neurogenic niche of the aged zebrafish telencephalon. These multiciliated cells appear predominantly in the dorsal part of the ventral telencephalic ventricular zone, which also contains clusters of migrating new neurons. Scanning electron microscopy and live imaging analyses indicated that these multiple cilia beat coordinately and generate constant fluid flow within the ventral telencephalic ventricle. Analysis of the cell composition by transmission electron microscopy revealed that the neurogenic niche in the aged zebrafish contains different types of cells, with ultrastructures similar to those of ependymal cells, transit-amplifying cells, and migrating new neurons in postnatal mice. These data suggest that the transformation capacity of radial glial cells is conserved but that its timing is different between fish and mice. J. Comp. Neurol. 524:2982-2992, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Utilization of ketone bodies by chick brain and spinal cord during embryonic and postnatal development.

PubMed

Linares, A; Caamaño, G J; Diaz, R; Gonzalez, F J; Garcia-Peregrin, E

1993-10-01

Lipid synthesis from acetoacetate and 3-hydroxybutyrate was studied in chick embryo from 15 to 21 days and in chick neonate from 1 to 21 days. Embryonic spinal cord showed higher ability than brain to incorporate acetoacetate into total lipids, although a sharp decrease was found at hatching. 3-Hydroxybutyrate incorporation into total lipids was also higher in spinal cord than in brain, especially during the embryonic period. Phospholipids were the main lipids formed in both tissues from both precursors. An appreciable percentage of radioactivity was also recovered as free cholesterol, especially during the embryonic phase. The developmental patterns of amino acid synthesis from acetoacetate and 3-hydroxybutyrate were similar in both tissues: a clear increase after hatching was followed by a decrease at day 4 of neonatal life. Acetoacetate was a better substrate for amino acid synthesis than 3-hydroxybutyrate during the embryonic development in both tissues. Oxidation of both precursors to CO2 strongly decreased between 15 and 21 days of embryonic development both in brain and spinal cord.

Zebrafish: a model animal for analyzing the impact of environmental pollutants on muscle and brain mitochondrial bioenergetics.

PubMed

Bourdineaud, Jean-Paul; Rossignol, R; Brèthes, D

2013-01-01

Mercury, anthropogenic release of uranium (U), and nanoparticles constitute hazardous environmental pollutants able to accumulate along the aquatic food chain with severe risk for animal and human health. The impact of such pollutants on living organisms has been up to now approached by classical toxicology in which huge doses of toxic compounds, environmentally irrelevant, are displayed through routes that never occur in the lifespan of organisms (for instance injecting a bolus of mercury to an animal although the main route is through prey and fish eating). We wanted to address the effect of such pollutants on the muscle and brain mitochondrial bioenergetics under realistic conditions, at unprecedented low doses, using an aquatic model animal, the zebrafish Danio rerio. We developed an original method to measure brain mitochondrial respiration: a single brain was put in 1.5 mL conical tube containing a respiratory buffer. Brains were gently homogenized by 13 strokes with a conical plastic pestle, and the homogenates were immediately used for respiration measurements. Skinned muscle fibers were prepared by saponin permeabilization. Zebrafish were contaminated with food containing 13 μg of methylmercury (MeHg)/g, an environmentally relevant dose. In permeabilized muscle fibers, we observed a strong inhibition of both state 3 mitochondrial respiration and cytochrome c oxidase activity after 49 days of MeHg exposure. We measured a dramatic decrease in the rate of ATP release by skinned muscle fibers. Contrarily to muscles, brain mitochondrial respiration was not modified by MeHg exposure although brain accumulated twice as much MeHg than muscles. When zebrafish were exposed to 30 μg/L of waterborne U, the basal mitochondrial respiratory control ratio was decreased in muscles after 28 days of exposure. This was due to an increase of the inner mitochondrial membrane permeability. The impact of a daily ration of food containing gold nanoparticles of two sizes (12 and

Simulated-microgravity induced G2/M arrest in zebrafish embryonic cell is regulated by dre-miR-22a and its target cep135

NASA Astrophysics Data System (ADS)

Hang, Xiaoming; Sun, Yeqing; Wu, Di; Li, Yixiao; Wang, Ruonan

2016-07-01

Microgravity has been recognized as a major environmental factor that can induce a number of adverse effects such as bone loss, skeletal muscle atrophy, cardiovascular problems and immune system dysregulation, etc. The underlying mechanisms are not absolutely identified yet. Our previous study demonstrated centrosomal protein of 135 kDa (CEP135) might be a microgravity sensitive molecule. In this study, the expression and regulation of CEP135 and its possible roles in cell cycle regulation under simulated microgravity (SMG) condition were investigated. SMG can induce significant increasing of cep135 in zebrafish embryos, detected by both in situ hybridization and RT-qPCR, while CEP135 protein level was significantly decreased, tested by western blot. The similar results were also obtained in zebrafish embryonic cells (ZF4) exposed to SMG. Accordingly, the expression level of dre-miR-22a, which might be the potential miRNA for targeting cep135, was significantly increased in SMG exposed ZF4 cells. By combining the results obtained from transfection and dual luciferase reporter assay, we firstly confirmed that dre-miR-22a regulated the expression of cep135 in ZF4 cells. Further investigation on cell cycle demonstrated SMG induced a significant arrest in G2/M phase. Transfection of dre-miR-22a also induced G2/M arrest in ZF4 cells. These results suggest that SMG induced G2/M arrest in ZF4 cells is via cep135, while dre-miR-22a plays a key role in modulating this effect. Key Words: Simulated-microgravity; cep135; dre-miR-22a; G2/M arrest; zebrafish embryonic cell

An approach to clarify the effect mechanism of glyphosate on body malformations during embryonic development of zebrafish (Daino rerio).

PubMed

Sulukan, Ekrem; Köktürk, Mine; Ceylan, Hamid; Beydemir, Şükrü; Işik, Mesut; Atamanalp, Muhammed; Ceyhun, Saltuk Buğrahan

2017-08-01

In this study, it has been investigated that the effects of glyphosate, which is a herbicide within organophosphate and unselective widely used in agriculture on enzyme activity of carbonic anhydrase, production of reactive oxygen species, cell apoptosis and body morphology during the embryonic development of zebrafish. To this end, it has been treated embryo with 1, 5, 10 and 100 mg/L gyphosate at 96 h. The embryos treated with glyphosate from 4 hpf were evaluated by considering the survival rates, hatching rates, body malformations under the stereo microscope in 24, 48, 72 and 96th hours. In order to clarify the mechanism of the abnormalities ROS, enzyme activity of carbonic anhydrase and cellular death were detected end of the 96th hour. The data obtained in the present study have shown that glyphosate treatment inhibited CA activity, caused production of ROS especially branchial regions, triggered cellular apoptosis and caused several types of malformations including pericardial edema, yolk sac edema, spinal curvature and body malformation in a dose-dependent manner. As a conclusion, in light of present and previous studies, we can deduce that (1) the probable reason of ROS production was CA inhibition via decreasing of CO 2 extraction and developing respiratory acidosis (however, one needs to clarify), (2) abundance of ROS triggered cellular apoptosis and (3) as a result of cellular apoptosis malformations increased. These data will enable us to further understand potential toxic mechanism of glyphosate on embryonic development stage of zebrafish and may be useful for assessment in the toxicology studies. Copyright © 2017 Elsevier Ltd. All rights reserved.

Identification of a brain center whose activity discriminates a choice behavior in zebrafish

PubMed Central

Lau, Billy Y. B.; Mathur, Priya; Gould, Georgianna G.; Guo, Su

2011-01-01

The ability to make choices and carry out appropriate actions is critical for individual survival and well-being. Choice behaviors, from hard-wired to experience-dependent, have been observed across the animal kingdom. Although differential engagement of sensory neuronal pathways is a known mechanism, neurobiological substrates in the brain that underlie choice making downstream of sensory perception are not well understood. Here, we report a behavioral paradigm in zebrafish in which a half-light/half-dark visual image evokes an innate choice behavior, light avoidance. Neuronal activity mapping using the immediate early gene c-fos reveals the engagement of distinct brain regions, including the medial zone of the dorsal telencephalic region (Dm) and the dorsal nucleus of the ventral telencephalic area (Vd), the teleost anatomical homologs of the mammalian amygdala and striatum, respectively. In animals that were subjected to the identical sensory stimulus but displayed little or no avoidance, strikingly, the Dm and Vd were not engaged, despite similar levels of activation in the brain nuclei involved in visual processing. Based on these findings and previous connectivity data, we propose a neural circuitry model in which the Dm serves as a brain center, the activity of which predicates this choice behavior in zebrafish. PMID:21262817

Early zebrafish development: It’s in the maternal genes

PubMed Central

Abrams, Elliott W.; Mullins, Mary C.

2009-01-01

Summary The earliest stages of embryonic development in all animals examined rely on maternal gene products that are generated during oogenesis and supplied to the egg. The period of maternal control of embryonic development varies among animals according to the onset of zygotic transcription and the persistence of maternal gene products. This maternal regulation has been little studied in vertebrates, due to the difficulty in manipulating maternal gene function and lack of basic molecular information. However, recent maternal-effect screens in the zebrafish have generated more than 40 unique mutants that are providing new molecular entry points to the maternal control of early vertebrate development. Here we discuss recent studies of 12 zebrafish mutant genes that illuminate the maternal molecular controls on embryonic development, including advances in the regulation of animal-vegetal polarity, egg activation, cleavage development, body plan formation, tissue morphogenesis, microRNA function and germ cell development. PMID:19608405

Relationships among msx gene structure and function in zebrafish and other vertebrates.

PubMed

Ekker, M; Akimenko, M A; Allende, M L; Smith, R; Drouin, G; Langille, R M; Weinberg, E S; Westerfield, M

1997-10-01

The zebrafish genome contains at least five msx homeobox genes, msxA, msxB, msxC, msxD, and the newly isolated msxE. Although these genes share structural features common to all Msx genes, phylogenetic analyses of protein sequences indicate that the msx genes from zebrafish are not orthologous to the Msx1 and Msx2 genes of mammals, birds, and amphibians. The zebrafish msxB and msxC are more closely related to each other and to the mouse Msx3. Similarly, although the combinatorial expression of the zebrafish msx genes in the embryonic dorsal neuroectoderm, visceral arches, fins, and sensory organs suggests functional similarities with the Msx genes of other vertebrates, differences in the expression patterns preclude precise assignment of orthological relationships. Distinct duplication events may have given rise to the msx genes of modern fish and other vertebrate lineages whereas many aspects of msx gene functions during embryonic development have been preserved.

Osteoblast Production by Reserved Progenitor Cells in Zebrafish Bone Regeneration and Maintenance.

PubMed

Ando, Kazunori; Shibata, Eri; Hans, Stefan; Brand, Michael; Kawakami, Atsushi

2017-12-04

Mammals cannot re-form heavily damaged bones as in large fracture gaps, whereas zebrafish efficiently regenerate bones even after amputation of appendages. However, the source of osteoblasts that mediate appendage regeneration is controversial. Several studies in zebrafish have shown that osteoblasts are generated by dedifferentiation of existing osteoblasts at injured sites, but other observations suggest that de novo production of osteoblasts also occurs. In this study, we found from cell-lineage tracing and ablation experiments that a group of cells reserved in niches serves as osteoblast progenitor cells (OPCs) and has a significant role in fin ray regeneration. Besides regeneration, OPCs also supply osteoblasts for normal bone maintenance. We further showed that OPCs are derived from embryonic somites, as is the case with embryonic osteoblasts, and are replenished from mesenchymal precursors in adult zebrafish. Our findings reveal that reserved progenitors are a significant and complementary source of osteoblasts for zebrafish bone regeneration. Copyright © 2017 Elsevier Inc. All rights reserved.

Delivery of Small Interfering RNA to Inhibit Vascular Endothelial Growth Factor in Zebrafish Using Natural Brain Endothelia Cell-Secreted Exosome Nanovesicles for the Treatment of Brain Cancer.

PubMed

Yang, Tianzhi; Fogarty, Brittany; LaForge, Bret; Aziz, Salma; Pham, Thuy; Lai, Leanne; Bai, Shuhua

2017-03-01

Although small interfering RNA (siRNA) holds great therapeutic promise, its delivery to the disease site remains a paramount obstacle. In this study, we tested whether brain endothelial cell-derived exosomes could deliver siRNA across the blood-brain barrier (BBB) in zebrafish. Natural exosomes were isolated from brain endothelial bEND.3 cell culture media and vascular endothelial growth factor (VEGF) siRNA was loaded in exosomes with the assistance of a transfection reagent. While fluorescence-activated cell flow cytometry and immunocytochemistry staining studies indicated that wild-type exosomes significantly increased the uptake of fluorescence-labeled siRNA in the autologous brain endothelial cells, decreased fluorescence intensity was observed in the cells treated with the tetraspanin CD63 antibody-blocked exosome-delivered formulation (p < 0.05). In the transport study, exosomes also enhanced the permeability of rhodamine 123 in a co-cultured monolayer of brain endothelial bEND.3 cell and astrocyte. Inhibition at the expression of VEGF RNA and protein levels was observed in glioblastoma-astrocytoma U-87 MG cells treated with exosome-delivered siRNAs. Imaging results showed that exosome delivered more siRNAs across the BBB in Tg(fli1:GFP) zebrafish. In a xenotransplanted brain tumor model, exosome-delivered VEGF siRNAs decreased the fluorescence intensity of labeled cancer cells in the brain of zebrafish. Brain endothelial cell-derived exosomes could be potentially used as a natural carrier for the brain delivery of exogenous siRNA.

3D MALDI Mass Spectrometry Imaging of a Single Cell: Spatial Mapping of Lipids in the Embryonic Development of Zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Dueñas, Maria Emilia; Essner, Jeffrey J.; Lee, Young Jin

The zebrafish ( Danio rerio) has been widely used as a model vertebrate system to study lipid metabolism, the roles of lipids in diseases, and lipid dynamics in embryonic development. Here, we applied high-spatial resolution matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) to map and visualize the three-dimensional spatial distribution of phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamines (PE), and phosphatidylinositol (PI), in newly fertilized individual zebrafish embryos. This is the first time MALDI-MSI has been applied for three dimensional chemical imaging of a single cell. PC molecular species are present inside the yolk in addition to the blastodisc, while PE andmore » PI species are mostly absent in the yolk. Two-dimensional MSI was also studied for embryos at different cell stages (1-, 2-, 4-, 8-, and 16-cell stage) to investigate the localization changes of some lipids at various cell developmental stages. Lastly, four different normalization approaches were compared to find reliable relative quantification in 2D- and 3D- MALDI MSI data sets.« less

3D MALDI Mass Spectrometry Imaging of a Single Cell: Spatial Mapping of Lipids in the Embryonic Development of Zebrafish

DOE PAGES

Dueñas, Maria Emilia; Essner, Jeffrey J.; Lee, Young Jin

2017-11-02

The zebrafish ( Danio rerio) has been widely used as a model vertebrate system to study lipid metabolism, the roles of lipids in diseases, and lipid dynamics in embryonic development. Here, we applied high-spatial resolution matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry imaging (MSI) to map and visualize the three-dimensional spatial distribution of phospholipid classes, phosphatidylcholine (PC), phosphatidylethanolamines (PE), and phosphatidylinositol (PI), in newly fertilized individual zebrafish embryos. This is the first time MALDI-MSI has been applied for three dimensional chemical imaging of a single cell. PC molecular species are present inside the yolk in addition to the blastodisc, while PE andmore » PI species are mostly absent in the yolk. Two-dimensional MSI was also studied for embryos at different cell stages (1-, 2-, 4-, 8-, and 16-cell stage) to investigate the localization changes of some lipids at various cell developmental stages. Lastly, four different normalization approaches were compared to find reliable relative quantification in 2D- and 3D- MALDI MSI data sets.« less

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function.

PubMed

Sarmah, Swapnalee; Marrs, James A

2016-12-16

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed.

Zebrafish as a Vertebrate Model System to Evaluate Effects of Environmental Toxicants on Cardiac Development and Function

PubMed Central

Sarmah, Swapnalee; Marrs, James A.

2016-01-01

Environmental pollution is a serious problem of the modern world that possesses a major threat to public health. Exposure to environmental pollutants during embryonic development is particularly risky. Although many pollutants have been verified as potential toxicants, there are new chemicals in the environment that need assessment. Heart development is an extremely sensitive process, which can be affected by environmentally toxic molecule exposure during embryonic development. Congenital heart defects are the most common life-threatening global health problems, and the etiology is mostly unknown. The zebrafish has emerged as an invaluable model to examine substance toxicity on vertebrate development, particularly on cardiac development. The zebrafish offers numerous advantages for toxicology research not found in other model systems. Many laboratories have used the zebrafish to study the effects of widespread chemicals in the environment on heart development, including pesticides, nanoparticles, and various organic pollutants. Here, we review the uses of the zebrafish in examining effects of exposure to external molecules during embryonic development in causing cardiac defects, including chemicals ubiquitous in the environment and illicit drugs. Known or potential mechanisms of toxicity and how zebrafish research can be used to provide mechanistic understanding of cardiac defects are discussed. PMID:27999267

Fatty acids of glycerophosphatides in developing chick embryonic brain and liver.

PubMed

Miyamoto, K; Stephanides, L M; Bernsohn, J

1966-09-01

Fatty acid compositions of glycerophosphatides of developing chick embryonic brain and liver were compared. In brain, ethanolamine and serine glycerophosphatides contained 30-40% polyunsaturated fatty acids, lecithin almost none (except for arachidonic). In the liver, these acids were equally distributed in the phospholipid fractions. The principal polyunsaturated fatty acids of the ethanolamine and serine glycerophosphatides in brain, liver, and yolk were 22:6, 20:4, and 18:2, respectively. During embryonic development of brain from the 8th day of incubation to hatching, the fatty acid composition of individual glycerophosphatide fractions remained constant. Because of the relative increase of ethanolamine glycerophosphatides and decrease of lecithin, total glycerophosphatides showed a decrease in 16:0 and an increase in 18:0. Substantial amounts of palmitaldehyde and stearaldehyde were present on the 8th day of incubation in the brain ethanolamine glycerophosphatide fraction. During the 3rd week of incubation, the liver showed a two-fold increase in the relative amount of 18:2 in all glycerophosphatide fractions. A decrease of 16:0 in the lecithin fraction and consequently in total glycerophosphatides was also observed during this period. No significant changes in glycerophosphatide fatty acids were observed in the yolk throughout incubation.

Fullerene C{sub 60} exposure elicits an oxidative stress response in embryonic zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Usenko, Crystal Y.; Harper, Stacey L.; Tanguay, Robert L.

2008-05-15

Due to its unique physicochemical and optical properties, C{sub 60} has raised interest in commercialization for a variety of products. While several reports have determined this nanomaterial to act as a powerful antioxidant, many other studies have demonstrated a strong oxidative potential through photoactivation. To directly address the oxidative potential of C{sub 60}, the effects of light and chemical supplementation and depletion of glutathione (GSH) on C{sub 60}-induced toxicity were evaluated. Embryonic zebrafish were used as a model organism to examine the potential of C{sub 60} to elicit oxidative stress responses. Reduced light during C{sub 60} exposure significantly decreased mortalitymore » and the incidence of fin malformations and pericardial edema at 200 and 300 ppb C{sub 60}. Embryos co-exposed to the glutathione precursor, N-acetylcysteine (NAC), also showed reduced mortality and pericardial edema; however, fin malformations were not reduced. Conversely, co-exposure to the GSH synthesis inhibitors, buthionine sulfoximine (BSO) and diethyl maleate (DEM), increased the sensitivity of zebrafish to C{sub 60} exposure. Co-exposure of C{sub 60} or its hydroxylated derivative, C{sub 60}(OH){sub 24}, with H{sub 2}O{sub 2} resulted in increased mortality along the concentration gradient of H{sub 2}O{sub 2} for both materials. Microarrays were used to examine the effects of C{sub 60} on the global gene expression at two time points, 36 and 48 h post fertilization (hpf). At both life stages there were alterations in the expression of several key stress response genes including glutathione-S-transferase, glutamate cysteine ligase, ferritin, {alpha}-tocopherol transport protein and heat shock protein 70. These results support the hypothesis that C{sub 60} induces oxidative stress in this model system.« less

The zebrafish bozozok locus encodes Dharma, a homeodomain protein essential for induction of gastrula organizer and dorsoanterior embryonic structures.

PubMed

Fekany, K; Yamanaka, Y; Leung, T; Sirotkin, H I; Topczewski, J; Gates, M A; Hibi, M; Renucci, A; Stemple, D; Radbill, A; Schier, A F; Driever, W; Hirano, T; Talbot, W S; Solnica-Krezel, L

1999-04-01

The dorsal gastrula organizer plays a fundamental role in establishment of the vertebrate axis. We demonstrate that the zebrafish bozozok (boz) locus is required at the blastula stages for formation of the embryonic shield, the equivalent of the gastrula organizer and expression of multiple organizer-specific genes. Furthermore, boz is essential for specification of dorsoanterior embryonic structures, including notochord, prechordal mesendoderm, floor plate and forebrain. We report that boz mutations disrupt the homeobox gene dharma. Overexpression of boz in the extraembryonic yolk syncytial layer of boz mutant embryos is sufficient for normal development of the overlying blastoderm, revealing an involvement of extraembryonic structures in anterior patterning in fish similarly to murine embryos. Epistatic analyses indicate that boz acts downstream of beta-catenin and upstream to TGF-beta signaling or in a parallel pathway. These studies provide genetic evidence for an essential function of a homeodomain protein in beta-catenin-mediated induction of the dorsal gastrula organizer and place boz at the top of a hierarchy of zygotic genes specifying the dorsal midline of a vertebrate embryo.

Ionic channels underlying the ventricular action potential in zebrafish embryo.

PubMed

Alday, Aintzane; Alonso, Hiart; Gallego, Monica; Urrutia, Janire; Letamendia, Ainhoa; Callol, Carles; Casis, Oscar

2014-06-01

Over the last years zebrafish has become a popular model in the study of cardiac physiology, pathology and pharmacology. Recently, the application of the 3Rs regulation and the characteristics of the embryo have reduced the use of adult zebrafish use in many studies. However, the zebrafish embryo cardiac physiology is poorly characterized since most works have used indirect techniques and direct recordings of cardiac action potential and ionic currents are scarce. In order to optimize the zebrafish embryo model, we used electrophysiological, pharmacological and immunofluorescence tools to identify the characteristics and the ionic channels involved in the ventricular action potentials of zebrafish embryos. The application of Na(+) or T-type Ca(+2) channel blockers eliminated the cardiac electrical activity, indicating that the action potential upstroke depends on Na(+) and T-type Ca(+2) currents. The plateau phase depends on L-type Ca(+2) channels since it is abolished by specific blockade. The direct channel blockade indicates that the action potential repolarization and diastolic potential depends on ERG K(+) channels. The presence in the embryonic heart of the Nav1.5, Cav1.2, Cav3.2 and ERG channels was also confirmed by immunofluorescence, while the absence of effect of specific blockers and immunostaining indicate that two K(+) repolarizing currents present in human heart, Ito and IKs, are absent in the embryonic zebrafish heart. Our results describe the ionic channels present and its role in the zebrafish embryo heart and support the use of zebrafish embryos to study human diseases and their use for drug testing. Copyright © 2014 Elsevier Ltd. All rights reserved.

Chitosan nanoparticles and their Tween 80 modified counterparts disrupt the developmental profile of zebrafish embryos.

PubMed

Yuan, Zhongyue; Li, Ying; Hu, Yulan; You, Jian; Higashisaka, Kazuma; Nagano, Kazuya; Tsutsumi, Yasuo; Gao, Jianqing

2016-12-30

Chitosan nanoparticles (CS-NPs) and their Tween 80 modified counterparts (TmCS-NPs) are among the most commonly used brain-targeted vehicles. However, their potential developmental toxicity is poorly understood. In this study, zebrafish embryos are introduced as an in vivo platform. Both NPs showed a dose-dependent increase in developmental toxicity (decreased hatching rate, increased mortality and incidences of malformation). Neurobehavioral changes included decreased spontaneous movement in TmCS-NP treated embryos and hyperactive effect in CS-NP treated larvae. Both NPs remarkably inhibited axonal development of primary and secondary motor neurons, and affected the muscle structure. Overall, this study demonstrated that CS-NPs and TmCS-NPs could affect embryonic development, disrupt neurobehavior of zebrafish larvae and affect muscle and neuron development, suggesting more attention on biodegradable chitosan nanoparticles. Copyright © 2016 Elsevier B.V. All rights reserved.

The gonadotropin-inhibitory hormone (Lpxrfa) system's regulation of reproduction in the brain-pituitary axis of the zebrafish (Danio rerio).

PubMed

Spicer, Olivia Smith; Zmora, Nilli; Wong, Ten-Tsao; Golan, Matan; Levavi-Sivan, Berta; Gothilf, Yoav; Zohar, Yonathan

2017-05-01

Gonadotropin-inhibitory hormone (GNIH) was discovered in quail with the ability to reduce gonadotropin expression/secretion in the pituitary. There have been few studies on GNIH orthologs in teleosts (LPXRFamide (Lpxrfa) peptides), which have provided inconsistent results. Therefore, the goal of this study was to determine the roles and modes of action by which Lpxrfa exerts its functions in the brain-pituitary axis of zebrafish (Danio rerio). We localized Lpxrfa soma to the ventral hypothalamus, with fibers extending throughout the brain and to the pituitary. In the preoptic area, Lpxrfa fibers interact with gonadotropin-releasing hormone 3 (Gnrh3) soma. In pituitary explants, zebrafish peptide Lpxrfa-3 downregulated luteinizing hormone beta subunit and common alpha subunit expression. In addition, Lpxrfa-3 reduced gnrh3 expression in brain slices, offering another pathway for Lpxrfa to exert its effects on reproduction. Receptor activation studies, in a heterologous cell-based system, revealed that all three zebrafish Lpxrfa peptides activate Lpxrf-R2 and Lpxrf-R3 via the PKA/cAMP pathway. Receptor activation studies demonstrated that, in addition to activating Lpxrf receptors, zebrafish Lpxrfa-2 and Lpxrfa-3 antagonize Kisspeptin-2 (Kiss2) activation of Kisspeptin receptor-1a (Kiss1ra). The fact that kiss1ra-expressing neurons in the preoptic area are innervated by Lpxrfa-ir fibers suggests an additional pathway for Lpxrfa action. Therefore, our results suggest that Lpxrfa may act as a reproductive inhibitory neuropeptide in the zebrafish that interacts with Gnrh3 neurons in the brain and with gonadotropes in the pituitary, while also potentially utilizing the Kiss2/Kiss1ra pathway. © The Authors 2017. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

Toxic effects of indoxacarb enantiomers on the embryonic development and induction of apoptosis in zebrafish larvae (Danio rerio).

PubMed

Fan, Yongmei; Feng, Qing; Lai, Kehua; Huang, Weikang; Zhang, Chenghui; Li, Qing X

2017-01-01

Indoxacarb is a highly potent insecticide widely used to control Lepidoptera insects in vegetable, tea, cotton, and rice fields. It can run off into aquatic environments. It is consisted of two enantiomers. Environmental risks and aquatic toxicity of indoxacarb enantiomers have not been fully investigated. In this study, zebrafish (Danio rerio) embryos were exposed to varying concentrations of (-)-R-indoxacarb and (+)-S-indoxacarb until 96-h post-fertilization (hpf) to assess the embryonic toxicity. (-)-R-indoxacarb was 1.3-fold more toxic than (+)-S-isomer to zebrafish embryos at 96 hpf. (-)-R-indoxacarb exhibited reduction in body length and pericardial edema compared with (+)-S-indoxacarb. (-)-R-indoxacarb decreased the hatching rate sixfold greater than (+)-S-indoxacarb. The rate of pericardial edema induced by (-)-R-indoxacarb was 2.5 times greater than that by (+)-S-indoxacarb. The heart rate of the larvae exposed to (-)-R-indoxacarb was 30% lower than that to (+)-S-indoxacarb. In addition, exposure to the chiral isomers resulted in significant increases in apoptosis; interestingly (-)-R-indoxacarb induced apoptosis in the heart area, whereas (+)-S-indoxacarb induced apoptosis in the head area. © 2015 Wiley Periodicals, Inc. Environ Toxicol 32: 7-16, 2017. © 2015 Wiley Periodicals, Inc.

Zebrafish models for translational neuroscience research: from tank to bedside

PubMed Central

Stewart, Adam Michael; Braubach, Oliver; Spitsbergen, Jan; Gerlai, Robert; Kalueff, Allan V.

2014-01-01

The zebrafish (Danio rerio) is emerging as a new important species for studying mechanisms of brain function and dysfunction. Focusing on selected central nervous system (CNS) disorders (brain cancer, epilepsy, and anxiety) and using them as examples, we discuss the value of zebrafish models in translational neuroscience. We further evaluate the contribution of zebrafish to neuroimaging, circuit level, and drug discovery research. Outlining the role of zebrafish in modeling a wide range of human brain disorders, we also summarize recent applications and existing challenges in this field. Finally, we emphasize the potential of zebrafish models in behavioral phenomics and high-throughput genetic/small molecule screening, which is critical for CNS drug discovery and identifying novel candidate genes. PMID:24726051

Zebrafish is a predictive model for identifying compounds that protect against brain toxicity in severe acute organophosphorus intoxication.

PubMed

Faria, Melissa; Prats, Eva; Padrós, Francesc; Soares, Amadeu M V M; Raldúa, Demetrio

2017-04-01

Acute organophosphorus (OP) intoxication is a worldwide clinical and public health problem. In addition to cholinergic crisis, neurodegeneration and brain damage are hallmarks of the severe form of this toxidrome. Recently, we generated a chemical model of severe acute OP intoxication in zebrafish that is characterized by altered head morphology and brain degeneration. The pathophysiological pathways resulting in brain toxicity in this model are similar to those described in humans. The aim of this study was to assess the predictive power of this zebrafish model by testing the effect of a panel of drugs that provide protection in mammalian models. The selected drugs included "standard therapy" drugs (atropine and pralidoxime), reversible acetylcholinesterase inhibitors (huperzine A, galantamine, physostigmine and pyridostigmine), N-methyl-D-aspartate (NMDA) receptor antagonists (MK-801 and memantine), dual-function NMDA receptor and acetylcholine receptor antagonists (caramiphen and benactyzine) and anti-inflammatory drugs (dexamethasone and ibuprofen). The effects of these drugs on zebrafish survival and the prevalence of abnormal head morphology in the larvae exposed to 4 µM chlorpyrifos oxon [1 × median lethal concentration (LC 50 )] were determined. Moreover, the neuroprotective effects of pralidoxime, memantine, caramiphen and dexamethasone at the gross morphological level were confirmed by histopathological and transcriptional analyses. Our results demonstrated that the zebrafish model for severe acute OP intoxication has a high predictive value and can be used to identify new compounds that provide neuroprotection against severe acute OP intoxication.

Effects of heavy ion radiation on the brain vascular system and embryonic development

NASA Technical Reports Server (NTRS)

Yang, T. C.; Tobias, C. A.

1984-01-01

The present investigation is concerned with the effects of heavy-ion radiation on the vascular system and the embryonic development, taking into account the results of experiments with neonatal rats and mouse embryos. It is found that heavy ions can be highly effective in producing brain hemorrhages and in causing body deformities. Attention is given to aspects of methodology, the induction of brain hemorrhages by X-rays and heavy ions, and the effect of iron particles on embryonic development. Reported results suggest that high linear energy transfer (LET) heavy ions can be very effective in producing developmental abnormalities.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain.

PubMed

Kuipers, Jeroen; Kalicharan, Ruby D; Wolters, Anouk H G; van Ham, Tjakko J; Giepmans, Ben N G

2016-05-25

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae(1-7). Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture(1-5). Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)(8) on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner.

Large-scale Scanning Transmission Electron Microscopy (Nanotomy) of Healthy and Injured Zebrafish Brain

PubMed Central

Kuipers, Jeroen; Kalicharan, Ruby D.; Wolters, Anouk H. G.

2016-01-01

Large-scale 2D electron microscopy (EM), or nanotomy, is the tissue-wide application of nanoscale resolution electron microscopy. Others and we previously applied large scale EM to human skin pancreatic islets, tissue culture and whole zebrafish larvae1-7. Here we describe a universally applicable method for tissue-scale scanning EM for unbiased detection of sub-cellular and molecular features. Nanotomy was applied to investigate the healthy and a neurodegenerative zebrafish brain. Our method is based on standardized EM sample preparation protocols: Fixation with glutaraldehyde and osmium, followed by epoxy-resin embedding, ultrathin sectioning and mounting of ultrathin-sections on one-hole grids, followed by post staining with uranyl and lead. Large-scale 2D EM mosaic images are acquired using a scanning EM connected to an external large area scan generator using scanning transmission EM (STEM). Large scale EM images are typically ~ 5 - 50 G pixels in size, and best viewed using zoomable HTML files, which can be opened in any web browser, similar to online geographical HTML maps. This method can be applied to (human) tissue, cross sections of whole animals as well as tissue culture1-5. Here, zebrafish brains were analyzed in a non-invasive neuronal ablation model. We visualize within a single dataset tissue, cellular and subcellular changes which can be quantified in various cell types including neurons and microglia, the brain's macrophages. In addition, nanotomy facilitates the correlation of EM with light microscopy (CLEM)8 on the same tissue, as large surface areas previously imaged using fluorescent microscopy, can subsequently be subjected to large area EM, resulting in the nano-anatomy (nanotomy) of tissues. In all, nanotomy allows unbiased detection of features at EM level in a tissue-wide quantifiable manner. PMID:27285162

Normal anatomy and histology of the adult zebrafish.

PubMed

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.

Making Waves: New Developments in Toxicology With the Zebrafish.

PubMed

Horzmann, Katharine A; Freeman, Jennifer L

2018-05-01

The laboratory zebrafish (Danio rerio) is now an accepted model in toxicologic research. The zebrafish model fills a niche between in vitro models and mammalian biomedical models. The developmental characteristics of the small fish are strategically being used by scientists to study topics ranging from high-throughput toxicity screens to toxicity in multi- and transgenerational studies. High-throughput technology has increased the utility of zebrafish embryonic toxicity assays in screening of chemicals and drugs for toxicity or effect. Additionally, advances in behavioral characterization and experimental methodology allow for observation of recognizable phenotypic changes after xenobiotic exposure. Future directions in zebrafish research are predicted to take advantage of CRISPR-Cas9 genome editing methods in creating models of disease and interrogating mechanisms of action with fluorescent reporters or tagged proteins. Zebrafish can also model developmental origins of health and disease and multi- and transgenerational toxicity. The zebrafish has many advantages as a toxicologic model and new methodologies and areas of study continue to expand the usefulness and application of the zebrafish.

Toxicity of Vascular Disrupting Chemicals to Developing Zebrafish

EPA Science Inventory

Vascular development is integral to proper embryonic development and disruption of that process can have serious developmental consequences. We performed static 48-hr exposures of transgenic TG(kdr:EGFP)s843 zebrafish (Danio rerio) embryos with the known vascular inhibitors Vatal...

Fast functional imaging of multiple brain regions in intact zebrafish larvae using selective plane illumination microscopy.

PubMed

Panier, Thomas; Romano, Sebastián A; Olive, Raphaël; Pietri, Thomas; Sumbre, Germán; Candelier, Raphaël; Debrégeas, Georges

2013-01-01

The optical transparency and the small dimensions of zebrafish at the larval stage make it a vertebrate model of choice for brain-wide in-vivo functional imaging. However, current point-scanning imaging techniques, such as two-photon or confocal microscopy, impose a strong limit on acquisition speed which in turn sets the number of neurons that can be simultaneously recorded. At 5 Hz, this number is of the order of one thousand, i.e., approximately 1-2% of the brain. Here we demonstrate that this limitation can be greatly overcome by using Selective-plane Illumination Microscopy (SPIM). Zebrafish larvae expressing the genetically encoded calcium indicator GCaMP3 were illuminated with a scanned laser sheet and imaged with a camera whose optical axis was oriented orthogonally to the illumination plane. This optical sectioning approach was shown to permit functional imaging of a very large fraction of the brain volume of 5-9-day-old larvae with single- or near single-cell resolution. The spontaneous activity of up to 5,000 neurons was recorded at 20 Hz for 20-60 min. By rapidly scanning the specimen in the axial direction, the activity of 25,000 individual neurons from 5 different z-planes (approximately 30% of the entire brain) could be simultaneously monitored at 4 Hz. Compared to point-scanning techniques, this imaging strategy thus yields a ≃20-fold increase in data throughput (number of recorded neurons times acquisition rate) without compromising the signal-to-noise ratio (SNR). The extended field of view offered by the SPIM method allowed us to directly identify large scale ensembles of neurons, spanning several brain regions, that displayed correlated activity and were thus likely to participate in common neural processes. The benefits and limitations of SPIM for functional imaging in zebrafish as well as future developments are briefly discussed.

From Whole-Brain Data to Functional Circuit Models: The Zebrafish Optomotor Response.

PubMed

Naumann, Eva A; Fitzgerald, James E; Dunn, Timothy W; Rihel, Jason; Sompolinsky, Haim; Engert, Florian

2016-11-03

Detailed descriptions of brain-scale sensorimotor circuits underlying vertebrate behavior remain elusive. Recent advances in zebrafish neuroscience offer new opportunities to dissect such circuits via whole-brain imaging, behavioral analysis, functional perturbations, and network modeling. Here, we harness these tools to generate a brain-scale circuit model of the optomotor response, an orienting behavior evoked by visual motion. We show that such motion is processed by diverse neural response types distributed across multiple brain regions. To transform sensory input into action, these regions sequentially integrate eye- and direction-specific sensory streams, refine representations via interhemispheric inhibition, and demix locomotor instructions to independently drive turning and forward swimming. While experiments revealed many neural response types throughout the brain, modeling identified the dimensions of functional connectivity most critical for the behavior. We thus reveal how distributed neurons collaborate to generate behavior and illustrate a paradigm for distilling functional circuit models from whole-brain data. Copyright © 2016 Elsevier Inc. All rights reserved.

Brain-wide neuronal dynamics during motor adaptation in zebrafish.

PubMed

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2012-05-09

A fundamental question in neuroscience is how entire neural circuits generate behaviour and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record the activity of large populations of neurons at the cellular level, throughout the brain of larval zebrafish expressing a genetically encoded calcium sensor, while the paralysed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neuronal response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioural adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behaviour.

Brain-wide neuronal dynamics during motor adaptation in zebrafish

PubMed Central

Ahrens, Misha B; Li, Jennifer M; Orger, Michael B; Robson, Drew N; Schier, Alexander F; Engert, Florian; Portugues, Ruben

2013-01-01

A fundamental question in neuroscience is how entire neural circuits generate behavior and adapt it to changes in sensory feedback. Here we use two-photon calcium imaging to record activity of large populations of neurons at the cellular level throughout the brain of larval zebrafish expressing a genetically-encoded calcium sensor, while the paralyzed animals interact fictively with a virtual environment and rapidly adapt their motor output to changes in visual feedback. We decompose the network dynamics involved in adaptive locomotion into four types of neural response properties, and provide anatomical maps of the corresponding sites. A subset of these signals occurred during behavioral adjustments and are candidates for the functional elements that drive motor learning. Lesions to the inferior olive indicate a specific functional role for olivocerebellar circuitry in adaptive locomotion. This study enables the analysis of brain-wide dynamics at single-cell resolution during behavior. PMID:22622571

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hinfray, N., E-mail: nathalie.hinfray@ineris.fr

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase genemore » under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. - Highlights: • Combined effects of EE2 and LNG were assessed on ER-dependent cyp19a1b expression. • EE2 and LNG alone induced brain aromatase in zebrafish specific bioassays. • Experimental ray design allowed complete concentration-response surfaces modeling. �

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish.

PubMed

Blechinger, Scott R; Kusch, Robin C; Haugo, Kristine; Matz, Carlyn; Chivers, Douglas P; Krone, Patrick H

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae. Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.

Brief embryonic cadmium exposure induces a stress response and cell death in the developing olfactory system followed by long-term olfactory deficits in juvenile zebrafish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Blechinger, Scott R.; Toxicology Group, University of Saskatchewan, Saskatoon, Saskatchewan; Kusch, Robin C.

2007-10-01

The toxic effects of cadmium and other metals have been well established. A primary target of these metals is known to be the olfactory system, and fish exposed to a number of different waterborne metals display deficiencies in olfaction. Importantly, exposure over embryonic/larval development periods can cause deficits in chemosensory function in juvenile fish, but the specific cell types affected are unknown. We have previously characterized a transgenic zebrafish strain expressing the green fluorescent protein (eGFP) gene linked to the hsp70 gene promoter, and shown it to be a useful tool for examining cell-specific toxicity in living embryos and larvae.more » Here we show that the hsp70/eGFP transgene is strongly and specifically upregulated within the olfactory sensory neurons (OSNs) of transgenic zebrafish larvae following a brief 3-h exposure to water-borne cadmium. This molecular response was closely correlated to an endpoint for tissue damage within the olfactory placode, namely cell death. Furthermore, cadmium-induced olfactory cytotoxicity in zebrafish larvae gives rise to more permanent effects. Juvenile zebrafish briefly exposed to cadmium during early larval development display deficits in olfactory-dependent predator avoidance behaviors 4-6 weeks after a return to clean water. Lateral line neuromasts of exposed zebrafish larvae also activate both the endogenous hsp70 gene and the hsp70/eGFP transgene. The data reveal that even a very brief exposure period that gives rise to cell death within the developing olfactory placode results in long-term deficits in olfaction, and that hsp70/eGFP may serve as an effective indicator of sublethal cadmium exposure in sensory cells.« less

Eye-Specific Gene Expression following Embryonic Ethanol Exposure in Zebrafish: Roles for Heat Shock Factor 1

PubMed Central

Kashyap, Bhavani; Pegorsch, Laurel; Frey, Ruth A.; Sun, Chi; Shelden, Eric A.; Stenkamp, Deborah L.

2014-01-01

The mechanisms through which ethanol exposure results in developmental defects remain unclear. We used the zebrafish model to elucidate eye-specific mechanisms that underlie ethanol-mediated microphthalmia (reduced eye size), through time-series microarray analysis of gene expression within eyes of embryos exposed to 1.5% ethanol. 62 genes were differentially expressed (DE) in ethanol-treated as compared to control eyes sampled during retinal neurogenesis (24-48 hours post-fertilization). The EDGE (extraction of differential gene expression) algorithm identified >3000 genes DE over developmental time in ethanol-exposed eyes as compared to controls. The DE lists included several genes indicating a mis-regulated cellular stress response due to ethanol exposure. Combined treatment with sub-threshold levels of ethanol and a morpholino targeting heat shock factor 1 mRNA resulted in microphthalmia, suggesting convergent molecular pathways. Thermal preconditioning partially prevented ethanol-mediated microphthalmia while maintaining Hsf-1 expression. These data suggest roles for reduced Hsf-1 in mediating microphthalmic effects of embryonic ethanol exposure. PMID:24355176

The Visual System of Zebrafish and its Use to Model Human Ocular Diseases

PubMed Central

Gestri, Gaia; Link, Brian A; Neuhauss, Stephan CF

2011-01-01

Free swimming zebrafish larvae depend mainly on their sense of vision to evade predation and to catch prey. Hence there is strong selective pressure on the fast maturation of visual function and indeed the visual system already supports a number of visually-driven behaviors in the newly hatched larvae. The ability to exploit the genetic and embryonic accessibility of the zebrafish in combination with a behavioral assessment of visual system function has made the zebrafish a popular model to study vision and its diseases. Here, we review the anatomy, physiology and development of the zebrafish eye as the basis to relate the contributions of the zebrafish to our understanding of human ocular diseases. PMID:21595048

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

PubMed

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.

A panel of recombinant monoclonal antibodies against zebrafish neural receptors and secreted proteins suitable for wholemount immunostaining.

PubMed

Staudt, Nicole; Müller-Sienerth, Nicole; Fane-Dremucheva, Alla; Yusaf, Shahnaz P; Millrine, David; Wright, Gavin J

2015-01-02

Cell surface receptors and secreted proteins play important roles in neural recognition processes, but because their site of action can be a long distance from neuron cell bodies, antibodies that label these proteins are valuable to understand their function. The zebrafish embryo is a popular vertebrate model for neurobiology, but suffers from a paucity of validated antibody reagents. Here, we use the entire ectodomain of neural zebrafish cell surface or secreted proteins expressed in mammalian cells to select monoclonal antibodies to ten different antigens. The antibodies were characterised by Western blotting and the sensitivity of their epitopes to formalin fixation was determined. The rearranged antigen binding regions of the antibodies were amplified and cloned which enabled expression in a recombinant form from a single plasmid. All ten antibodies gave specific staining patterns within formalin-treated embryonic zebrafish brains, demonstrating that this generalised approach is particularly efficient to elicit antibodies that stain native antigen in fixed wholemount tissue. Finally, we show that additional tags can be easily added to the recombinant antibodies for convenient multiplex staining. The antibodies and the approaches described here will help to address the lack of well-defined antibody reagents in zebrafish research. Copyright © 2014 The Authors. Published by Elsevier Inc. All rights reserved.

Generation and characterization of Kctd15 mutations in zebrafish

PubMed Central

Heffer, Alison; Marquart, Gregory D.; Aquilina-Beck, Allisan; Saleem, Nabil; Burgess, Harold A.

2017-01-01

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. PMID:29216270

In silico predicted reproductive endocrine transcriptional regulatory networks during zebrafish (Danio rerio) development.

PubMed

Hala, D

2017-03-21

The interconnected topology of transcriptional regulatory networks (TRNs) readily lends to mathematical (or in silico) representation and analysis as a stoichiometric matrix. Such a matrix can be 'solved' using the mathematical method of extreme pathway (ExPa) analysis, which identifies uniquely activated genes subject to transcription factor (TF) availability. In this manuscript, in silico multi-tissue TRN models of brain, liver and gonad were used to study reproductive endocrine developmental programming in zebrafish (Danio rerio) from 0.25h post fertilization (hpf; zygote) to 90 days post fertilization (dpf; adult life stage). First, properties of TRN models were studied by sequentially activating all genes in multi-tissue models. This analysis showed the brain to exhibit lowest proportion of co-regulated genes (19%) relative to liver (23%) and gonad (32%). This was surprising given that the brain comprised 75% and 25% more TFs than liver and gonad respectively. Such 'hierarchy' of co-regulatory capability (brainbrain, alluding to its role as 'master controller' of endocrine function. Second, TRN models were constrained with varying TF availabilities during zebrafish development. Normalized numbers of genes active during development showed concomitant activations between brain and gonad from 10 to 12 hpf (embryonic life stage) up to 30-90 dpf (adult life stage). This indicated a putative 'syncing' between the brain and gonad, and initiation of an early reproductive endocrine developmental program. Finally, comparison of in vivo active genes with those predicted in silico showed relatively good agreement for brain (49%), liver (27%) and gonad (32%). The multi-tissue TRN models presented can lend diagnostic insights into the effects of changing environmental and/or genetic constraints on reproductive endocrine function. Copyright © 2017 Elsevier Ltd. All rights reserved.

Developmental Toxicity of Louisiana Crude Oiled Sediment to Zebrafish

EPA Science Inventory

Embryonic exposures to polycyclic aromatic hydrocarbons (PAHs) and petroleum products cause a characteristic suite of developmental defects in a variety of fish species. We exposed zebrafish embryos to sediment mixed with laboratory weathered South Louisiana crude oil. Oiled sedi...

Zebrafish as a model for monocarboxyl transporter 8-deficiency.

PubMed

Vatine, Gad David; Zada, David; Lerer-Goldshtein, Tali; Tovin, Adi; Malkinson, Guy; Yaniv, Karina; Appelbaum, Lior

2013-01-04

Allan-Herndon-Dudley syndrome (AHDS) is a severe psychomotor retardation characterized by neurological impairment and abnormal thyroid hormone (TH) levels. Mutations in the TH transporter, monocarboxylate transporter 8 (MCT8), are associated with AHDS. MCT8 knock-out mice exhibit impaired TH levels; however, they lack neurological defects. Here, the zebrafish mct8 gene and promoter were isolated, and mct8 promoter-driven transgenic lines were used to show that, similar to humans, mct8 is primarily expressed in the nervous and vascular systems. Morpholino-based knockdown and rescue experiments revealed that MCT8 is strictly required for neural development in the brain and spinal cord. This study shows that MCT8 is a crucial regulator during embryonic development and establishes the first vertebrate model for MCT8 deficiency that exhibits a neurological phenotype.

PFOS, PFNA, and PFOA Sub-Lethal Exposure to Embryonic Zebrafish Have Different Toxicity Profiles in Terms of Morphometrics, Behavior and Gene Expression

PubMed Central

Jantzen, Carrie E.; Annunziato, Kate A.; Bugel, Sean M.; Cooper, Keith R.

2016-01-01

Polyfluorinated compounds (PFC) are a class of anthropogenic, persistent and toxic chemicals. PFCs are detected worldwide and consist of fluorinated carbon chains of varying length, terminal groups, and industrial uses. Previous zebrafish studies in the literature as well as our own studies have shown that exposure to these chemicals at a low range of concentrations (0.02 µM – 2.0 µM; 20–2000 ppb) resulted in chemical specific developmental defects and reduced post hatch survival. It was hypothesized that sub-lethal embryonic exposure to perfluorooctanesulfonic acid (PFOS), perfluorononanoic acid (PFNA), or perfluorooctanoic acid (PFOA) would result in different responses with regard to morphometric, behavior, and gene expression in both yolk sac fry and larval zebrafish. Zebrafish were exposed to PFOS, PFOA, and PFNA (0.02, 0.2, 2.0 µM) for the first five days post fertilization (dpf) and analyzed for morphometrics (5 dpf, 14 dpf), targeted gene expression (5 dpf, 14 dpf), and locomotive behavior (14 dpf). All three PFCs commonly resulted in a decrease in total body length, increased tfc3a (muscle development) expression and decreased ap1s (protein transport) expression at 5dpf, and hyperactive locomotor activity 14 dpf. All other endpoints measured at both life-stage time points varied between each of the PFCs. PFOS, PFNA, and PFOA exposure resulted in significantly altered responses in terms of morphometric, locomotion, and gene expression endpoints, which could be manifested in field exposed teleosts. PMID:27058923

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yan, Lifeng; Key Laboratory of Modern Toxicology of Ministry of Education, School of Public Health, Nanjing Medical University, Nanjing 210029; Zhou, Yong

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 andmore » 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.« less

Treatment with Myf5-morpholino results in somite patterning and brain formation defects in zebrafish.

PubMed

Chen, Yau-Hung; Tsai, Huai-Jen

2002-10-01

Myf-5 is a stage-dependent transcription factor associated with somitogenesis. To study its biological functions in zebrafish, we injected the Myf5-morpholinos ZMF-MO (antisense nucleotides 28 to 52) and ZMF-OTHER (antisense nucleotides 3 to 27) into zebrafish embryos to establish a myf-5 gene knockdown. No phenotypic abnormalities were observed following injection with 0.2 ng of ZMF-MO, but defects were displayed in 2 of 118 (1.7%) surviving embryos injected with 1 ng ZMF-MO. Morphological defects became more severe with increased dosages: 105 of 270 (38.9%) surviving embryos injected with 4.5 ng of ZMF-MO displayed such abnormalities as the absence of eyes or brains in addition to the following low-dosage defects in 24 hpf embryos: longitudinal yolk sacs, incomplete epiboly coverage, abnormal and suspended tail buds, diffused somite boundaries, and head shrinkage. Similar results were observed in the 4.5 ng ZMF-OTHER injection group. However, when fish were co-injected with 4.5 ng ZMF-MO and 4.5 ng myf-5 mRNA, abnormality rates decreased from 49.6% to 5.5%. Our results show that the brain krox20 gene was down-regulated at rhombomere 3; the pax2.1 gene was completely down-regulated; myoD was expressed normally; myogenin was substantially down-regulated in whole somites; and desmin was partly inhibited in newly forming somites. Our conclusion is that zebrafish Myf-5 may play important roles in brain formation and in the convergence and extension of shield epiblasts and tail buds during early embryogenesis, in addition to its well-understood role as a muscle regulatory factor in somites.

Embryonic Ethanol Exposure Dysregulates BMP and Notch Signaling, Leading to Persistent Atrio-Ventricular Valve Defects in Zebrafish

PubMed Central

Sarmah, Swapnalee; Muralidharan, Pooja

2016-01-01

Fetal alcohol spectrum disorder (FASD), birth defects associated with ethanol exposure in utero, includes a wide spectrum of congenital heart defects (CHDs), the most prevalent of which are septal and conotruncal defects. Zebrafish FASD model was used to dissect the mechanisms underlying FASD-associated CHDs. Embryonic ethanol exposure (3–24 hours post fertilization) led to defects in atrio-ventricular (AV) valvulogenesis beginning around 37 hpf, a morphogenetic event that arises long after ethanol withdrawal. Valve leaflets of the control embryos comprised two layers of cells confined at the compact atrio-ventricular canal (AVC). Ethanol treated embryos had extended AVC and valve forming cells were found either as rows of cells spanning the AVC or as unorganized clusters near the AV boundary. Ethanol exposure reduced valve precursors at the AVC, but some ventricular cells in ethanol treated embryos exhibited few characteristics of valve precursors. Late staged larvae and juvenile fish exposed to ethanol during embryonic development had faulty AV valves. Examination of AVC morphogenesis regulatory networks revealed that early ethanol exposure disrupted the Bmp signaling gradient in the heart during valve formation. Bmp signaling was prominent at the AVC in controls, but ethanol-exposed embryos displayed active Bmp signaling throughout the ventricle. Ethanol exposure also led to mislocalization of Notch signaling cells in endocardium during AV valve formation. Normally, highly active Notch signaling cells were organized at the AVC. In ethanol-exposed embryos, highly active Notch signaling cells were dispersed throughout the ventricle. At later stages, ethanol-exposed embryos exhibited reduced Wnt/β-catenin activity at the AVC. We conclude that early embryonic ethanol exposure alters Bmp, Notch and other signaling activities during AVC differentiation leading to faulty valve morphogenesis and valve defects persist in juvenile fish. PMID:27556898

Embryonic Ethanol Exposure Dysregulates BMP and Notch Signaling, Leading to Persistent Atrio-Ventricular Valve Defects in Zebrafish.

PubMed

Sarmah, Swapnalee; Muralidharan, Pooja; Marrs, James A

2016-01-01

Fetal alcohol spectrum disorder (FASD), birth defects associated with ethanol exposure in utero, includes a wide spectrum of congenital heart defects (CHDs), the most prevalent of which are septal and conotruncal defects. Zebrafish FASD model was used to dissect the mechanisms underlying FASD-associated CHDs. Embryonic ethanol exposure (3-24 hours post fertilization) led to defects in atrio-ventricular (AV) valvulogenesis beginning around 37 hpf, a morphogenetic event that arises long after ethanol withdrawal. Valve leaflets of the control embryos comprised two layers of cells confined at the compact atrio-ventricular canal (AVC). Ethanol treated embryos had extended AVC and valve forming cells were found either as rows of cells spanning the AVC or as unorganized clusters near the AV boundary. Ethanol exposure reduced valve precursors at the AVC, but some ventricular cells in ethanol treated embryos exhibited few characteristics of valve precursors. Late staged larvae and juvenile fish exposed to ethanol during embryonic development had faulty AV valves. Examination of AVC morphogenesis regulatory networks revealed that early ethanol exposure disrupted the Bmp signaling gradient in the heart during valve formation. Bmp signaling was prominent at the AVC in controls, but ethanol-exposed embryos displayed active Bmp signaling throughout the ventricle. Ethanol exposure also led to mislocalization of Notch signaling cells in endocardium during AV valve formation. Normally, highly active Notch signaling cells were organized at the AVC. In ethanol-exposed embryos, highly active Notch signaling cells were dispersed throughout the ventricle. At later stages, ethanol-exposed embryos exhibited reduced Wnt/β-catenin activity at the AVC. We conclude that early embryonic ethanol exposure alters Bmp, Notch and other signaling activities during AVC differentiation leading to faulty valve morphogenesis and valve defects persist in juvenile fish.

Novel function of vitamin E in regulation of zebrafish (Danio rerio) brain lysophospholipids discovered using lipidomics

PubMed Central

Choi, Jaewoo; Leonard, Scott W.; Kasper, Katherine; McDougall, Melissa; Stevens, Jan F.; Tanguay, Robert L.; Traber, Maret G.

2015-01-01

We hypothesized that brains from vitamin E-deficient (E−) zebrafish (Danio rerio) would undergo increased lipid peroxidation because they contain highly polyunsaturated fatty acids, thus susceptible lipids could be identified. Brains from zebrafish fed for 9 months defined diets without (E−) or with (E+) added vitamin E (500 mg RRR-α-tocopheryl acetate per kilogram diet) were studied. Using an untargeted approach, 1-hexadecanoyl-2-docosahexaenoyl-sn-glycero-3-phosphocholine [DHA-PC 38:6, PC 16:0/22:6]was the lipid that showed the most significant and greatest fold-differences between groups. DHA-PC concentrations were approximately 1/3 lower in E− (4.3 ± 0.6 mg/g) compared with E+ brains (6.5 ± 0.9 mg/g, mean ± SEM, n = 10 per group, P = 0.04). Using lipidomics, 155 lipids in brain extracts were identified. Only four phospholipids (PLs) were different (P < 0.05) between groups; they were lower in E− brains and contained DHA with DHA-PC 38:6 at the highest abundances. Moreover, hydroxy-DHA-PC 38:6 was increased in E− brains (P = 0.0341) supporting the hypothesis of DHA peroxidation. More striking was the depletion in E− brains of nearly 60% of 19 different lysophospholipids (lysoPLs) (combined P = 0.0003), which are critical for membrane PL remodeling. Thus, E− brains contained fewer DHA-PLs, more hydroxy-DHA-PCs, and fewer lysoPLs, suggesting that lipid peroxidation depletes membrane DHA-PC and homeostatic mechanisms to repair the damage resulting in lysoPL depletion. PMID:25855633

Chronic vitamin E deficiency impairs cognitive function in adult zebrafish via dysregulation of brain lipids and energy metabolism.

PubMed

McDougall, Melissa; Choi, Jaewoo; Magnusson, Kathy; Truong, Lisa; Tanguay, Robert; Traber, Maret G

2017-11-01

Zebrafish (Danio rerio) are a recognized model for studying the pathogenesis of cognitive deficits and the mechanisms underlying behavioral impairments, including the consequences of increased oxidative stress within the brain. The lipophilic antioxidant vitamin E (α-tocopherol; VitE) has an established role in neurological health and cognitive function, but the biological rationale for this action remains unknown. In the present study, we investigated behavioral perturbations due to chronic VitE deficiency in adult zebrafish fed from 45 days to 18-months of age diets that were either VitE-deficient (E-) or VitE-sufficient (E+). We hypothesized that E- zebrafish would display cognitive impairments associated with elevated lipid peroxidation and metabolic disruptions in the brain. Quantified VitE levels at 18-months in E- brains (5.7 ± 0.1 nmol/g tissue) were ~20-times lower than in E+ (122.8 ± 1.1; n = 10/group). Using assays of both associative (avoidance conditioning) and non-associative (habituation) learning, we found E- vs E+ fish were learning impaired. These functional deficits occurred concomitantly with the following observations in adult E- brains: decreased concentrations of and increased peroxidation of polyunsaturated fatty acids (especially docosahexaenoic acid, DHA), altered brain phospholipid and lysophospholipid composition, as well as perturbed energy (glucose/ketone), phosphatidylcholine and choline/methyl-donor metabolism. Collectively, these data suggest that chronic VitE deficiency leads to neurological dysfunction through multiple mechanisms that become dysregulated secondary to VitE deficiency. Apparently, the E- animals alter their metabolism to compensate for the VitE deficiency, but these compensatory mechanisms are insufficient to maintain cognitive function. Copyright © 2017 Elsevier Inc. All rights reserved.

[Effect of rhynchophylline on behaviors of methamphetamine-dependent zebrafish and the mechanism].

PubMed

Chen, Yi-Fei; Peng, Ju; Fang, Miao; Liu, Yi; Nie, Ling-Hui; Mo, Zhi-Xian; Zhu, Ling-Ling

2016-11-20

To observe the effect of rhynchophylline on methamphetamine-dependent zebrafish and explore the possible mechanism. Zebrafish were divided into control group, amphetamine group, low- (50 mg/kg) and high (100 mg/kg)-dose rhynchophylline groups, and ketamine (150 mg/kg) group. Conditioned place preference (CPP) was induced in zebrafish with methamphetamine, and the staying time in the drug box and the tracking map of the zebrafish were observed with Noldus Ethovision XT system. The protein expressions of TH, NR2B and GLUR2 in the brain of zebrafish with CPP were detected with Western blotting. Compared with the control group, zebrafish in methamphetamine group showed significant variations in the staying time and swimming distance in the drug box after conditioning (P<0.05) with obvious alterations of NR2B, TH and GLUR2 expressions in the brain (P<0.05). Treatment of methamphetamine-dependent zebrafish with high-dose rhynchophylline significantly reduced the variations in the staying time and swimming distance in the drug box (P<0.05) and in the expressions of NR2B, TH and GLUR2 in the brain (P<0.05). Rhynchophylline can inhibit methamphetamine dependence in zebrafish, the mechanism of which may involve the expressions of TH, NR2B and GLUR2 proteins in the brain.

Gene expression profiles of brain endothelial cells during embryonic development at bulk and single-cell levels.

PubMed

Hupe, Mike; Li, Minerva Xueting; Kneitz, Susanne; Davydova, Daria; Yokota, Chika; Kele-Olovsson, Julianna; Hot, Belma; Stenman, Jan M; Gessler, Manfred

2017-07-11

The blood-brain barrier is a dynamic interface that separates the brain from the circulatory system, and it is formed by highly specialized endothelial cells. To explore the molecular mechanisms defining the unique nature of vascular development and differentiation in the brain, we generated high-resolution gene expression profiles of mouse embryonic brain endothelial cells using translating ribosome affinity purification and single-cell RNA sequencing. We compared the brain vascular translatome with the vascular translatomes of other organs and analyzed the vascular translatomes of the brain at different time points during embryonic development. Because canonical Wnt signaling is implicated in the formation of the blood-brain barrier, we also compared the brain endothelial translatome of wild-type mice with that of mice lacking the transcriptional cofactor β-catenin ( Ctnnb1 ). Our analysis revealed extensive molecular changes during the embryonic development of the brain endothelium. We identified genes encoding brain endothelium-specific transcription factors ( Foxf2 , Foxl2 , Foxq1 , Lef1 , Ppard , Zfp551 , and Zic3 ) that are associated with maturation of the blood-brain barrier and act downstream of the Wnt-β-catenin signaling pathway. Profiling of individual brain endothelial cells revealed substantial heterogeneity in the population. Nevertheless, the high abundance of Foxf2 , Foxq1 , Ppard , or Zic3 transcripts correlated with the increased expression of genes encoding markers of brain endothelial cell differentiation. Expression of Foxf2 and Zic3 in human umbilical vein endothelial cells induced the production of blood-brain barrier differentiation markers. This comprehensive data set may help to improve the engineering of in vitro blood-brain barrier models. Copyright © 2017 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

Comparison of molecular marker expression in early zebrafish brain development following chronic ethanol or morpholino treatment.

PubMed

Zhang, Chengjin; Boa-Amponsem, Oswald; Cole, Gregory J

2017-08-01

This study was undertaken to ascertain whether defined markers of early zebrafish brain development are affected by chronic ethanol exposure or morpholino knockdown of agrin, sonic hedgehog, retinoic acid, and fibroblast growth factors, four signaling molecules that are suggested to be ethanol sensitive. Zebrafish embryos were exposed to 2% ethanol from 6 to 24 hpf or injected with agrin, shha, aldh1a3, or fgf8a morpholinos. In situ hybridization was employed to analyze otx2, pax6a, epha4a, krx20, pax2a, fgf8a, wnt1, and eng2b expression during early brain development. Our results showed that pax6a mRNA expression was decreased in eye, forebrain, and hindbrain of both chronic ethanol exposed and select MO treatments. Epha4a expression in rhombomere R1 boundary was decreased in chronic ethanol exposure and aldh1a3 morphants, lost in fgf8a morphants, but largely unaffected in agrin and shha morphants. Ectopic pax6a and epha4a expression in midbrain was only found in fgf8a morphants. These results suggest that while chronic ethanol induces obvious morphological change in brain architecture, many molecular markers of these brain structures are relatively unaffected by ethanol exposure.

Intraperitoneal Exposure to Nano/Microparticles of Fullerene (C60) Increases Acetylcholinesterase Activity and Lipid Peroxidation in Adult Zebrafish (Danio rerio) Brain

PubMed Central

Dal Forno, Gonzalo Ogliari; Kist, Luiza Wilges; de Azevedo, Mariana Barbieri; Fritsch, Rachel Seemann; Pereira, Talita Carneiro Brandão; Britto, Roberta Socoowski; Guterres, Sílvia Stanisçuaski; Külkamp-Guerreiro, Irene Clemes; Bonan, Carla Denise; Monserrat, José María; Bogo, Maurício Reis

2013-01-01

Even though technologies involving nano/microparticles have great potential, it is crucial to determine possible toxicity of these technological products before extensive use. Fullerenes C60 are nanomaterials with unique physicochemical and biological properties that are important for the development of many technological applications. The aim of this study was to evaluate the consequences of nonphotoexcited fullerene C60 exposure in brain acetylcholinesterase expression and activity, antioxidant responses, and oxidative damage using adult zebrafish as an animal model. None of the doses tested (7.5, 15, and 30 mg/kg) altered AChE activity, antioxidant responses, and oxidative damage when zebrafish were exposed to nonphotoexcited C60 nano/microparticles during 6 and 12 hours. However, adult zebrafish exposed to the 30 mg/kg dose for 24 hours have shown enhanced AChE activity and augmented lipid peroxidation (TBARS assays) in brain. In addition, the up-regulation of brain AChE activity was neither related to the transcriptional control (RT-qPCR analysis) nor to the direct action of nonphotoexcited C60 nano/microparticles on the protein (in vitro results) but probably involved a posttranscriptional or posttranslational modulation of this enzymatic activity. Taken together these findings provided further evidence of toxic effects on brain after C60 exposure. PMID:23865059

Developmental Toxicity of Louisiana Crude Oil-Spiked Sediment to Zebrafish

EPA Science Inventory

Embryonic exposures to the components of petroleum, including polycyclic aromatic hydrocarbons (PAHs), cause a characteristic suite of developmental defects and cardiotoxicity in a variety of fish species. We exposed zebrafish embryos to reference sediment mixed with laboratory w...

Colonizing the embryonic zebrafish gut with anaerobic bacteria derived from the human gastrointestinal tract.

PubMed

Toh, Michael C; Goodyear, Mara; Daigneault, Michelle; Allen-Vercoe, Emma; Van Raay, Terence J

2013-06-01

The zebrafish has become increasingly popular for microbiological research. It has been used as an infection model for a variety of pathogens, and is also emerging as a tool for studying interactions between a host and its resident microbial communities. The mouse microbiota has been transplanted into the zebrafish gut, but to our knowledge, there has been no attempt to introduce a bacterial community derived from the human gut. We explored two methods for colonizing the developing gut of 5-day-old germ-free zebrafish larvae with a defined anaerobic microbial community derived from a single human fecal sample. Both environmental exposure (static immersion) and direct microinjection into the gut resulted in the establishment of two species-Lactobacillus paracasei and Eubacterium limosum-from a community of 30 strains consisting of 22 anaerobic species. Of particular interest is E. limosum, which, as a strict anaerobe, represents a group of bacteria which until now have not been shown to colonize the developing zebrafish gut. Our success here indicates that further investigation of zebrafish as a tool for studying human gut microbial communities is warranted.

Whole-brain serial-section electron microscopy in larval zebrafish.

PubMed

Hildebrand, David Grant Colburn; Cicconet, Marcelo; Torres, Russel Miguel; Choi, Woohyuk; Quan, Tran Minh; Moon, Jungmin; Wetzel, Arthur Willis; Scott Champion, Andrew; Graham, Brett Jesse; Randlett, Owen; Plummer, George Scott; Portugues, Ruben; Bianco, Isaac Henry; Saalfeld, Stephan; Baden, Alexander David; Lillaney, Kunal; Burns, Randal; Vogelstein, Joshua Tzvi; Schier, Alexander Franz; Lee, Wei-Chung Allen; Jeong, Won-Ki; Lichtman, Jeff William; Engert, Florian

2017-05-18

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Whole-brain serial-section electron microscopy in larval zebrafish

NASA Astrophysics Data System (ADS)

Hildebrand, David Grant Colburn; Cicconet, Marcelo; Torres, Russel Miguel; Choi, Woohyuk; Quan, Tran Minh; Moon, Jungmin; Wetzel, Arthur Willis; Scott Champion, Andrew; Graham, Brett Jesse; Randlett, Owen; Plummer, George Scott; Portugues, Ruben; Bianco, Isaac Henry; Saalfeld, Stephan; Baden, Alexander David; Lillaney, Kunal; Burns, Randal; Vogelstein, Joshua Tzvi; Schier, Alexander Franz; Lee, Wei-Chung Allen; Jeong, Won-Ki; Lichtman, Jeff William; Engert, Florian

2017-05-01

High-resolution serial-section electron microscopy (ssEM) makes it possible to investigate the dense meshwork of axons, dendrites, and synapses that form neuronal circuits. However, the imaging scale required to comprehensively reconstruct these structures is more than ten orders of magnitude smaller than the spatial extents occupied by networks of interconnected neurons, some of which span nearly the entire brain. Difficulties in generating and handling data for large volumes at nanoscale resolution have thus restricted vertebrate studies to fragments of circuits. These efforts were recently transformed by advances in computing, sample handling, and imaging techniques, but high-resolution examination of entire brains remains a challenge. Here, we present ssEM data for the complete brain of a larval zebrafish (Danio rerio) at 5.5 days post-fertilization. Our approach utilizes multiple rounds of targeted imaging at different scales to reduce acquisition time and data management requirements. The resulting dataset can be analysed to reconstruct neuronal processes, permitting us to survey all myelinated axons (the projectome). These reconstructions enable precise investigations of neuronal morphology, which reveal remarkable bilateral symmetry in myelinated reticulospinal and lateral line afferent axons. We further set the stage for whole-brain structure-function comparisons by co-registering functional reference atlases and in vivo two-photon fluorescence microscopy data from the same specimen. All obtained images and reconstructions are provided as an open-access resource.

Electrochemical quantification of serotonin in the live embryonic zebrafish intestine

PubMed Central

Njagi, John; Ball, Michael; Best, Marc; Wallace, Kenneth N.; Andreescu, Silvana

2010-01-01

We monitored real-time in vivo levels of serotonin release in the digestive system of intact zebrafish embryos during early development (5 dpf) using differential pulse voltammetry with implanted carbon fiber microelectrodes modified with carbon nanotubes dispersed in nafion. A detection limit of 1 nM, a linear range between 5 to 200 nM and a sensitivity of 83.65 nA·μM−1 were recorded. The microelectrodes were implanted at various locations in the intestine of zebrafish embryos. Serotonin levels of up to 29.9(±1.13) nM were measured in vivo in normal physiological conditions. Measurements were performed in intact live embryos without additional perturbation beyond electrode insertion. The sensor was able to quantify pharmacological alterations in serotonin release and provide the longitudinal distribution of this neurotransmitter along the intestine with high spatial resolution. In the presence of fluvoxamine, a selective serotonin reuptake inhibitor (SSRI), concentrations of 54.1(±1.05) nM were recorded while in the presence of p-chloro-phenylalanine (PCPA), a tryptophan hydroxylase inhibitor, the serotonin levels decreased to 7.2(±0.45) nM. The variation of serotonin levels was correlated with immunohistochemical analysis. We have demonstrated the first use of electrochemical microsensors for in vivo monitoring of intestinal serotonin levels in intact zebrafish embryos. PMID:20148518

Nicotine increases fear responses and brain acetylcholinesterase activity in a context-dependent manner in zebrafish.

PubMed

Ziani, Paola R; Müller, Talise E; Stefanello, Flavia V; Fontana, Barbara D; Duarte, Tâmie; Canzian, Julia; Rosemberg, Denis B

2018-07-01

Nicotine is an alkaloid with positive effects on learning and memory processes. Exposure to conspecific alarm substance (CAS) elicits fear responses in zebrafish, but the effects of nicotine on aversive behaviors and associative learning in this species remain unclear. Here, we evaluated whether nicotine enhances contextual fear responses in zebrafish and investigated a putative involvement of brain acetylcholinesterase (AChE) in associative learning. Fish were exposed to 1 mg/L nicotine for 3 min and then kept in non-chlorinated water for 20 min. Later, animals were transferred to experimental tanks in the absence or presence of 3.5 mL/L CAS for 5 min (training session). After 24 h, fish were tested in tanks with similar or altered context in the absence of CAS (post-training session) and brain AChE activity was further assessed. At training, CAS increased freezing, erratic movements, and decreased the time spent in top area, while nicotine abolished the effects of CAS on erratic movements. Nicotine/CAS group tested in a similar context showed exacerbated freezing and reduced transitions to top area. Moreover, a decrease in distance traveled was observed in control, nicotine, and nicotine/CAS groups at post-training. Nicotine also stimulated brain AChE activity in CAS-exposed animals reintroduced in tanks with similar context. Although freezing bouts and time spent in top could serve as behavioral endpoints that reflect CAS-induced sensitization, the effects of nicotine occurred in a context-dependent manner. Collectively, our data suggest an involvement of cholinergic signaling in aversive learning, reinforcing the growing utility of zebrafish models to explore the neurobehavioral effects of nicotine in vertebrates. Copyright © 2018 Elsevier Inc. All rights reserved.

Development of the zebrafish mesonephros.

PubMed

Diep, Cuong Q; Peng, Zhenzhen; Ukah, Tobechukwu K; Kelly, Paul M; Daigle, Renee V; Davidson, Alan J

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. © 2015 Wiley Periodicals, Inc.

Development of the zebrafish mesonephros

PubMed Central

Diep, Cuong Q.; Peng, Zhenzhen; Ukah, Tobechukwu K.; Kelly, Paul M.; Daigle, Renee V.; Davidson, Alan J.

2015-01-01

The vertebrate kidney plays an essential role in removing metabolic waste and balancing water and salt. This is carried out by nephrons, which comprise a blood filter attached to an epithelial tubule with proximal and distal segments. In zebrafish, two nephrons are first formed as part of the embryonic kidney (pronephros) and hundreds are formed later to make up the adult kidney (mesonephros). Previous studies have focused on the development of the pronephros while considerably less is known about how the mesonephros is formed. Here, we characterize mesonephros development in zebrafish and examine the nephrons that form during larval metamorphosis. These nephrons, arising from proliferating progenitor cells that express the renal transcription factor genes wt1b, pax2a, and lhx1a, form on top of the pronephric tubules and develop a segmentation pattern similar to pronephric nephrons. We find that the pronephros acts as a scaffold for the mesonephros, where new nephrons fuse with the distal segments of the pronephric tubules to form the final branching network that characterizes the adult zebrafish kidney. PMID:25677367

A review of monoaminergic neuropsychopharmacology in zebrafish.

PubMed

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.

Direct measurement of local material properties within living embryonic tissues

NASA Astrophysics Data System (ADS)

Serwane, Friedhelm; Mongera, Alessandro; Rowghanian, Payam; Kealhofer, David; Lucio, Adam; Hockenbery, Zachary; Campàs, Otger

The shaping of biological matter requires the control of its mechanical properties across multiple scales, ranging from single molecules to cells and tissues. Despite their relevance, measurements of the mechanical properties of sub-cellular, cellular and supra-cellular structures within living embryos pose severe challenges to existing techniques. We have developed a technique that uses magnetic droplets to measure the mechanical properties of complex fluids, including in situ and in vivo measurements within living embryos ,across multiple length and time scales. By actuating the droplets with magnetic fields and recording their deformation we probe the local mechanical properties, at any length scale we choose by varying the droplets' diameter. We use the technique to determine the subcellular mechanics of individual blastomeres of zebrafish embryos, and bridge the gap to the tissue scale by measuring the local viscosity and elasticity of zebrafish embryonic tissues. Using this technique, we show that embryonic zebrafish tissues are viscoelastic with a fluid-like behavior at long time scales. This technique will enable mechanobiology and mechano-transduction studies in vivo, including the study of diseases correlated with tissue stiffness, such as cancer.

Zebrafish knockout of Down syndrome gene, DYRK1A, shows social impairments relevant to autism.

PubMed

Kim, Oc-Hee; Cho, Hyun-Ju; Han, Enna; Hong, Ted Inpyo; Ariyasiri, Krishan; Choi, Jung-Hwa; Hwang, Kyu-Seok; Jeong, Yun-Mi; Yang, Se-Yeol; Yu, Kweon; Park, Doo-Sang; Oh, Hyun-Woo; Davis, Erica E; Schwartz, Charles E; Lee, Jeong-Soo; Kim, Hyung-Goo; Kim, Cheol-Hee

2017-01-01

DYRK1A maps to the Down syndrome critical region at 21q22. Mutations in this kinase-encoding gene have been reported to cause microcephaly associated with either intellectual disability or autism in humans. Intellectual disability accompanied by microcephaly was recapitulated in a murine model by overexpressing Dyrk1a which mimicked Down syndrome phenotypes. However, given embryonic lethality in homozygous knockout (KO) mice, no murine model studies could present sufficient evidence to link Dyrk1a dysfunction with autism. To understand the molecular mechanisms underlying microcephaly and autism spectrum disorders (ASD), we established an in vivo dyrk1aa KO model using zebrafish. We identified a patient with a mutation in the DYRK1A gene using microarray analysis. Circumventing the barrier of murine model studies, we generated a dyrk1aa KO zebrafish using transcription activator-like effector nuclease (TALEN)-mediated genome editing. For social behavioral tests, we have established a social interaction test, shoaling assay, and group behavior assay. For molecular analysis, we examined the neuronal activity in specific brain regions of dyrk1aa KO zebrafish through in situ hybridization with various probes including c-fos and crh which are the molecular markers for stress response. Microarray detected an intragenic microdeletion of DYRK1A in an individual with microcephaly and autism. From behavioral tests of social interaction and group behavior, dyrk1aa KO zebrafish exhibited social impairments that reproduce human phenotypes of autism in a vertebrate animal model. Social impairment in dyrk1aa KO zebrafish was further confirmed by molecular analysis of c-fos and crh expression. Transcriptional expression of c-fos and crh was lower than that of wild type fish in specific hypothalamic regions, suggesting that KO fish brains are less activated by social context. In this study, we established a zebrafish model to validate a candidate gene for autism in a vertebrate

Suppression of the endoplasmic reticulum calcium pump during zebrafish gastrulation affects left-right asymmetry of the heart and brain.

PubMed

Kreiling, Jill A; Balantac, Zaneta L; Crawford, Andrew R; Ren, Yuexin; Toure, Jamal; Zchut, Sigalit; Kochilas, Lazaros; Creton, Robbert

2008-01-01

Vertebrate embryos generate striking Ca(2+) patterns, which are unique regulators of dynamic developmental events. In the present study, we used zebrafish embryos as a model system to examine the developmental roles of Ca(2+) during gastrulation. We found that gastrula stage embryos maintain a distinct pattern of cytosolic Ca(2+) along the dorsal-ventral axis, with higher Ca(2+) concentrations in the ventral margin and lower Ca(2+) concentrations in the dorsal margin and dorsal forerunner cells. Suppression of the endoplasmic reticulum Ca(2+) pump with 0.5 microM thapsigargin elevates cytosolic Ca(2+) in all embryonic regions and induces a randomization of laterality in the heart and brain. Affected hearts, visualized in living embryos by a subtractive imaging technique, displayed either a reversal or loss of left-right asymmetry. Brain defects include a left-right reversal of pitx2 expression in the dorsal diencephalon and a left-right reversal of the prominent habenular nucleus in the brain. Embryos are sensitive to inhibition of the endoplasmic reticulum Ca(2+) pump during early and mid gastrulation and lose their sensitivity during late gastrulation and early segmentation. Suppression of the endoplasmic reticulum Ca(2+) pump during gastrulation inhibits expression of no tail (ntl) and left-right dynein related (lrdr) in the dorsal forerunner cells and affects development of Kupffer's vesicle, a ciliated organ that generates a counter-clockwise flow of fluid. Previous studies have shown that Ca(2+) plays a role in Kupffer's vesicle function, influencing ciliary motility and translating the vesicle's counter-clockwise flow into asymmetric patterns of gene expression. The present results suggest that Ca(2+) plays an additional role in the formation of Kupffer's vesicle.

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions.

PubMed

Lindsey, Benjamin W; Douek, Alon M; Loosli, Felix; Kaslin, Jan

2017-01-01

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish ( Danio rerio ) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2'-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a

A Whole Brain Staining, Embedding, and Clearing Pipeline for Adult Zebrafish to Visualize Cell Proliferation and Morphology in 3-Dimensions

PubMed Central

Lindsey, Benjamin W.; Douek, Alon M.; Loosli, Felix; Kaslin, Jan

2018-01-01

The field of macro-imaging has grown considerably with the appearance of innovative clearing methods and confocal microscopes with lasers capable of penetrating increasing tissue depths. The ability to visualize and model the growth of whole organs as they develop from birth, or with manipulation, disease or injury, provides new ways of thinking about development, tissue-wide signaling, and cell-to-cell interactions. The zebrafish (Danio rerio) has ascended from a predominantly developmental model to a leading adult model of tissue regeneration. The unmatched neurogenic and regenerative capacity of the mature central nervous system, in particular, has received much attention, however tools to interrogate the adult brain are sparse. At present there exists no straightforward methods of visualizing changes in the whole adult brain in 3-dimensions (3-D) to examine systemic patterns of cell proliferation or cell populations of interest under physiological, injury, or diseased conditions. The method presented here is the first of its kind to offer an efficient step-by-step pipeline from intraperitoneal injections of the proliferative marker, 5-ethynyl-2′-deoxyuridine (EdU), to whole brain labeling, to a final embedded and cleared brain sample suitable for 3-D imaging using optical projection tomography (OPT). Moreover, this method allows potential for imaging GFP-reporter lines and cell-specific antibodies in the presence or absence of EdU. The small size of the adult zebrafish brain, the highly consistent degree of EdU labeling, and the use of basic clearing agents, benzyl benzoate, and benzyl alcohol, makes this method highly tractable for most laboratories interested in understanding the vertebrate central nervous system in health and disease. Post-processing of OPT-imaged adult zebrafish brains injected with EdU illustrate that proliferative patterns in EdU can readily be observed and analyzed using IMARIS and/or FIJI/IMAGEJ software. This protocol will be a

Response mechanisms to joint exposure of triclosan and its chlorinated derivatives on zebrafish (Danio rerio) behavior.

PubMed

Liu, Jinfeng; Sun, Limei; Zhang, Hongqin; Shi, Mengru; Dahlgren, Randy A; Wang, Xuedong; Wang, Huili

2018-02-01

Triclosan (TCS), 2,4,6-trichlorophenol (2,4,6-TCP) and 2,4-dichlorophenol (2,4-DCP) frequently co-exist in real-world aquatic environments; the latter two contaminants contributing to TCS photolytic products or chlorinated derivatives. There is a paucity of information regarding their joint toxicity to aquatic organisms leading us to study their effects on the swimming behavior of zebrafish (Danio rerio). Herein, we reported that 0.28 mg/L TDT exposure (mixtures of TCS, 2,4,6-TCP and 2,4-DCP) enhanced 24-hpf embryonic spontaneous movement frequency, 96-hpf larval activity; however, the 0.56 and 1.12 mg/L TDT treatments decreased all of these behavioral endpoints. All adult behavioral tests demonstrated that chronic TDT exposure (0.14 mg/L) led to hyperactivity and restlessness in adult zebrafish. A 0.14 mg/L TD DATE /@ "M/d/yyyy" 11/21/2017T treatment led to anxiety-like behavior in a bottom dwelling test and excessive panic and low hedging capacity in a conditioned place preference test. Social interaction test demonstrated that zebrafish preferred quiet and isolated space in response to TDT stress. Zebrafish memory was significantly decreased in a T-maze experiment. Whole mount in situ hybridization of pax2a and bcl2l11 genes revealed that their differential expression in the brain and skeleton were related to the corresponding phenotypic behavioral abnormality. A series of biomarker and estrogen receptor assays demonstrated that TDT acute exposure caused abnormal energy metabolism and neurological diseases. AO staining revealed that TDT exposure produced vascular ablation in the head, as well as the occurrence of massive apoptosis in the brain. TEM observation showed pyknosis of nucleus following TDT exposure. These results allow assessment of mechanisms for zebrafish abnormal behavior in response to TDT exposure, and are useful for early intervention and gene therapy of contaminant-induced diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

Next generation mothers: Maternal control of germline development in zebrafish.

PubMed

Dosch, Roland

2015-01-01

In many animals, factors deposited by the mother into the egg control the earliest events in development of the zygote. These maternal RNAs and proteins play critical roles in oocyte development and the earliest steps of embryogenesis such as fertilization, cell division and embryonic patterning. Here, this article summarizes recent discoveries made on the maternal control of germline specification in zebrafish. Moreover, this review will discuss the major gaps remaining in our understanding of this process and highlight recent technical innovations in zebrafish, which allow tackling some of these questions in the near future.

ZebraBeat: a flexible platform for the analysis of the cardiac rate in zebrafish embryos

NASA Astrophysics Data System (ADS)

de Luca, Elisa; Zaccaria, Gian Maria; Hadhoud, Marwa; Rizzo, Giovanna; Ponzini, Raffaele; Morbiducci, Umberto; Santoro, Massimo Mattia

2014-05-01

Heartbeat measurement is important in assesssing cardiac function because variations in heart rhythm can be the cause as well as an effect of hidden pathological heart conditions. Zebrafish (Danio rerio) has emerged as one of the most useful model organisms for cardiac research. Indeed, the zebrafish heart is easily accessible for optical analyses without conducting invasive procedures and shows anatomical similarity to the human heart. In this study, we present a non-invasive, simple, cost-effective process to quantify the heartbeat in embryonic zebrafish. To achieve reproducibility, high throughput and flexibility (i.e., adaptability to any existing confocal microscope system and with a user-friendly interface that can be easily used by researchers), we implemented this method within a software program. We show here that this platform, called ZebraBeat, can successfully detect heart rate variations in embryonic zebrafish at various developmental stages, and it can record cardiac rate fluctuations induced by factors such as temperature and genetic- and chemical-induced alterations. Applications of this methodology may include the screening of chemical libraries affecting heart rhythm and the identification of heart rhythm variations in mutants from large-scale forward genetic screens.

The neurogenetic frontier--lessons from misbehaving zebrafish.

PubMed

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.

MiR-145 mediates zebrafish hepatic outgrowth through progranulin A signaling

PubMed Central

Li, Ya-Wen; Chiang, Keng-Yu; Li, Yen-Hsing; Wu, Sung-Yu; Liu, Wangta; Lin, Chia-Ray

2017-01-01

MicroRNAs (miRs) are mRNA-regulatory molecules that fine-tune gene expression and modulate both processes of development and tumorigenesis. Our previous studies identified progranulin A (GrnA) as a growth factor which induces zebrafish hepatic outgrowth through MET signaling. We also found that miR-145 is one of potential fine-tuning regulators of GrnA involved in embryonic hepatic outgrowth. The low level of miR-145 seen in hepatocarinogenesis has been shown to promote pathological liver growth. However, little is known about the regulatory mechanism of miR-145 in embryonic liver development. In this study, we demonstrate a significant decrease in miR-145 expression during hepatogenesis. We modulate miR-145 expression in zebrafish embryos by injection with a miR-145 mimic or a miR-145 hairpin inhibitor. Altered embryonic liver outgrowth is observed in response to miR-145 expression modulation. We also confirm a critical role of miR-145 in hepatic outgrowth by using whole-mount in situ hybridization. Loss of miR-145 expression in embryos results in hepatic cell proliferation, and vice versa. Furthermore, we demonstrate that GrnA is a target of miR-145 and GrnA-induced MET signaling is also regulated by miR-145 as determined by luciferase reporter assay and gene expression analysis, respectively. In addition, co-injection of GrnA mRNA with miR-145 mimic or MO-GrnA with miR-145 inhibitor restores the liver defects caused by dysregulation of miR-145 expression. In conclusion, our findings suggest an important role of miR-145 in regulating GrnA-dependent hepatic outgrowth in zebrafish embryonic development. PMID:28531199

Embryonic exposure to sodium arsenite perturbs vascular development in zebrafish.

PubMed

McCollum, Catherine W; Hans, Charu; Shah, Shishir; Merchant, Fatima A; Gustafsson, Jan-Åke; Bondesson, Maria

2014-07-01

Exposure to arsenic in its inorganic form, arsenite, causes adverse effects to many different organs and tissues. Here, we have investigated arsenite-induced adverse effects on vascular tissues in the model organism zebrafish, Danio rerio. Zebrafish embryos were exposed to arsenite at different exposure windows and the susceptibility to vascular tissue damage was recorded at 72hours post fertilization (hpf). Intersegmental vessel sprouting and growth was most perturbed by exposure to arsenite during the 24-48hpf window, while disruption in the condensation of the caudal vein plexus was more often observed at the 48-72hpf exposure window, reflecting when these structures develop during normal embryogenesis. The vascular growth rate was decreased by arsenite exposure, and deviated from that of control embryos at around 24-26.5hpf. We further mapped changes in expression of key regulators of angiogenesis and vasculogenesis. Downregulation of vascular endothelial growth factor receptor 1/fms-related tyrosine kinase 1 (vegfr1/flt1) expression was evident already at 24hpf, coinciding with the decreased vascular growth rate. At later time points, matrix metalloproteinase 9 (mmp9) expression was upregulated, suggesting that arsenite affects the composition of the extracellular matrix. In total, the expression of eight key factors involved in different aspects of vascularization was significantly altered by arsenic exposure. In conclusion, our results show that arsenite is a potent vascular disruptor in the developing zebrafish embryo, a finding that calls for an evaluation of arsenite as a developmental vascular toxicant in mammalian model systems. Copyright © 2014 Elsevier B.V. All rights reserved.

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

PubMed

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.

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

PubMed

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.

The role of Fanconi anemia/BRCA genes in zebrafish sex determination.

PubMed

Rodríguez-Marí, Adriana; Postlethwait, John H

2011-01-01

Fanconi anemia (FA) is a human disease of bone marrow failure, leukemia, squamous cell carcinoma, and developmental anomalies, including hypogonadism and infertility. Bone marrow transplants improve hematopoietic phenotypes but do not prevent other cancers. FA arises from mutation in any of the 15 FANC genes that cooperate to repair double stranded DNA breaks by homologous recombination. Zebrafish has a single ortholog of each human FANC gene and unexpectedly, mutations in at least two of them (fancl and fancd1(brca2)) lead to female-to-male sex reversal. Investigations show that, as in human, zebrafish fanc genes are required for genome stability and for suppressing apoptosis in tissue culture cells, in embryos treated with DNA damaging agents, and in meiotic germ cells. The sex reversal phenotype requires the action of Tp53 (p53), an activator of apoptosis. These results suggest that in normal sex determination, zebrafish oocytes passing through meiosis signal the gonadal soma to maintain expression of aromatase, an enzyme that converts androgen to estrogen, thereby feminizing the gonad and the individual. According to this model, normal male and female zebrafish differ in genetic factors that control the strength of the late meiotic oocyte-derived signal, probably by regulating the number of meiotic oocytes, which environmental factors can also alter. Transcripts from fancd1(brca2) localize at the animal pole of the zebrafish oocyte cytoplasm and are required for normal oocyte nuclear architecture, for normal embryonic development, and for preventing ovarian tumors. Embryonic DNA repair and sex reversal phenotypes provide assays for the screening of small molecule libraries for therapeutic substances for FA. Copyright © 2011 Elsevier Inc. All rights reserved.

Gene expression changes in female zebrafish (Danio rerio) brain in response to acute exposure to methylmercury

USGS Publications Warehouse

Richter, Catherine A.; Garcia-Reyero, Natàlia; Martyniuk, Chris; Knoebl, Iris; Pope, Marie; Wright-Osment, Maureen K.; Denslow, Nancy D.; Tillitt, Donald E.

2011-01-01

Methylmercury (MeHg) is a potent neurotoxicant and endocrine disruptor that accumulates in aquatic systems. Previous studies have shown suppression of hormone levels in both male and female fish, suggesting effects on gonadotropin regulation in the brain. The gene expression profile in adult female zebrafish whole brain induced by acute (96 h) MeHg exposure was investigated. Fish were exposed by injection to 0 or 0.5(mu or u)g MeHg/g. Gene expression changes in the brain were examined using a 22,000-feature zebrafish microarray. At a significance level of pbrain. Future studies will compare the gene expression profile induced in response to MeHg with that induced by other toxicants and will investigate responsive genes as potential biomarkers of MeHg exposure.

Elevated nitrate alters the metabolic activity of embryonic zebrafish.

PubMed

Conlin, Sarah M; Tudor, M Scarlett; Shim, Juyoung; Gosse, Julie A; Neilson, Andrew; Hamlin, Heather J

2018-04-01

Nitrate accumulation in aquatic reservoirs from agricultural pollution has often been overlooked as a water quality hazard, yet a growing body of literature suggests negative effects on human and wildlife health following nitrate exposure. This research seeks to understand differences in oxygen consumption rates between different routes of laboratory nitrate exposure, whether via immersion or injection, in zebrafish (Danio rerio) embryos. Embryos were exposed within 1 h post fertilization (hpf) to 0, 10, and 100 mg/L NO 3 -N with sodium nitrate, or to counter ion control (CIC) treatments using sodium chloride. Embryos in the immersion treatments received an injection of 4 nL of appropriate treatment solution into the perivitelline space. At 24 hpf, Oxygen Consumption Rates (OCR) were measured and recorded in vivo using the Agilent Technologies XF e 96 Extracellular Flux Analyzer and Spheroid Microplate. Immersion exposures did not induce significant changes in OCR, yet nitrate induced significant changes when injected through the embryo chorion. Injection of 10 and 100 mg/L NO 3 -N down-regulated OCR compared to the control treatment group. Injection of the 100 mg/L CIC also significantly down-regulated OCR compared to the control treatment group. Interestingly, the 100 mg/L NO 3 -N treatment further down-regulated OCR compared to the 100 mg/L CIC treatment, suggesting the potential for additive effects between the counter ion and the ion of interest. These data support that elevated nitrate exposure can alter normal metabolic activity by changing OCR in 24 hpf embryos. These results highlight the need for regularly examining the counter ion of laboratory nitrate compounds while conducting research with developing zebrafish, and justify examining different routes of laboratory nitrate exposure, as the chorion may act as an effective barrier to nitrate penetration in zebrafish, which may lead to conservative estimates of significant effects in other

Zebrafish: an important tool for liver disease research.

PubMed

Goessling, Wolfram; Sadler, Kirsten C

2015-11-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

Zebrafish: An Important Tool for Liver Disease Research

PubMed Central

Goessling, Wolfram; Sadler, Kirsten C.

2016-01-01

As the incidence of hepatobiliary diseases increases, we must improve our understanding of the molecular, cellular, and physiological factors that contribute to the pathogenesis of liver disease. Animal models help us identify disease mechanisms that might be targeted therapeutically. Zebrafish (Danio rerio) have traditionally been used to study embryonic development but are also important to the study of liver disease. Zebrafish embryos develop rapidly; all of their digestive organs are mature in larvae by 5 days of age. At this stage, they can develop hepatobiliary diseases caused by developmental defects or toxin- or ethanol-induced injury and manifest premalignant changes within weeks. Zebrafish are similar to humans in hepatic cellular composition, function, signaling, and response to injury as well as the cellular processes that mediate liver diseases. Genes are highly conserved between humans and zebrafish, making them a useful system to study the basic mechanisms of liver disease. We can perform genetic screens to identify novel genes involved in specific disease processes and chemical screens to identify pathways and compounds that act on specific processes. We review how studies of zebrafish have advanced our understanding of inherited and acquired liver diseases as well as liver cancer and regeneration. PMID:26319012

The neurogenetic frontier—lessons from misbehaving zebrafish

PubMed Central

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

Neurobehavioural Changes and Brain Oxidative Stress Induced by Acute Exposure to GSM900 Mobile Phone Radiations in Zebrafish (Danio rerio)

PubMed Central

Nirwane, Abhijit; Sridhar, Vinay; Majumdar, Anuradha

2016-01-01

The impact of mobile phone (MP) radiation on the brain is of specific interest to the scientific community and warrants investigations, as MP is held close to the head. Studies on humans and rodents revealed hazards MP radiation associated such as brain tumors, impairment in cognition, hearing etc. Melatonin (MT) is an important modulator of CNS functioning and is a neural antioxidant hormone. Zebrafish has emerged as a popular model organism for CNS studies. Herein, we evaluated the impact of GSM900MP (GSM900MP) radiation exposure daily for 1 hr for 14 days with the SAR of 1.34W/Kg on neurobehavioral and oxidative stress parameters in zebrafish. Our study revealed that, GSM900MP radiation exposure, significantly decreased time spent near social stimulus zone and increased total distance travelled, in social interaction test. In the novel tank dive test, the GSM900MP radiation exposure elicited anxiety as revealed by significantly increased time spent in bottom half; freezing bouts and duration and decreased distance travelled, average velocity, and number of entries to upper half of the tank. Exposed zebrafish spent less time in the novel arm of the Y-Maze, corroborating significant impairment in learning as compared to the control group. Exposure decreased superoxide dismutase (SOD), catalase (CAT) activities whereas, increased levels of reduced glutathione (GSH) and lipid peroxidation (LPO) was encountered showing compromised antioxidant defense. Treatment with MT significantly reversed the above neurobehavioral and oxidative derangements induced by GSM900MP radiation exposure. This study traced GSM900MP radiation exposure induced neurobehavioral aberrations and alterations in brain oxidative status. Furthermore, MT proved to be a promising therapeutic candidate in ameliorating such outcomes in zebrafish. PMID:27123163

Neurobehavioral impairments produced by developmental lead exposure persisted for generations in zebrafish (Danio rerio).

PubMed

Xu, Xiaojuan; Weber, Daniel; Burge, Rebekah; VanAmberg, Kelsey

2016-01-01

The zebrafish has become a useful animal model for studying the effects of environmental contaminants on neurobehavioral development due to its ease of breeding, high number of eggs per female, short generation times, and a well-established avoidance conditioning paradigm. Using avoidance conditioning as the behavioral paradigm, the present study investigated the effects of embryonic exposure to lead (Pb) on learning in adult zebrafish and the third (F3) generation of those fish. In Experiment 1, adult zebrafish that were developmentally exposed to 0.0, 0.1, 1.0 or 10.0μM Pb (2-24h post fertilization) as embryos were trained and tested for avoidance responses. The results showed that adult zebrafish hatched from embryos exposed to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while those hatched from embryos exposed to 1.0 or 10.0μM Pb displayed no significant increases in avoidance responses from training to testing. In Experiment 2, the F3 generation of zebrafish that were developmentally exposed to an identical exposure regimen as in Experiment 1 were trained and tested for avoidance responses. The results showed that the F3 generation of zebrafish developmentally exposed as embryos to 0.0 or 0.1μM Pb learned avoidance responses during training and displayed significantly increased avoidance responses during testing, while the F3 generation of zebrafish developmentally exposed as embryos to 1.0 or 10.0μM Pb displayed no significant changes in avoidance responses from training to testing. Thus, developmental Pb exposure produced learning impairments that persisted for at least three generations, demonstrating trans-generational effects of embryonic exposure to Pb. Copyright © 2015. Published by Elsevier B.V.

Using Zebrafish to Study Podocyte Genesis During Kidney Development and Regeneration

PubMed Central

Kroeger, Paul T.; Wingert, Rebecca A.

2014-01-01

SUMMARY During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. PMID:24920186

In Vivo Assessment of Drug Efficacy against Mycobacterium abscessus Using the Embryonic Zebrafish Test System

PubMed Central

Bernut, Audrey; Le Moigne, Vincent; Lesne, Tiffany; Lutfalla, Georges; Herrmann, Jean-Louis

2014-01-01

Mycobacterium abscessus is responsible for a wide spectrum of clinical syndromes and is one of the most intrinsically drug-resistant mycobacterial species. Recent evaluation of the in vivo therapeutic efficacy of the few potentially active antibiotics against M. abscessus was essentially performed using immunocompromised mice. Herein, we assessed the feasibility and sensitivity of fluorescence imaging for monitoring the in vivo activity of drugs against acute M. abscessus infection using zebrafish embryos. A protocol was developed where clarithromycin and imipenem were directly added to water containing fluorescent M. abscessus-infected embryos in a 96-well plate format. The status of the infection with increasing drug concentrations was visualized on a spatiotemporal level. Drug efficacy was assessed quantitatively by measuring the index of protection, the bacterial burden (CFU), and the number of abscesses through fluorescence measurements. Both drugs were active in infected embryos and were capable of significantly increasing embryo survival in a dose-dependent manner. Protection from bacterial killing correlated with restricted mycobacterial growth in the drug-treated larvae and with reduced pathophysiological symptoms, such as the number of abscesses within the brain. In conclusion, we present here a new and efficient method for testing and compare the in vivo activity of two clinically relevant drugs based on a fluorescent reporter strain in zebrafish embryos. This approach could be used for rapid determination of the in vivo drug susceptibility profile of clinical isolates and to assess the preclinical efficacy of new compounds against M. abscessus. PMID:24798271

Comparative toxicological assessment of PAMAM and thiophosphoryl dendrimers using embryonic zebrafish

PubMed Central

Pryor, Joseph B; Harper, Bryan J; Harper, Stacey L

2014-01-01

Dendrimers are well-defined, polymeric nanomaterials currently being investigated for biomedical applications such as medical imaging, gene therapy, and tissue targeted therapy. Initially, higher generation (size) dendrimers were of interest because of their drug carrying capacity. However, increased generation was associated with increased toxicity. The majority of studies exploring dendrimer toxicity have focused on a small range of materials using cell culture methods, with few studies investigating the toxicity across a wide range of materials in vivo. The objective of the present study was to investigate the role of surface charge and generation in dendrimer toxicity using embryonic zebrafish (Danio rerio) as a model vertebrate. Due to the generational and charge effects observed at the cellular level, higher generation cationic dendrimers were hypothesized to be more toxic than lower generation anionic or neutral dendrimers with the same core composition. Polyamidoamine (PAMAM) dendrimers elicited significant morbidity and mortality as generation was decreased. No significant adverse effects were observed from the suite of thiophosphoryl dendrimers studied. Exposure to ≥50 ppm cationic PAMAM dendrimers G3-amine, G4-amine, G5-amine, and G6-amine caused 100% mortality by 24 hours post-fertilization. Cationic PAMAM G6-amine at 250 ppm was found to be statistically more toxic than both neutral PAMAM G6-amidoethanol and anionic PAMAM G6-succinamic acid at the same concentration. The toxicity observed within the suite of varying dendrimers provides evidence that surface charge may be the best indicator of dendrimer toxicity. Dendrimer class and generation are other potential contributors to the toxicity of dendrimers. Further studies are required to better understand the relative role each plays in driving the toxicity of dendrimers. To the best of our knowledge, this is the first in vivo study to address such a broad range of dendrimers. PMID:24790436

It's time to swim! Zebrafish and the circadian clock.

PubMed

Vatine, Gad; Vallone, Daniela; Gothilf, Yoav; Foulkes, Nicholas S

2011-05-20

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research. Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

Subchronic atrazine exposure changes defensive behaviour profile and disrupts brain acetylcholinesterase activity of zebrafish.

PubMed

Schmidel, Ademir J; Assmann, Karla L; Werlang, Chariane C; Bertoncello, Kanandra T; Francescon, Francini; Rambo, Cassiano L; Beltrame, Gabriela M; Calegari, Daiane; Batista, Cibele B; Blaser, Rachel E; Roman Júnior, Walter A; Conterato, Greicy M M; Piato, Angelo L; Zanatta, Leila; Magro, Jacir Dal; Rosemberg, Denis B

2014-01-01

Animal behaviour is the interaction between environment and an individual organism, which also can be influenced by its neighbours. Variations in environmental conditions, as those caused by contaminants, may lead to neurochemical impairments altering the pattern of the behavioural repertoire of the species. Atrazine (ATZ) is an herbicide widely used in agriculture that is frequently detected in surface water, affecting non-target species. The zebrafish is a valuable model organism to assess behavioural and neurochemical effects of different contaminants since it presents a robust behavioural repertoire and also all major neurotransmitter systems described for mammalian species. The goal of this study was to evaluate the effects of subchronic ATZ exposure in defensive behaviours of zebrafish (shoaling, thigmotaxis, and depth preference) using the split depth tank. Furthermore, to investigate a putative role of cholinergic signalling on ATZ-mediated effects, we tested whether this herbicide alters acetylcholinesterase (AChE) activity in brain and muscle preparations. Fish were exposed to ATZ for 14days and the following groups were tested: control (0.2% acetone) and ATZ (10 and 1000μg/L). The behaviour of four animals in the same tank was recorded for 6min and biological samples were prepared. Our results showed that 1000μg/L ATZ significantly increased the inter-fish distance, as well as the nearest and farthest neighbour distances. This group also presented an increase in the shoal area with decreased social interaction. No significant differences were detected for the number of animals in the shallow area, latency to enter the shallow and time spent in shallow and deep areas of the apparatus, but the ATZ 1000 group spent significantly more time near the walls. Although ATZ did not affect muscular AChE, it significantly reduced AChE activity in brain. Exposure to 10μg/L ATZ did not affect behaviour or AChE activity. These data suggest that ATZ impairs defensive

Cardiac Development in Zebrafish and Human Embryonic Stem Cells Is Inhibited by Exposure to Tobacco Cigarettes and E-Cigarettes

PubMed Central

Palpant, Nathan J.; Hofsteen, Peter; Pabon, Lil; Reinecke, Hans; Murry, Charles E.

2015-01-01

Background Maternal smoking is a risk factor for low birth weight and other adverse developmental outcomes. Objective We sought to determine the impact of standard tobacco cigarettes and e-cigarettes on heart development in vitro and in vivo. Methods 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. Results 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. Conclusion 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. PMID:25978043

An assay for lateral line regeneration in adult zebrafish.

PubMed

Pisano, Gina C; Mason, Samantha M; Dhliwayo, Nyembezi; Intine, Robert V; Sarras, Michael P

2014-04-08

Due to the clinical importance of hearing and balance disorders in man, model organisms such as the zebrafish have been used to study lateral line development and regeneration. The zebrafish is particularly attractive for such studies because of its rapid development time and its high regenerative capacity. To date, zebrafish studies of lateral line regeneration have mainly utilized fish of the embryonic and larval stages because of the lower number of neuromasts at these stages. This has made quantitative analysis of lateral line regeneration/and or development easier in the earlier developmental stages. Because many zebrafish models of neurological and non-neurological diseases are studied in the adult fish and not in the embryo/larvae, we focused on developing a quantitative lateral line regenerative assay in adult zebrafish so that an assay was available that could be applied to current adult zebrafish disease models. Building on previous studies by Van Trump et al. that described procedures for ablation of hair cells in adult Mexican blind cave fish and zebrafish (Danio rerio), our assay was designed to allow quantitative comparison between control and experimental groups. This was accomplished by developing a regenerative neuromast standard curve based on the percent of neuromast reappearance over a 24 hr time period following gentamicin-induced necrosis of hair cells in a defined region of the lateral line. The assay was also designed to allow extension of the analysis to the individual hair cell level when a higher level of resolution is required.

IL4/STAT6 Signaling Activates Neural Stem Cell Proliferation and Neurogenesis upon Amyloid-β42 Aggregation in Adult Zebrafish Brain.

PubMed

Bhattarai, Prabesh; Thomas, Alvin Kuriakose; Cosacak, Mehmet Ilyas; Papadimitriou, Christos; Mashkaryan, Violeta; Froc, Cynthia; Reinhardt, Susanne; Kurth, Thomas; Dahl, Andreas; Zhang, Yixin; Kizil, Caghan

2016-10-18

Human brains are prone to neurodegeneration, given that endogenous neural stem/progenitor cells (NSPCs) fail to support neurogenesis. To investigate the molecular programs potentially mediating neurodegeneration-induced NSPC plasticity in regenerating organisms, we generated an Amyloid-β42 (Aβ42)-dependent neurotoxic model in adult zebrafish brain through cerebroventricular microinjection of cell-penetrating Aβ42 derivatives. Aβ42 deposits in neurons and causes phenotypes reminiscent of amyloid pathophysiology: apoptosis, microglial activation, synaptic degeneration, and learning deficits. Aβ42 also induces NSPC proliferation and enhanced neurogenesis. Interleukin-4 (IL4) is activated primarily in neurons and microglia/macrophages in response to Aβ42 and is sufficient to increase NSPC proliferation and neurogenesis via STAT6 phosphorylation through the IL4 receptor in NSPCs. Our results reveal a crosstalk between neurons and immune cells mediated by IL4/STAT6 signaling, which induces NSPC plasticity in zebrafish brains. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Two-photon-based photoactivation in live zebrafish embryos.

PubMed

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.

Heart-specific expression of laminopathic mutations in transgenic zebrafish.

PubMed

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.

Whole-body and multispectral photoacoustic imaging of adult zebrafish

NASA Astrophysics Data System (ADS)

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.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals.

PubMed

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC(10)) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate "pattern-only" PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress.

Proteomic Signatures of the Zebrafish (Danio rerio) Embryo: Sensitivity and Specificity in Toxicity Assessment of Chemicals

PubMed Central

Hanisch, Karen; Küster, Eberhard; Altenburger, Rolf; Gündel, Ulrike

2010-01-01

Studies using embryos of the zebrafish Danio rerio (DarT) instead of adult fish for characterising the (eco-) toxic potential of chemicals have been proposed as animal replacing methods. Effect analysis at the molecular level might enhance sensitivity, specificity, and predictive value of the embryonal studies. The present paper aimed to test the potential of toxicoproteomics with zebrafish eleutheroembryos for sensitive and specific toxicity assessment. 2-DE-based toxicoproteomics was performed applying low-dose (EC10) exposure for 48 h with three-model substances Rotenone, 4,6-dinitro-o-cresol (DNOC) and Diclofenac. By multivariate “pattern-only” PCA and univariate statistical analyses, alterations in the embryonal proteome were detectable in nonetheless visibly intact organisms and treatment with the three substances was distinguishable at the molecular level. Toxicoproteomics enabled the enhancement of sensitivity and specificity of the embryonal toxicity assay and bear the potency to identify protein markers serving as general stress markers and early diagnosis of toxic stress. PMID:22084678

Egfl6 is involved in zebrafish notochord development.

PubMed

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.

Advanced Morphological — Behavioral Test Platform Reveals Neurodevelopmental Defects in Embryonic Zebrafish Exposed to Comprehensive Suite of Halogenated and Organophosphate Flame Retardants

PubMed Central

Noyes, Pamela D.; Haggard, Derik E.; Gonnerman, Greg D.; Tanguay, Robert L.

2015-01-01

The increased use of flammable plastics and electronic devices along with stricter fire safety standards has led to the heavy use of flame retardant chemicals in many consumer, commercial, and industrial products. Although flame retardant use has increased, a great deal of uncertainty surrounds their safety with some evidence showing toxicity and risk to human and environmental health. Recent efforts have focused on designing high-throughput biological platforms with nonmammalian models to evaluate and prioritize chemicals with limited hazard information. To complement these efforts, this study used a new morphological and behavioral testing platform with embryonic zebrafish to characterize the developmental toxicity of 44 halogenated and organophosphate flame retardants, including several of their known metabolites. Zebrafish were exposed to flame retardants from 6 to 120 h post fertilization (hpf) across concentrations spanning 4 orders of magnitude (eg, 6.4 nM to 64 µM). Flame retardant effects on survival and development were evaluated at 24 and 120 hpf, and neurobehavioral changes were measured using 2 photomotor response (PMR) assays. Compared to controls, 93% (41/44) of flame retardants studied elicited adverse effects among one or more of the bioassays and concentrations tested with the aryl phosphate ester (APE)-based mono-isopropylated triaryl phosphate and the brominated-bisphenol-A analog tetrabromobisphenol-A producing the greatest array of malformations. Hierarchical clustering showed that APE flame retardants with isopropyl, butyl, and cresyl substituents on phenyl rings clustered tightly and were particularly potent. Both PMR assays were highly predictive of morphological defects supporting their use as nonlethal means of evaluating teratogenicity that could allow for additional evaluations of long-term or delayed effects in older animals. Taken together, evidence presented here indicates that zebrafish neurodevelopment is highly sensitive to

A chronological expression profile of gene activity during embryonic mouse brain development.

PubMed

Goggolidou, P; Soneji, S; Powles-Glover, N; Williams, D; Sethi, S; Baban, D; Simon, M M; Ragoussis, I; Norris, D P

2013-12-01

The brain is a functionally complex organ, the patterning and development of which are key to adult health. To help elucidate the genetic networks underlying mammalian brain patterning, we conducted detailed transcriptional profiling during embryonic development of the mouse brain. A total of 2,400 genes were identified as showing differential expression between three developmental stages. Analysis of the data identified nine gene clusters to demonstrate analogous expression profiles. A significant group of novel genes of as yet undiscovered biological function were detected as being potentially relevant to brain development and function, in addition to genes that have previously identified roles in the brain. Furthermore, analysis for genes that display asymmetric expression between the left and right brain hemispheres during development revealed 35 genes as putatively asymmetric from a combined data set. Our data constitute a valuable new resource for neuroscience and neurodevelopment, exposing possible functional associations between genes, including novel loci, and encouraging their further investigation in human neurological and behavioural disorders.

Aspp2 negatively regulates body growth but not developmental timing by modulating IRS signaling in zebrafish embryos.

PubMed

Liu, Chengdong; Luan, Jing; Bai, Yan; Li, Yun; Lu, Ling; Liu, Yunzhang; Hakuno, Fumihiko; Takahashi, Shin-Ichiro; Duan, Cunming; Zhou, Jianfeng

2014-02-01

The growth and developmental rate of developing embryos and fetus are tightly controlled and coordinated to maintain proper body shape and size. The insulin receptor substrate (IRS) proteins, key intracellular transducers of insulin and insulin-like growth factor signaling, play essential roles in the regulation of growth and development. A short isoform of apoptosis-stimulating protein of p53 2 (ASPP2) was recently identified as a binding partner of IRS-1 and IRS-2 in mammalian cells in vitro. However, it is unclear whether ASPP2 plays any role in vertebrate embryonic growth and development. Here, we show that zebrafish Aspp2a and Aspp2b negatively regulate embryonic growth without affecting developmental rate. Human ASPP2 had similar effects on body growth in zebrafish embryos. Aspp2a and 2b inhibit Akt signaling. This inhibition was reversed by coinjection of myr-Akt1, a constitutively active form of Akt1. Zebrafish Aspp2a and Aspp2b physically bound with Irs-1, and the growth inhibitory effects of ASPP2/Aspp2 depend on the presence of their ankyrin repeats and SH3 domains. These findings uncover a novel role of Aspp2 in regulating vertebrate embryonic growth. Copyright © 2013 Elsevier Inc. All rights reserved.

CNNM2 Mutations Cause Impaired Brain Development and Seizures in Patients with Hypomagnesemia

PubMed Central

Lameris, Anke L. L.; van Wijk, Erwin; Flik, Gert; Regele, Sabrina; Korenke, G. Christoph; Neophytou, Birgit; Rust, Stephan; Reintjes, Nadine; Konrad, Martin; Bindels, René J. M.; Hoenderop, Joost G. J.

2014-01-01

Intellectual disability and seizures are frequently associated with hypomagnesemia and have an important genetic component. However, to find the genetic origin of intellectual disability and seizures often remains challenging because of considerable genetic heterogeneity and clinical variability. In this study, we have identified new mutations in CNNM2 in five families suffering from mental retardation, seizures, and hypomagnesemia. For the first time, a recessive mode of inheritance of CNNM2 mutations was observed. Importantly, patients with recessive CNNM2 mutations suffer from brain malformations and severe intellectual disability. Additionally, three patients with moderate mental disability were shown to carry de novo heterozygous missense mutations in the CNNM2 gene. To elucidate the physiological role of CNNM2 and explain the pathomechanisms of disease, we studied CNNM2 function combining in vitro activity assays and the zebrafish knockdown model system. Using stable Mg2+ isotopes, we demonstrated that CNNM2 increases cellular Mg2+ uptake in HEK293 cells and that this process occurs through regulation of the Mg2+-permeable cation channel TRPM7. In contrast, cells expressing mutated CNNM2 proteins did not show increased Mg2+ uptake. Knockdown of cnnm2 isoforms in zebrafish resulted in disturbed brain development including neurodevelopmental impairments such as increased embryonic spontaneous contractions and weak touch-evoked escape behaviour, and reduced body Mg content, indicative of impaired renal Mg2+ absorption. These phenotypes were rescued by injection of mammalian wild-type Cnnm2 cRNA, whereas mammalian mutant Cnnm2 cRNA did not improve the zebrafish knockdown phenotypes. We therefore concluded that CNNM2 is fundamental for brain development, neurological functioning and Mg2+ homeostasis. By establishing the loss-of-function zebrafish model for CNNM2 genetic disease, we provide a unique system for testing therapeutic drugs targeting CNNM2 and for

Transforming growth factor-β signalling controls human breast cancer metastasis in a zebrafish xenograft model.

PubMed

Drabsch, Yvette; He, Shuning; Zhang, Long; Snaar-Jagalska, B Ewa; ten Dijke, Peter

2013-11-07

The transforming growth factor beta (TGF-β) signalling pathway is known to control human breast cancer invasion and metastasis. We demonstrate that the zebrafish xenograft assay is a robust and dependable animal model for examining the role of pharmacological modulators and genetic perturbation of TGF-β signalling in human breast tumour cells. We injected cancer cells into the embryonic circulation (duct of cuvier) and examined their invasion and metastasis into the avascular collagenous tail. Various aspects of the TGF-β signalling pathway were blocked by chemical inhibition, small interfering RNA (siRNA), or small hairpin RNA (shRNA). Analysis was conducted using fluorescent microscopy. Breast cancer cells with different levels of malignancy, according to in vitro and in vivo mouse studies, demonstrated invasive and metastatic properties within the embryonic zebrafish model that nicely correlated with their differential tumourigenicity in mouse models. Interestingly, MCF10A M2 and M4 cells invaded into the caudal hematopoietic tissue and were visible as a cluster of cells, whereas MDA MB 231 cells invaded into the tail fin and were visible as individual cells. Pharmacological inhibition with TGF-β receptor kinase inhibitors or tumour specific Smad4 knockdown disturbed invasion and metastasis in the zebrafish xenograft model and closely mimicked the results we obtained with these cells in a mouse metastasis model. Inhibition of matrix metallo proteinases, which are induced by TGF-β in breast cancer cells, blocked invasion and metastasis of breast cancer cells. The zebrafish-embryonic breast cancer xenograft model is applicable for the mechanistic understanding, screening and development of anti-TGF-β drugs for the treatment of metastatic breast cancer in a timely and cost-effective manner.

Recent Advances in Elucidating the Genetic Mechanisms of Nephrogenesis Using Zebrafish

PubMed Central

Cheng, Christina N.; Verdun, Valerie A.; Wingert, Rebecca A.

2015-01-01

The kidney is comprised of working units known as nephrons, which are epithelial tubules that contain a series of specialized cell types organized into a precise pattern of functionally distinct segment domains. There is a limited understanding of the genetic mechanisms that establish these discrete nephron cell types during renal development. The zebrafish embryonic kidney serves as a simplified yet conserved vertebrate model to delineate how nephron segments are patterned from renal progenitors. Here, we provide a concise review of recent advances in this emerging field, and discuss how continued research using zebrafish genetics can be applied to gain insightsabout nephrogenesis. PMID:26024215

Quantification of Estradiol Uptake in Zebrafish Embryos and Larvae.

PubMed

Souder, Jaclyn Paige; Gorelick, Daniel A

2017-08-01

Zebrafish are a powerful model system to assess the molecular and cellular effects of exposure to toxic chemicals during embryonic development. To study the effects of environmental endocrine disruptors, embryos and larvae are commonly exposed to supraphysiologic concentrations of these compounds in the water, but their bioavailability in zebrafish is largely unknown. One hypothesis is that supraphysiologic concentrations of estrogens in the water are required to achieve physiologic levels in vivo; however, this has not been directly tested. To test this hypothesis, we developed an assay using radiolabeled estradiol ([3H]E2) to measure uptake from water at multiple concentrations and exposure durations in developing zebrafish from 0 to 5 days postfertilization (dpf). We found that [3H]E2 uptake increased with increasing concentration, duration, and developmental stage. Percent uptake from the total volume of treatment solution increased with increasing exposure duration and developmental stage, but remained constant with increasing concentration. We also found that the chorion, an acellular envelope surrounding embryos through 3 dpf, did not substantially affect [3H]E2 uptake. Finally, we found that at 1 dpf, E2 was preferentially taken up by the yolk at multiple exposure durations, while at 2 dpf E2 was preferentially taken up into the embryonic body. Our results support the hypothesis that exposing zebrafish embryos and larvae to supraphysiologic concentrations of estrogens is required to achieve physiologically relevant doses in vivo. The isotopic assay reported here will provide a foundation for determining the uptake of other compounds for teratogenicity, toxicology and drug discovery studies. © 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.

Developmental Toxicity of Dextromethorphan in Zebrafish Embryos/Larvae

PubMed Central

Xu, Zheng; Williams, Frederick E.; Liu, Ming-Cheh

2012-01-01

Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use the zebrafish as a model to investigate the potential toxicity of dextromethorphan during the embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24 hours post fertilization (hpf), 48 hpf, and 72 hpf, respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder, and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the Phase I and Phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction (RT-PCR) analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. PMID:20737414

The Identification of Zebrafish Mutants Showing Alterations in Senescence-Associated Biomarkers

PubMed Central

Uchiyama, Junzo; Koshimizu, Eriko; Qi, Jie; Nanjappa, Purushothama; Imamura, Shintaro; Islam, Asiful; Neuberg, Donna; Amsterdam, Adam; Roberts, Thomas M.

2008-01-01

There is an interesting overlap of function in a wide range of organisms between genes that modulate the stress responses and those that regulate aging phenotypes and, in some cases, lifespan. We have therefore screened mutagenized zebrafish embryos for the altered expression of a stress biomarker, senescence-associated β-galactosidase (SA-β-gal) in our current study. We validated the use of embryonic SA-β-gal production as a screening tool by analyzing a collection of retrovirus-insertional mutants. From a pool of 306 such mutants, we identified 11 candidates that showed higher embryonic SA-β-gal activity, two of which were selected for further study. One of these mutants is null for a homologue of Drosophila spinster, a gene known to regulate lifespan in flies, whereas the other harbors a mutation in a homologue of the human telomeric repeat binding factor 2 (terf2) gene, which plays roles in telomere protection and telomere-length regulation. Although the homozygous spinster and terf2 mutants are embryonic lethal, heterozygous adult fish are viable and show an accelerated appearance of aging symptoms including lipofuscin accumulation, which is another biomarker, and shorter lifespan. We next used the same SA-β-gal assay to screen chemically mutagenized zebrafish, each of which was heterozygous for lesions in multiple genes, under the sensitizing conditions of oxidative stress. We obtained eight additional mutants from this screen that, when bred to homozygosity, showed enhanced SA-β-gal activity even in the absence of stress, and further displayed embryonic neural and muscular degenerative phenotypes. Adult fish that are heterozygous for these mutations also showed the premature expression of aging biomarkers and the accelerated onset of aging phenotypes. Our current strategy of mutant screening for a senescence-associated biomarker in zebrafish embryos may thus prove to be a useful new tool for the genetic dissection of vertebrate stress response and

MOLECULAR AND MORPHOLOGICAL CHANGES IN ZEBRAFISH FOLLOWING TRANSIENT ETHANOL EXPOSURE DURING DEFINED DEVELOPMENTAL STAGES

PubMed Central

Zhang, Chengjin; Frazier, Jared M.; Chen, Hao; Liu, Yao; Lee, Ju-Ahng; Cole, Gregory J.

2014-01-01

Alcohol is a teratogen that has diverse effects on brain and craniofacial development, leading to a constellation of developmental disorders referred to as fetal alcohol spectrum disorder (FASD). The molecular basis of ethanol insult remains poorly understood, as does the relationship between molecular and behavioral changes as a consequence of prenatal ethanol exposure. Zebrafish embryos were exposed to a range of ethanol concentrations (0.5–5.0%) during defined developmental stages, and examined for morphological phenotypes characteristic of FASD. Embryos were also analyzed by in situ hybridization for changes in expression of defined cell markers for neural cell types that are sonic hedgehog-dependent. We show that transient binge-like ethanol exposures during defined developmental stages, such as early gastrulation and early neurulation, result in a range of phenotypes and changes in expression of Shh-dependent genes. The severity of fetal alcohol syndrome (FAS) morphological phenotypes, such as microphthalmia, depends on the embryonic stage and concentration of alcohol exposure, as does diminution of retinal Pax6a or forebrain and hindbrain GAD1 gene expression. We also show that changes in eye and brain morphology correlate with changes in Pax6a and GAD1 gene expression. Our results therefore show that transient binge-like ethanol exposures in zebrafish embryos produce the stereotypical morphological phenotypes of FAS, with the severity of phenotypes depending on the developmental stage and alcohol concentration of exposure. PMID:24929233

Additive effects of levonorgestrel and ethinylestradiol on brain aromatase (cyp19a1b) in zebrafish specific in vitro and in vivo bioassays.

PubMed

Hinfray, N; Tebby, C; Garoche, C; Piccini, B; Bourgine, G; Aït-Aïssa, S; Kah, O; Pakdel, F; Brion, F

2016-09-15

Estrogens and progestins are widely used in combination in human medicine and both are present in aquatic environment. Despite the joint exposure of aquatic wildlife to estrogens and progestins, very little information is available on their combined effects. In the present study we investigated the effect of ethinylestradiol (EE2) and Levonorgestrel (LNG), alone and in mixtures, on the expression of the brain specific ER-regulated cyp19a1b gene. For that purpose, recently established zebrafish-derived tools were used: (i) an in vitro transient reporter gene assay in a human glial cell line (U251-MG) co-transfected with zebrafish estrogen receptors (zfERs) and the luciferase gene under the control of the zebrafish cyp19a1b gene promoter and (ii) an in vivo bioassay using a transgenic zebrafish expressing GFP under the control of the zebrafish cyp19a1b gene promoter (cyp19a1b-GFP). Concentration-response relationships for single chemicals were modeled and used to design the mixture experiments following a ray design. The results from mixture experiments were analyzed to predict joint effects according to concentration addition and statistical approaches were used to characterize the potential interactions between the components of the mixtures (synergism/antagonism). We confirmed that some progestins could elicit estrogenic effects in fish brain. In mixtures, EE2 and LNG exerted additive estrogenic effects both in vitro and in vivo, suggesting that some environmental progestin could exert effects that will add to those of environmental (xeno-)estrogens. Moreover, our zebrafish specific assays are valuable tools that could be used in risk assessment for both single chemicals and their mixtures. Copyright © 2016 Elsevier Inc. All rights reserved.

Zebrafish neurobehavioral phenomics for aquatic neuropharmacology and toxicology research.

PubMed

Kalueff, Allan V; Echevarria, David J; Homechaudhuri, Sumit; Stewart, Adam Michael; Collier, Adam D; Kaluyeva, Aleksandra A; Li, Shaomin; Liu, Yingcong; Chen, Peirong; Wang, JiaJia; Yang, Lei; Mitra, Anisa; Pal, Subharthi; Chaudhuri, Adwitiya; Roy, Anwesha; Biswas, Missidona; Roy, Dola; Podder, Anupam; Poudel, Manoj K; Katare, Deepshikha P; Mani, Ruchi J; Kyzar, Evan J; Gaikwad, Siddharth; Nguyen, Michael; Song, Cai

2016-01-01

Zebrafish (Danio rerio) are rapidly emerging as an important model organism for aquatic neuropharmacology and toxicology research. The behavioral/phenotypic complexity of zebrafish allows for thorough dissection of complex human brain disorders and drug-evoked pathological states. As numerous zebrafish models become available with a wide spectrum of behavioral, genetic, and environmental methods to test novel drugs, here we discuss recent zebrafish phenomics methods to facilitate drug discovery, particularly in the field of biological psychiatry. Additionally, behavioral, neurological, and endocrine endpoints are becoming increasingly well-characterized in zebrafish, making them an inexpensive, robust and effective model for toxicology research and pharmacological screening. We also discuss zebrafish behavioral phenotypes, experimental considerations, pharmacological candidates and relevance of zebrafish neurophenomics to other 'omics' (e.g., genomic, proteomic) approaches. Finally, we critically evaluate the limitations of utilizing this model organism, and outline future strategies of research in the field of zebrafish phenomics. Copyright © 2015 Elsevier B.V. All rights reserved.

Comparative hazard analysis and toxicological modeling of diverse nanomaterials using the embryonic zebrafish (EZ) metric of toxicity

NASA Astrophysics Data System (ADS)

Harper, Bryan; Thomas, Dennis; Chikkagoudar, Satish; Baker, Nathan; Tang, Kaizhi; Heredia-Langner, Alejandro; Lins, Roberto; Harper, Stacey

2015-06-01

The integration of rapid assays, large datasets, informatics, and modeling can overcome current barriers in understanding nanomaterial structure-toxicity relationships by providing a weight-of-the-evidence mechanism to generate hazard rankings for nanomaterials. Here, we present the use of a rapid, low-cost assay to perform screening-level toxicity evaluations of nanomaterials in vivo. Calculated EZ Metric scores, a combined measure of morbidity and mortality in developing embryonic zebrafish, were established at realistic exposure levels and used to develop a hazard ranking of diverse nanomaterial toxicity. Hazard ranking and clustering analysis of 68 diverse nanomaterials revealed distinct patterns of toxicity related to both the core composition and outermost surface chemistry of nanomaterials. The resulting clusters guided the development of a surface chemistry-based model of gold nanoparticle toxicity. Our findings suggest that risk assessments based on the size and core composition of nanomaterials alone may be wholly inappropriate, especially when considering complex engineered nanomaterials. Research should continue to focus on methodologies for determining nanomaterial hazard based on multiple sub-lethal responses following realistic, low-dose exposures, thus increasing the availability of quantitative measures of nanomaterial hazard to support the development of nanoparticle structure-activity relationships.

Small GTPase R-Ras participates in neural tube formation in zebrafish embryonic spinal cord.

PubMed

Ohata, Shinya; Uga, Hideko; Okamoto, Hitoshi; Katada, Toshiaki

2018-06-27

Ras related (R-Ras), a small GTPase, is involved in the maintenance of apico-basal polarity in neuroepithelial cells of the zebrafish hindbrain, axonal collapse in cultured murine hippocampal neurons, and maturation of blood vessels in adult mice. However, the role of R-Ras in neural tube formation remains unknown. Using antisense morpholino oligonucleotides (AMOs), we found that in the spinal cord of zebrafish embryos, the lumen was formed bilaterally in rras morphants, whereas it was formed at the midline in control embryos. As AMO can cause off-target effects, we generated rras mutant zebrafish lines using CRISPR/Cas9 technology. Although these rras mutant embryos did not have a bilateral lumen in the spinal cord, the following findings suggest that the phenotype is unlikely due to an off-target effect of rras AMO: 1) The rras morphant phenotype was rescued by an injection of AMO-resistant rras mRNA, and 2) a bilaterally segregated spinal cord was not observed in rras mutant embryos injected with rras AMO. The results suggest that the function of other ras family genes may be redundant in rras mutants. Previous research reported a bilaterally formed lumen in the spinal cord of zebrafish embryos with a mutation in a planar cell polarity (PCP) gene, van gogh-like 2 (vangl2). In the present study, in cultured cells, R-Ras was co-immunoprecipitated with Vangl2 but not with another PCP regulator, Pricke1. Interestingly, the interaction between R-Ras and Vangl2 was stronger in guanine-nucleotide free point mutants of R-Ras than in wild-type or constitutively active (GTP-bound) forms of R-Ras. R-Ras may regulate neural tube formation in cooperation with Vangl2 in the developing zebrafish spinal cord. Copyright © 2018 Elsevier Inc. All rights reserved.

Interordinal chimera formation between medaka and zebrafish for analyzing stem cell differentiation.

PubMed

Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing; Yi, Meisheng; Hong, Yunhan

2012-08-10

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos.

Interordinal Chimera Formation Between Medaka and Zebrafish for Analyzing Stem Cell Differentiation

PubMed Central

Hong, Ni; Chen, Songlin; Ge, Ruowen; Song, Jianxing

2012-01-01

Chimera formation is a standard test for pluripotency of stem cells in vivo. Interspecific chimera formation between distantly related organisms offers also an attractive approach for propagating endangered species. Parameters influencing interspecies chimera formation have remained poorly elucidated. Here, we report interordinal chimera formation between medaka and zebrafish, which separated ∼320 million years ago and exhibit a more than 2-fold difference in developmental speed. We show that, on transplantation into zebrafish blastulae, both noncultivated blastomeres and long-term cultivated embryonic stem (ES) cells of medaka adopted the zebrafish developmental program and differentiated into physiologically functional cell types including pigment cells, blood cells, and cardiomyocytes. We also show that medaka ES cells express differentiation gene markers during chimeric embryogenesis. Therefore, the evolutionary distance and different embryogenesis speeds do not produce donor-host incompatibility to compromise chimera formation between medaka and zebrafish, and molecular markers are valuable for analyzing lineage commitment and cell differentiation in interspecific chimeric embryos. PMID:22204449

Strategies for Analyzing Cardiac Phenotypes in the Zebrafish Embryo

PubMed Central

Houk, Andrew R.; Yelon, Deborah

2017-01-01

The molecular mechanisms underlying cardiogenesis are of critical biomedical importance due to the high prevalence of cardiac birth defects. Over the past two decades, the zebrafish has served as a powerful model organism for investigating heart development, facilitated by its powerful combination of optical access to the embryonic heart and plentiful opportunities for genetic analysis. Work in zebrafish has identified numerous factors that are required for various aspects of heart formation, including the specification and differentiation of cardiac progenitor cells, the morphogenesis of the heart tube, cardiac chambers, and atrioventricular canal, and the establishment of proper cardiac function. However, our current roster of regulators of cardiogenesis is by no means complete. It is therefore valuable for ongoing studies to continue pursuit of additional genes and pathways that control the size, shape, and function of the zebrafish heart. An extensive arsenal of techniques is available to distinguish whether particular mutations, morpholinos, or small molecules disrupt specific processes during heart development. In this chapter, we provide a guide to the experimental strategies that are especially effective for the characterization of cardiac phenotypes in the zebrafish embryo. PMID:27312497

Zebrafish: an animal model for research in veterinary medicine.

PubMed

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

A Zebrafish Loss-of-Function Model for Human CFAP53 Mutations Reveals Its Specific Role in Laterality Organ Function.

PubMed

Noël, Emily S; Momenah, Tarek S; Al-Dagriri, Khalid; Al-Suwaid, Abdulrahman; Al-Shahrani, Safar; Jiang, Hui; Willekers, Sven; Oostveen, Yara Y; Chocron, Sonja; Postma, Alex V; Bhuiyan, Zahurul A; Bakkers, Jeroen

2016-02-01

Establishing correct left-right asymmetry during embryonic development is crucial for proper asymmetric positioning of the organs. Congenital heart defects, such as dextrocardia, transposition of the arteries, and inflow or outflow tract malformations, comprise some of the most common birth defects and may be attributed to incorrect establishment of body laterality. Here, we identify new patients with dextrocardia who have mutations in CFAP53, a coiled-coil domain containing protein. To elucidate the mechanism by which CFAP53 regulates embryonic asymmetry, we used genome editing to generate cfap53 zebrafish mutants. Zebrafish cfap53 mutants have specific defects in organ laterality and randomization of asymmetric gene expression. We show that cfap53 is required for cilia rotation specifically in Kupffer's vesicle, the zebrafish laterality organ, providing a mechanism by which patients with CFAP53 mutations develop dextrocardia and heterotaxy, and confirming previous evidence that left-right asymmetry in humans is regulated through cilia-driven fluid flow in a laterality organ. © 2015 WILEY PERIODICALS, INC.

Zebrabow: multispectral cell labeling for cell tracing and lineage analysis in zebrafish

PubMed Central

Pan, Y. Albert; Freundlich, Tom; Weissman, Tamily A.; Schoppik, David; Wang, X. Cindy; Zimmerman, Steve; Ciruna, Brian; Sanes, Joshua R.; Lichtman, Jeff W.; Schier, Alexander F.

2013-01-01

Advances in imaging and cell-labeling techniques have greatly enhanced our understanding of developmental and neurobiological processes. Among vertebrates, zebrafish is uniquely suited for in vivo imaging owing to its small size and optical translucency. However, distinguishing and following cells over extended time periods remains difficult. Previous studies have demonstrated that Cre recombinase-mediated recombination can lead to combinatorial expression of spectrally distinct fluorescent proteins (RFP, YFP and CFP) in neighboring cells, creating a ‘Brainbow’ of colors. The random combination of fluorescent proteins provides a way to distinguish adjacent cells, visualize cellular interactions and perform lineage analyses. Here, we describe Zebrabow (Zebrafish Brainbow) tools for in vivo multicolor imaging in zebrafish. First, we show that the broadly expressed ubi:Zebrabow line provides diverse color profiles that can be optimized by modulating Cre activity. Second, we find that colors are inherited equally among daughter cells and remain stable throughout embryonic and larval stages. Third, we show that UAS:Zebrabow lines can be used in combination with Gal4 to generate broad or tissue-specific expression patterns and facilitate tracing of axonal processes. Fourth, we demonstrate that Zebrabow can be used for long-term lineage analysis. Using the cornea as a model system, we provide evidence that embryonic corneal epithelial clones are replaced by large, wedge-shaped clones formed by centripetal expansion of cells from the peripheral cornea. The Zebrabow tool set presented here provides a resource for next-generation color-based anatomical and lineage analyses in zebrafish. PMID:23757414

The zebrafish orphan nuclear receptor genes nr2e1 and nr2e3 are expressed in developing eye and forebrain.

PubMed

Kitambi, Satish Srinivas; Hauptmann, Giselbert

2007-02-01

Mammalian Nr2e1 (Tailless, Mtll or Tlx) and Nr2e3 (photoreceptor-specific nuclear receptor, Pnr) are highly related orphan nuclear receptors, that are expressed in eye and forebrain-derived structures. In this study, we analyzed the developmental expression patterns of zebrafish nr2e1 and nr2e3. RT-PCR analysis showed that nr2e1 and nr2e3 are both expressed during embryonic and post-embryonic development. To examine the spatial distribution of nr2e1 and nr2e3 during development whole-mount in situ hybridization was performed. At tailbud stage, initial nr2e1 expression was localized to the rostral brain rudiment anterior to pax2.1 and eng2 expression at the prospective midbrain-hindbrain boundary. During subsequent stages, nr2e1 became widely expressed in fore- and midbrain primordia, eye and olfactory placodes. At 24hpf, strong nr2e1 expression was detected in telencephalon, hypothalamus, dorsal thalamus, pretectum, midbrain tectum, and retina. At 2dpf, the initially widespread nr2e1 expression became more restricted to distinct regions within the fore- and midbrain and to the retinal ciliary margin, the germinal zone which gives rise to retina and presumptive iris. Expression of nr2e3 was exclusively found in the developing retina and epiphysis. In both structures, nr2e3 expression was found in photoreceptor cells. The developmental expression profile of zebrafish nr2e1 and nr2e3 is consistent with evolutionary conserved functions in eye and rostral brain structures.

Zebrafish models in neuropsychopharmacology and CNS drug discovery.

PubMed

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.

Imaging Subcellular Structures in the Living Zebrafish Embryo.

PubMed

Engerer, Peter; Plucinska, Gabriela; Thong, Rachel; Trovò, Laura; Paquet, Dominik; Godinho, Leanne

2016-04-02

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Zebrafish numb and numblike are involved in primitive erythrocyte differentiation.

PubMed

Bresciani, Erica; Confalonieri, Stefano; Cermenati, Solei; Cimbro, Simona; Foglia, Efrem; Beltrame, Monica; Di Fiore, Pier Paolo; Cotelli, Franco

2010-12-13

Notch signaling is an evolutionarily conserved regulatory circuitry implicated in cell fate determination in various developmental processes including hematopoietic stem cell self-renewal and differentiation of blood lineages. Known endogenous inhibitors of Notch activity are Numb-Nb and Numblike-Nbl, which play partially redundant functions in specifying and maintaining neuronal differentiation. Nb and Nbl are expressed in most tissues including embryonic and adult hematopoietic tissues in mice and humans, suggesting possible roles for these proteins in hematopoiesis. We employed zebrafish to investigate the possible functional role of Numb and Numblike during hematopoiesis, as this system allows a detailed analysis even in embryos with severe defects that would be lethal in other organisms. Here we describe that nb/nbl knockdown results in severe reduction or absence of embryonic erythrocytes in zebrafish. Interestingly, nb/nbl knocked-down embryos present severe downregulation of the erythroid transcription factor gata1. This results in erythroblasts which fail to mature and undergo apoptosis. Our results indicate that Notch activity is increased in embryos injected with nb/nbl morpholino, and we show that inhibition of Notch activation can partially rescue the hematopoietic phenotype. Our results provide the first in vivo evidence of an involvement of Numb and Numblike in zebrafish erythroid differentiation during primitive hematopoiesis. Furthermore, we found that, at least in part, the nb/nbl morphant phenotype is due to enhanced Notch activation within hematopoietic districts, which in turn results in primitive erythroid differentiation defects.

Textile dyes induce toxicity on zebrafish early life stages.

PubMed

de Oliveira, Gisele Augusto Rodrigues; de Lapuente, Joaquín; Teixidó, Elisabet; Porredón, Constança; Borràs, Miquel; de Oliveira, Danielle Palma

2016-02-01

Textile manufacturing is one of the most polluting industrial sectors because of the release of potentially toxic compounds, such as synthetic dyes, into the environment. Depending on the class of the dyes, their loss in wastewaters can range from 2% to 50% of the original dye concentration. Consequently, uncontrolled use of such dyes can negatively affect human health and the ecological balance. The present study assessed the toxicity of the textile dyes Direct Black 38 (DB38), Reactive Blue 15 (RB15), Reactive Orange 16 (RO16), and Vat Green 3 (VG3) using zebrafish (Danio rerio) embryos for 144 h postfertilization (hpf). At the tested conditions, none of the dyes caused significant mortality. The highest RO16 dose significantly delayed or inhibited the ability of zebrafish embryos to hatch from the chorion after 96 hpf. From 120 hpf to 144 hpf, all the dyes impaired the gas bladder inflation of zebrafish larvae, DB38 also induced curved tail, and VG3 led to yolk sac edema in zebrafish larvae. Based on these data, DB38, RB15, RO16, and VG3 can induce malformations during embryonic and larval development of zebrafish. Therefore, it is essential to remove these compounds from wastewater or reduce their concentrations to safe levels before discharging textile industry effluents into the aquatic environment. © 2015 SETAC.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development

PubMed Central

David, Aldana P.; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B.

2016-01-01

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. PMID:26773060

Developmental toxicity of dextromethorphan in zebrafish embryos/larvae.

PubMed

Xu, Zheng; Williams, Frederick E; Liu, Ming-Cheh

2011-03-01

Dextromethorphan is widely used in over-the-counter cough and cold medications. Its efficacy and safety for infants and young children remains to be clarified. The present study was designed to use zebrafish as a model to investigate the potential toxicity of dextromethorphan during embryonic and larval development. Three sets of zebrafish embryos/larvae were exposed to dextromethorphan at 24, 48 and 72 h post fertilization (hpf), respectively, during the embryonic/larval development. Compared with the 48 and 72 hpf exposure sets, the embryos/larvae in the 24 hpf exposure set showed much higher mortality rates which increased in a dose-dependent manner. Bradycardia and reduced blood flow were observed for the embryos/larvae treated with increasing concentrations of dextromethorphan. Morphological effects of dextromethorphan exposure, including yolk sac and cardiac edema, craniofacial malformation, lordosis, non-inflated swim bladder and missing gill, were also more frequent and severe among zebrafish embryos/larvae exposed to dextromethorphan at 24 hpf. Whether the more frequent and severe developmental toxicity of dextromethorphan observed among the embryos/larvae in the 24 hpf exposure set, as compared with the 48 and 72 hpf exposure sets, is due to the developmental expression of the phase I and phase II enzymes involved in the metabolism of dextromethorphan remains to be clarified. A reverse transcription-polymerase chain reaction analysis, nevertheless, revealed developmental stage-dependent expression of mRNAs encoding SULT3 ST1 and SULT3 ST3, two enzymes previously shown to be capable of sulfating dextrorphan, an active metabolite of dextromethorphan. Copyright © 2010 John Wiley & Sons, Ltd.

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development

PubMed Central

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W.; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2015-01-01

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. PMID:26427652

Developmental Toxicity of Zinc Oxide Nanoparticles to Zebrafish (Danio rerio): A Transcriptomic Analysis

PubMed Central

Choi, Jin Soo; Kim, Ryeo-Ok; Yoon, Seokjoo

2016-01-01

Zinc oxide nanoparticles (ZnO NPs) are being utilized in an increasing number of fields and commercial applications. While their general toxicity and associated oxidative stress have been extensively studied, the toxicological pathways that they induce in developmental stages are still largely unknown. In this study, the developmental toxicity of ZnO NPs to embryonic/larval zebrafish was investigated. The transcriptional expression profiles induced by ZnO NPs were also investigated to ascertain novel genomic responses related to their specific toxicity pathway. Zebrafish embryos were exposed to 0.01, 0.1, 1, and 10 mg/L ZnO NPs for 96 h post-fertilization. The toxicity of ZnO NPs, based on their Zn concentration, was quite similar to that in embryonic/larval zebrafish exposed to corresponding ZnSO4 concentrations. Pericardial edema and yolk-sac edema were the principal malformations induced by ZnO NPs. Gene-expression profiling using microarrays demonstrated 689 genes that were differentially regulated (fold change >1.5) following exposure to ZnO NPs (498 upregulated, 191 downregulated). Several genes that were differentially regulated following ZnO NP exposure shared similar biological pathways with those observed with ZnSO4 exposure, but six genes (aicda, cyb5d1, edar, intl2, ogfrl2 and tnfsf13b) associated with inflammation and the immune system responded specifically to ZnO NPs (either in the opposite direction or were unchanged in ZnSO4 exposure). Real-time reverse-transcription quantitative polymerase chain reaction confirmed that the responses of these genes to ZnO NPs were significantly different from their response to ZnSO4 exposure. ZnO NPs may affect genes related to inflammation and the immune system, resulting in yolk-sac edema and pericardia edema in embryonic/larval developmental stages. These results will assist in elucidating the mechanisms of toxicity of ZnO NPs during development of zebrafish. PMID:27504894

Zebrafish collagen XII is present in embryonic connective tissue sheaths (fascia) and basement membranes.

PubMed

Bader, Hannah L; Keene, Douglas R; Charvet, Benjamin; Veit, Guido; Driever, Wolfgang; Koch, Manuel; Ruggiero, Florence

2009-01-01

Connective tissues ensure the cohesion of the tissues of the body, but also form specialized structures such as tendon and bone. Collagen XII may enhance the stability of connective tissues by bridging collagen fibrils, but its function is still unclear. Here, we used the zebrafish model to visualize its expression pattern in the whole organism. The zebrafish col12a1 gene is homologous to the small isoform of the tetrapod col12a1 gene. In agreement with the biochemical data reported for the small isoform, the zebrafish collagen XII alpha1 chain was characterized as a collagenase sensitive band migrating at approximately 200 kDa. Using newly generated polyclonal antibodies and anti-sense probes, we performed a comprehensive analysis of its expression in developing zebrafish. Collagen XII exhibited a much broader expression pattern than previously thought: it was ubiquitously expressed in the connective tissue sheaths (fascia) that encase the tissues and organs of the body. For example, it was found in sclera, meninges, epimysia and horizontal and vertical myosepta. Collagen XII was also detected in head mesenchyme, pharyngeal arches and within the spinal cord, where it was first expressed within and then at the lateral borders of the floor plate and at the dorsal midline. Furthermore, double immunofluorescence staining with laminin and immunogold electron microscopy revealed that collagen XII is associated with basement membranes. These data suggest that collagen XII is implicated in tissue cohesion by stabilizing fascia and by linking fascia to basement membranes.

Microbiota colonization status influences developmental toxicity of bisphenol A in embryonic zebrafish

EPA Science Inventory

There is growing evidence that microbiota can modify the toxicokinetics and/or toxicodynamics of environmental chemicals. Commonly used mammalian systems have limited ability to link phenotypic effects in exposed animals to colonization status. Here, we used gnotobiotic zebrafish...

The effects of rearing light level and duration differences on the optic nerve, brain, and associated structures in developing zebrafish larvae: a light and transmission electron microscope study.

PubMed

Chapman, George B; Tarboush, Rania; Connaughton, Victoria P

2012-03-01

The ultrastructure of the optic nerve, brain, and some associated structures of larval zebrafish, grown under three different light regimens were studied. Fish grown under cyclic light (control), constant dark (CD), and constant light (CL) were studied for 4 and 8 days postfertilization (dpf). We also studied the control and CD fish at 15 dpf. The brains of the control and CL fish were larger at 4 dpf than at 8 dpf. In all 4 dpf fish, the brain occupied the entire expanse between the two retinas and the optic nerve extended the shortest distance between the retina and the brain. The 15 dpf zebrafish had the smallest brain size. Groups of skeletal muscle cells associated with the optic nerves became visible in all older larvae. In the 15 dpf larvae, bulges and dilations in the optic nerve occurred as it reached the brain and optic chiasms occurred proximal to the brain. Electron microscopy yielded information about myelinated and unmyelinated axons in the optic nerve, the dimensions of neurotubules, neurofilaments, and myofilaments, including a unique variation in actin myofilaments, and a confirmation of reported myosin myofilament changes (but with dimensions). We also describe the ultrastructure of a sheath-like structure that is confluent over the optic nerve and the brain, which has not been described before in zebrafish. Also presented are images of associated fibroblasts, epithelial cells lining the mouth, cartilage plates, blood vessels, nerve bundles, and skeletal muscle cells, most of which have not been previously described in the literature. Copyright © 2012 Wiley Periodicals, Inc.

Temperature during embryonic development has persistent effects on thermal acclimation capacity in zebrafish

PubMed Central

Scott, Graham R.; Johnston, Ian A.

2012-01-01

Global warming is intensifying interest in the mechanisms enabling ectothermic animals to adjust physiological performance and cope with temperature change. Here we show that embryonic temperature can have dramatic and persistent effects on thermal acclimation capacity at multiple levels of biological organization. Zebrafish embryos were incubated until hatching at control temperature (TE = 27 °C) or near the extremes for normal development (TE = 22 °C or 32 °C) and were then raised to adulthood under common conditions at 27 °C. Short-term temperature challenge affected aerobic exercise performance (Ucrit), but each TE group had reduced thermal sensitivity at its respective TE. In contrast, unexpected differences arose after long-term acclimation to 16 °C, when performance in the cold was ∼20% higher in both 32 °C and 22 °C TE groups compared with 27 °C TE controls. Differences in performance after acclimation to cold or warm (34 °C) temperatures were partially explained by variation in fiber type composition in the swimming muscle. Cold acclimation changed the abundance of 3,452 of 19,712 unique and unambiguously identified transcripts detected in the fast muscle using RNA-Seq. Principal components analysis differentiated the general transcriptional responses to cold of the 27 °C and 32 °C TE groups. Differences in expression were observed for individual genes involved in energy metabolism, angiogenesis, cell stress, muscle contraction and remodeling, and apoptosis. Therefore, thermal acclimation capacity is not fixed and can be modified by temperature during early development. Developmental plasticity may thus help some ectothermic organisms cope with the more variable temperatures that are expected under future climate-change scenarios. PMID:22891320

Zebrafish antipredatory responses: A future for translational research?

PubMed Central

Gerlai, Robert

2011-01-01

Human neuropsychiatric conditions associated with abnormally exaggerated or misdirected fear (anxiety disorders and phobias) still represent a large unmet medical need because the biological mechanisms underlying these diseases are not well understood. Animal models have been proposed to facilitate this research. Here I review the literature with a focus on zebrafish, an upcoming laboratory organism in behavioral brain research. I argue that abnormal human fear responses are likely the result of the malfunction of neurobiological mechanisms (brain areas, circuits and/or molecular mechanisms) that originally evolved to support avoidance of predators or other harm in nature. I also argue that the understanding of the normal as well as pathological functioning of such mechanisms may be best achieved if one utilizes naturalistic experimental approaches. In case of laboratory model organisms, this may entail presenting stimuli associated with predators and measuring species-specific antipredatory responses. Although zebrafish is a relatively new subject of such inquiry, I review the recently rapidly increasing number of zebrafish studies in this area, and conclude that zebrafish is a promising research tool for the analysis of the neurobiology and genetics of vertebrate fear responses. PMID:19836422

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

PubMed

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.

Several synthetic progestins disrupt the glial cell specific-brain aromatase expression in developing zebra fish

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cano-Nicolau, Joel

+ 24 progestins were tested on embryonic brain aromatase expression in zebrafish. • 19 nor-testosterone derivatives induced cyp19a1b expression. • cyp19a1b up-regulation involved functional zfERs. • 19 nor-testosterone derivatives are pro-estrogenic compounds. • Effect of progestins should be further investigated at the brain level.« less

MK-801 increases locomotor activity in a context-dependent manner in zebrafish.

PubMed

Tran, Steven; Muraleetharan, Arrujyan; Fulcher, Niveen; Chatterjee, Diptendu; Gerlai, Robert

2016-01-01

Zebrafish have become a popular animal model for behavioral neuroscience with an increasing number of studies examining the effects of pharmacological compounds targeting the brain. Exposure to MK-801, a non-competitive N-methyl-d-aspartate receptor antagonist has been shown to increase locomotor activity in zebrafish. However, others have failed to replicate this finding as several contradicting studies report no changes in locomotor activity following exposure to similar doses. In the current study we reconcile these behavioral reports by demonstrating that zebrafish do not exhibit changes in locomotor activity during exposure to non-sedative doses of MK-801. Interestingly, zebrafish do exhibit significant increases in locomotion if pre-treated with MK-801 followed by subsequent testing in a novel environment, which suggests the effects of MK-801 are context-dependent. In addition, we examine the potential role of the dopaminergic system in mediating MK-801's locomotor stimulant effect by quantifying the levels of dopamine and its metabolite 3,4-dihydroxyphenylacetic acid (DOPAC) in the brains of zebrafish following a 30 min exposure to 10 μM of MK-801 (the dose found to induce the largest increase in locomotor activity). Our findings indicate that the MK-801-induced increase in locomotor activity is not accompanied by changes in whole-brain levels of dopamine or DOPAC. Overall, our results suggest that MK-801's context-dependent locomotor stimulant effect may be independent of whole-brain dopaminergic activation. Copyright © 2015 Elsevier B.V. All rights reserved.

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework

PubMed Central

Ruiter, Sander; Sippel, Josefine; Bouwmeester, Manon C.; Lommelaars, Tobias; Beekhof, Piet; Hodemaekers, Hennie M.; Bakker, Frank; van den Brandhof, Evert-Jan; Pennings, Jeroen L. A.; van der Ven, Leo T. M.

2016-01-01

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50–3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S-adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes. PMID:27827847

Programmed Effects in Neurobehavior and Antioxidative Physiology in Zebrafish Embryonically Exposed to Cadmium: Observations and Hypothesized Adverse Outcome Pathway Framework.

PubMed

Ruiter, Sander; Sippel, Josefine; Bouwmeester, Manon C; Lommelaars, Tobias; Beekhof, Piet; Hodemaekers, Hennie M; Bakker, Frank; van den Brandhof, Evert-Jan; Pennings, Jeroen L A; van der Ven, Leo T M

2016-11-02

Non-communicable diseases (NCDs) are a major cause of premature mortality. Recent studies show that predispositions for NCDs may arise from early-life exposure to low concentrations of environmental contaminants. This developmental origins of health and disease (DOHaD) paradigm suggests that programming of an embryo can be disrupted, changing the homeostatic set point of biological functions. Epigenetic alterations are a possible underlying mechanism. Here, we investigated the DOHaD paradigm by exposing zebrafish to subtoxic concentrations of the ubiquitous contaminant cadmium during embryogenesis, followed by growth under normal conditions. Prolonged behavioral responses to physical stress and altered antioxidative physiology were observed approximately ten weeks after termination of embryonal exposure, at concentrations that were 50-3200-fold below the direct embryotoxic concentration, and interpreted as altered developmental programming. Literature was explored for possible mechanistic pathways that link embryonic subtoxic cadmium to the observed apical phenotypes, more specifically, the probability of molecular mechanisms induced by cadmium exposure leading to altered DNA methylation and subsequently to the observed apical phenotypes. This was done using the adverse outcome pathway model framework, and assessing key event relationship plausibility by tailored Bradford-Hill analysis. Thus, cadmium interaction with thiols appeared to be the major contributor to late-life effects. Cadmium-thiol interactions may lead to depletion of the methyl donor S -adenosyl-methionine, resulting in methylome alterations, and may, additionally, result in oxidative stress, which may lead to DNA oxidation, and subsequently altered DNA methyltransferase activity. In this way, DNA methylation may be affected at a critical developmental stage, causing the observed apical phenotypes.

Comparative hazard analysis and toxicological modeling of diverse nanomaterials using the embryonic zebrafish (EZ) metric of toxicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Harper, Bryan; Thomas, Dennis G.; Chikkagoudar, Satish

The integration of rapid assays, large data sets, informatics and modeling can overcome current barriers in understanding nanomaterial structure-toxicity relationships by providing a weight-of-the-evidence mechanism to generate hazard rankings for nanomaterials. Here we present the use of a rapid, low-cost assay to perform screening-level toxicity evaluations of nanomaterials in vivo. Calculated EZ Metric scores, a combined measure of morbidity and mortality, were established at realistic exposure levels and used to develop a predictive model of nanomaterial toxicity. Hazard ranking and clustering analysis of 68 diverse nanomaterials revealed distinct patterns of toxicity related to both core composition and outermost surface chemistrymore » of nanomaterials. The resulting clusters guided the development of a predictive model of gold nanoparticle toxicity to embryonic zebrafish. In addition, our findings suggest that risk assessments based on the size and core composition of nanomaterials alone may be wholly inappropriate, especially when considering complex engineered nanomaterials. These findings reveal the need to expeditiously increase the availability of quantitative measures of nanomaterial hazard and broaden the sharing of that data and knowledge to support predictive modeling. In addition, research should continue to focus on methodologies for developing predictive models of nanomaterial hazard based on sub-lethal responses to low dose exposures.« less

Comparative hazard analysis and toxicological modeling of diverse nanomaterials using the embryonic zebrafish (EZ) metric of toxicity

DOE PAGES

Harper, Bryan; Thomas, Dennis G.; Chikkagoudar, Satish; ...

2015-06-04

The integration of rapid assays, large data sets, informatics and modeling can overcome current barriers in understanding nanomaterial structure-toxicity relationships by providing a weight-of-the-evidence mechanism to generate hazard rankings for nanomaterials. Here we present the use of a rapid, low-cost assay to perform screening-level toxicity evaluations of nanomaterials in vivo. Calculated EZ Metric scores, a combined measure of morbidity and mortality, were established at realistic exposure levels and used to develop a predictive model of nanomaterial toxicity. Hazard ranking and clustering analysis of 68 diverse nanomaterials revealed distinct patterns of toxicity related to both core composition and outermost surface chemistrymore » of nanomaterials. The resulting clusters guided the development of a predictive model of gold nanoparticle toxicity to embryonic zebrafish. In addition, our findings suggest that risk assessments based on the size and core composition of nanomaterials alone may be wholly inappropriate, especially when considering complex engineered nanomaterials. These findings reveal the need to expeditiously increase the availability of quantitative measures of nanomaterial hazard and broaden the sharing of that data and knowledge to support predictive modeling. In addition, research should continue to focus on methodologies for developing predictive models of nanomaterial hazard based on sub-lethal responses to low dose exposures.« less

Study of effects of radio-wave frequency radiation emitted from cellular telephones on embryonic development of danio rerio

NASA Astrophysics Data System (ADS)

Vagula, Mary; Harkless, Ryan

2013-05-01

Radio wave frequency (RF) radiation emitted from cellular telephones has become increasingly ubiquitous as a result of the popularity of these phones. With the increasing and unavoidable exposure to RF radiation a reality, it is imperative that the effects of such radiation on living tissue be well understood. In particular, it is critical to understand any effects that RF radiation may have as a carcinogen and on embryonic development, as pregnant women are not exempt from such exposure. As a model organism, zebrafish (Danio rerio) have been studied extensively, and their value in studies of gene expression cannot be overstated. This study observed the effects of RF radiation on the embryonic development of zebrafish. The expression of two genes, shha and hoxb9a, that are key to the early development of the fish was examined. Both genes have homologs in humans as well as in other model organisms. Preliminary results suggest that exposure to cell phone radiation might have an effect on the expression of shha in zebrafish embryos, causing under expression. More trials are necessary to validate these results.

Halogenated Bisphenol-A Analogs Act as Obesogens in Zebrafish Larvae (Danio rerio)

PubMed Central

Pinto, Caroline L.; Grimaldi, Marina; Hillenweck, Anne; Perdu, Elisabeth; Zalko, Daniel; Bernard, Laure; Laudet, Vincent; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Ake

2014-01-01

Obesity has increased dramatically over the past decades, reaching epidemic proportions. The reasons are likely multifactorial. One of the suggested causes is the accelerated exposure to obesity-inducing chemicals (obesogens). However, out of the tens of thousands of industrial chemicals humans are exposed to, very few have been tested for their obesogenic potential, mostly due to the limited availability of appropriate in vivo screening models. In this study, we investigated whether two commonly used flame retardants, the halogenated bisphenol-A (BPA) analogs tetrabromobisphenol-A (TBBPA) and tetrachlorobisphenol-A (TCBPA), could act as obesogens using zebrafish larvae as an in vivo animal model. The effect of embryonic exposure to these chemicals on lipid accumulation was analyzed by Oil Red-O staining, and correlated to their capacity to activate human and zebrafish peroxisome proliferator-activated receptor gamma (PPARγ) in zebrafish and in reporter cell lines. Then, the metabolic fate of TBBPA and TCBPA in zebrafish larvae was analyzed by high-performance liquid chromatography (HPLC) . TBBPA and TCBPA were readily taken up by the fish embryo and both compounds were biotransformed to sulfate-conjugated metabolites. Both halogenated-BPAs, as well as TBBPA-sulfate induced lipid accumulation in zebrafish larvae. TBBPA and TCBPA also induced late-onset weight gain in juvenile zebrafish. These effects correlated to their capacity to act as zebrafish PPARγ agonists. Screening of chemicals for inherent obesogenic capacities through the zebrafish lipid accumulation model could facilitate prioritizing chemicals for further investigations in rodents, and ultimately, help protect humans from exposure to environmental obesogens. PMID:24591153

Using the zebrafish to understand tendon development and repair

PubMed Central

Chen, Jessica W.; Galloway, Jenna L.

2017-01-01

Tendons are important components of our musculoskeletal system. Injuries to these tissues are very common, resulting from occupational-related injuries, sports-related trauma, and age-related degeneration. Unfortunately, there are few treatment options, and current therapies rarely restore injured tendons to their original function. An improved understanding of the pathways regulating their development and repair would have significant impact in stimulating the formulation of regenerative-based approaches for tendon injury. The zebrafish provides an ideal system in which to perform genetic and chemical screens to identify new pathways involved in tendon biology. Until recently, there had been few descriptions of tendons and ligaments in the zebrafish and their similarity to mammalian tendon tissues. In this chapter, we describe the development of the zebrafish tendon and ligament tissues in the context of their gene expression, structure, and interactions with neighboring musculoskeletal tissues. We highlight the similarities with tendon development in higher vertebrates, showing that the craniofacial tendons and ligaments in zebrafish morphologically, molecularly, and structurally resemble mammalian tendons and ligaments from embryonic to adult stages. We detail methods for fluorescent in situ hybridization and immunohistochemistry as an assay to examine morphological changes in the zebrafish musculoskeleton. Staining assays such as these could provide the foundation for screen-based approaches to identify new regulators of tendon development, morphogenesis, and repair. These discoveries would provide new targets and pathways to study in the context of regenerative medicine-based approaches to improve tendon healing. PMID:28129848

Lxr regulates lipid metabolic and visual perception pathways during zebrafish development.

PubMed

Pinto, Caroline Lucia; Kalasekar, Sharanya Maanasi; McCollum, Catherine W; Riu, Anne; Jonsson, Philip; Lopez, Justin; Swindell, Eric C; Bouhlatouf, Abdel; Balaguer, Patrick; Bondesson, Maria; Gustafsson, Jan-Åke

2016-01-05

The Liver X Receptors (LXRs) play important roles in multiple metabolic pathways, including fatty acid, cholesterol, carbohydrate and energy metabolism. To expand the knowledge of the functions of LXR signaling during embryonic development, we performed a whole-genome microarray analysis of Lxr target genes in zebrafish larvae treated with either one of the synthetic LXR ligands T0901317 or GW3965. Assessment of the biological processes enriched by differentially expressed genes revealed a prime role for Lxr in regulating lipid metabolic processes, similarly to the function of LXR in mammals. In addition, exposure to the Lxr ligands induced changes in expression of genes in the neural retina and lens of the zebrafish eye, including the photoreceptor guanylate cyclase activators and lens gamma crystallins, suggesting a potential novel role for Lxr in modulating the transcription of genes associated with visual function in zebrafish. The regulation of expression of metabolic genes was phenotypically reflected in an increased absorption of yolk in the zebrafish larvae, and changes in the expression of genes involved in visual perception were associated with morphological alterations in the retina and lens of the developing zebrafish eye. The regulation of expression of both lipid metabolic and eye specific genes was sustained in 1 month old fish. The transcriptional networks demonstrated several conserved effects of LXR activation between zebrafish and mammals, and also identified potential novel functions of Lxr, supporting zebrafish as a promising model for investigating the role of Lxr during development. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

G-quadruplexes as novel cis-elements controlling transcription during embryonic development.

PubMed

David, Aldana P; Margarit, Ezequiel; Domizi, Pablo; Banchio, Claudia; Armas, Pablo; Calcaterra, Nora B

2016-05-19

G-quadruplexes are dynamic structures folded in G-rich single-stranded DNA regions. These structures have been recognized as a potential nucleic acid based mechanism for regulating multiple cellular processes such as replication, transcription and genomic maintenance. So far, their transcriptional role in vivo during vertebrate embryonic development has not yet been addressed. Here, we performed an in silico search to find conserved putative G-quadruplex sequences (PQSs) within proximal promoter regions of human, mouse and zebrafish developmental genes. Among the PQSs able to fold in vitro as G-quadruplex, those present in nog3, col2a1 and fzd5 promoters were selected for further studies. In cellulo studies revealed that the selected G-quadruplexes affected the transcription of luciferase controlled by the SV40 nonrelated promoter. G-quadruplex disruption in vivo by microinjection in zebrafish embryos of either small ligands or DNA oligonucleotides complementary to the selected PQSs resulted in lower transcription of the targeted genes. Moreover, zebrafish embryos and larvae phenotypes caused by the presence of complementary oligonucleotides fully resembled those ones reported for nog3, col2a1 and fzd5 morphants. To our knowledge, this is the first work revealing in vivo the role of conserved G-quadruplexes in the embryonic development, one of the most regulated processes of the vertebrates biology. © The Author(s) 2016. Published by Oxford University Press on behalf of Nucleic Acids Research.

Effect of chilling and cryopreservation on expression of Pax genes in zebrafish (Danio rerio) embryos and blastomeres.

PubMed

Lin, C; Spikings, E; Zhang, T; Rawson, D M

2009-08-01

Cryopreservation is now common practice in the fields of aquaculture, conservation and biomedicine. However, there is a lack of information on the effect of chilling and cryopreservation at the molecular level. In the present study, we used real-time RT-PCR analysis to determine the effect of chilling and cryopreservation on expression of Pax2a, Pax2b, Pax5 and Pax8 which constitute one subgroup of the Pax gene family. As intact embryos of zebrafish have not yet been successfully cryopreserved, we have used two alternatives: chilling of intact embryos and cryopreservation of isolated blastomeres. Cryopreservation was found to affect the normal pattern of gene expression in zebrafish embryonic blastomeres. The trends, profile changes, in expression of Pax2a and Pax5 occurred to a lesser extent in frozen-thawed blastomeres than in fresh blastomeres whilst the opposite was true for Pax8. The trends in expression of Pax2b were delayed in frozen-thawed blastomeres compared to fresh blastomeres. Cryopreservation can therefore disrupt normal gene expression patterns in zebrafish embryonic blastomeres which could have a detrimental effect on embryo development.

Tumor suppressor Lzap regulates cell cycle progression, doming and zebrafish epiboly

PubMed Central

Liu, Dan; Wang, Wen-Der; Melville, David B.; Cha, Yong I.; Yin, Zhirong; Issaeva, Natalia; Knapik, Ela W.; Yarbrough, Wendell G.

2012-01-01

Initial stages of embryonic development rely on rapid, synchronized cell divisions of the fertilized egg followed by a set of morphogenetic movements collectively called epiboly and gastrulation. Lzap is a putative tumor suppressor whose expression is lost in 30% of head and neck squamous cell carcinomas. Lzap activities include regulation of cell cycle progression and response to therapeutic agents. Here we explore developmental roles of the lzap gene during zebrafish morphogenesis. Lzap is highly conserved among vertebrates and is maternally deposited. Expression is initially ubiquitous during gastrulation, and later becomes more prominent in the pharyngeal arches, digestive tract and brain. Antisense morpholino-mediated depletion of Lzap resulted in delayed cell divisions and apoptosis during blastomere formation, resulting in fewer, larger cells. Cell cycle analysis suggested that Lzap loss in early embryonic cells resulted in a G2/M arrest. Furthermore, the Lzap-deficient embryos failed to initiate epiboly – the earliest morphogenetic movement in animal development – which has been shown to be dependent on cell adhesion and migration of epithelial sheets. Our results strongly implicate Lzap in regulation of cell cycle progression, adhesion and migratory activity of epithelial cell sheets during early development. These functions provide further insight into Lzap activity that may contribute not only to development, but also to tumor formation. PMID:21523853

Developmental Neurotoxicity of Methamidophos in the Embryo-Larval Stages of Zebrafish.

PubMed

He, Xiaowei; Gao, Jiawei; Dong, Tianyu; Chen, Minjian; Zhou, Kun; Chang, Chunxin; Luo, Jia; Wang, Chao; Wang, Shoulin; Chen, Daozhen; Zhou, Zuomin; Tian, Ying; Xia, Yankai; Wang, Xinru

2016-12-28

Methamidophos is a representative organophosphate insecticide. The knowledge of its developmental neurotoxicity is limited, especially for zebrafish in the early stages of their life. Four hour post-fertilization (hpf) zebrafish embryos were exposed to several environmentally relevant concentrations of methamidophos (0, 25, and 500 μg/L) for up to 72 hpf. Locomotor behavior was then studied in the zebrafish larvae at this timepoint. Acridine orange (AO) staining was carried out in the zebrafish larvae, and the mRNA levels of genes associated with neural development ( mbp and syn2a ) were analyzed by reverse transcription-polymerase chain reaction (RT-PCR). The number of escape responders for mechanical stimulation was significantly decreased in exposed groups. AO staining showed noticeable signs of apoptosis mainly in the brain. In addition, the mRNA levels of mbp and syn2a were both significantly down-regulated in exposed groups. Our study provides the first evidence that methamidophos exposure can cause developmental neurotoxicity in the early stages of zebrafish life, which may be caused by the effect of methamidophos on neurodevelopmental genes and the activation of cell apoptosis in the brain.

Can zebrafish be used as animal model to study Alzheimer's disease?

PubMed Central

Santana, Soraya; Rico, Eduardo P; Burgos, Javier S

2012-01-01

Zebrafish is rapidly emerging as a promising model organism to study various central nervous system (CNS) disorders, including Alzheimer’s disease (AD). AD is the main cause of dementia in the human population and there is an urgency to understand the causes of this neurodegenerative disease. In this respect, the development of new animal models to study the underlying neurodegenerative mechanisms of AD is an urgent need. In this review we analyze the current situation in the use of zebrafish as a model for AD, discussing the reasons to use this experimental paradigm in CNS investigation and analyzing the several strategies adopted to induce an AD-like pathology in zebrafish. We discuss the strategies of performing interventions to cause damage in the zebrafish brain by altering the major neurotransmitter systems (such as cholinergic, glutamatergic or GABAergic circuits). We also analyze the several transgenic zebrafish constructed for the AD study, discussing both the familial-AD models based on APP processing pathway (APP and presenilins) and in the TAU hyperphosphorylation, together with the genes involved in sporadic-AD, as apolipoprotein E. We conclude that zebrafish is in a preliminary stage of development in the AD field, and that the transgenic animals must be improved to use this fish as an optimal model for AD research. Furthermore, a deeper knowledge of the zebrafish brain and a better characterization of the injury caused by alterations in the major neurotransmitter systems are needed. PMID:23383380

Smad4 is required for the development of cardiac and skeletal muscle in zebrafish.

PubMed

Yang, Jie; Wang, Junnai; Zeng, Zhen; Qiao, Long; Zhuang, Liang; Jiang, Lijun; Wei, Juncheng; Ma, Quanfu; Wu, Mingfu; Ye, Shuangmei; Gao, Qinglei; Ma, Ding; Huang, Xiaoyuan

Transforming growth factor-beta (TGF-beta) regulates cellular functions and plays key roles in development and carcinogenesis. Smad4 is the central intracellular mediator of TGF-beta signaling and plays crucial roles in tissue regeneration, cell differentiation, embryonic development, regulation of the immune system and tumor progression. To clarify the role of smad4 in development, we examined both the pattern of smad4 expression in zebrafish embryos and the effect of smad4 suppression on embryonic development using smad4-specific antisense morpholino-oligonucleotides. We show that smad4 is expressed in zebrafish embryos at all developmental stages examined and that embryonic knockdown of smad4 results in pericardial edema, decreased heartbeat and defects in the trunk structure. Additionally, these phenotypes were associated with abnormal expression of the two heart-chamber markers, cmlc2 and vmhc, as well as abnormal expression of three makers of myogenic terminal differentiation, mylz2, smyhc1 and mck. Furthermore, a notable increase in apoptosis was apparent in the smad4 knockdown embryos, while no obvious reduction in cell proliferation was observed. Collectively, these data suggest that smad4 plays an important role in heart and skeletal muscle development. Copyright © 2016 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

Expression of sall4 in taste buds of zebrafish.

PubMed

Jackson, Robyn; Braubach, Oliver R; Bilkey, Jessica; Zhang, Jing; Akimenko, Marie-Andrée; Fine, Alan; Croll, Roger P; Jonz, Michael G

2013-07-01

We characterized the expression of sall4, a gene encoding a zinc finger transcription factor involved in the maintenance of embryonic stem cells, in taste buds of zebrafish (Danio rerio). Using an enhancer trap line (ET5), we detected enhanced green fluorescent protein (EGFP) in developing and adult transgenic zebrafish in regions containing taste buds: the lips, branchial arches, and the nasal and maxillary barbels. Localization of EGFP to taste cells of the branchial arches and lips was confirmed by co-immunolabeling with antibodies against calretinin and serotonin, and a zebrafish-derived neuronal marker (zn-12). Transgenic insertion of the ET construct into the zebrafish genome was evaluated and mapped to chromosome 23 in proximity (i.e. 23 kb) to the sall4 gene. In situ hybridization and expression analysis between 24 and 96 h post-fertilization (hpf) demonstrated that transgenic egfp expression in ET5 zebrafish was correlated with the spatial and temporal pattern of expression of sall4 in the wild-type. Expression was first observed in the central nervous system and branchial arches at 24 hpf. At 48 hpf, sall4 and egfp expression was observed in taste bud primordia surrounding the mouth and branchial arches. At 72 and 96 hpf, expression was detected in the upper and lower lips and branchial arches. Double fluorescence in situ hybridization at 3 and 10 dpf confirmed colocalization of sall4 and egfp in the lips and branchial arches. These studies reveal sall4 expression in chemosensory cells and implicate this transcription factor in the development and renewal of taste epithelia in zebrafish. Copyright © 2013 Wiley Periodicals, Inc.

New isocoumarins from a cold-adapted fungal strain mucor sp. and their developmental toxicity to zebrafish embryos.

PubMed

Feng, Chun-Chi; Chen, Guo-Dong; Zhao, Yan-Qiu; Xin, Sheng-Chang; Li, Song; Tang, Jin-Shan; Li, Xiao-Xia; Hu, Dan; Liu, Xing-Zhong; Gao, Hao

2014-07-01

Three new isocoumarin derivatives, mucorisocoumarins A-C (1-3, resp.), together with seven known compounds, 4-10, were isolated from the cold-adapted fungal strain Mucor sp. (No. XJ07027-5). The structures of the new compounds were identified by detailed IR, MS, and 1D- and 2D-NMR analyses. It was noteworthy that compounds 1, 2, 4, and 5 were successfully resolved by chiral HPLC, indicating that 1-7 should exist as enantiomers. In an embryonic developmental toxicity assay using a zebrafish model, compound 3 produced developmental abnormalities in the zebrafish embryos. This is the first report of isocoumarins with developmental toxicity to zebrafish embryos. Copyright © 2014 Verlag Helvetica Chimica Acta AG, Zürich.

Developmental toxicity of CdTe QDs in zebrafish embryos and larvae

NASA Astrophysics Data System (ADS)

Duan, Junchao; Yu, Yongbo; Li, Yang; Yu, Yang; Li, Yanbo; Huang, Peili; Zhou, Xianqing; Peng, Shuangqing; Sun, Zhiwei

2013-07-01

Quantum dots (QDs) have widely been used in biomedical and biotechnological applications. However, few studies focus on the assessing toxicity of QDs exposure in vivo. In this study, zebrafish embryos were treated with CdTe QDs (4 nm) during 4-96 h post-fertilization (hpf). Mortality, hatching rate, malformation, heart rate, and QDs uptake were detected. We also measured the larval behavior to analyze whether QDs had persistent effects on larvae locomotor activity at 144 hpf. The results showed that as the exposure dosages increased, the hatching rate and heart rate of zebrafish embryos were decreased, while the mortality increased. Exposure to QDs caused embryonic malformations, including head malformation, pericardial edema, yolk sac edema, bent spine, and yolk not depleted. QDs fluorescence was mainly localized in the intestines region. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lowest dose (2.5 nM QDs) produced substantial hyperactivity while the higher doses groups (5, 10, and 20 nM QDs) elicited remarkably hypoactivity in dark periods. In summary, the data of this article indicated that QDs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior.

Optimisation of Embryonic and Larval ECG Measurement in Zebrafish for Quantifying the Effect of QT Prolonging Drugs

PubMed Central

Dhillon, Sundeep Singh; Dóró, Éva; Magyary, István; Egginton, Stuart; Sík, Attila; Müller, Ferenc

2013-01-01

Effective chemical compound toxicity screening is of paramount importance for safe cardiac drug development. Using mammals in preliminary screening for detection of cardiac dysfunction by electrocardiography (ECG) is costly and requires a large number of animals. Alternatively, zebrafish embryos can be used as the ECG waveform is similar to mammals, a minimal amount of chemical is necessary for drug testing, while embryos are abundant, inexpensive and represent replacement in animal research with reduced bioethical concerns. We demonstrate here the utility of pre-feeding stage zebrafish larvae in detection of cardiac dysfunction by electrocardiography. We have optimised an ECG recording system by addressing key parameters such as the form of immobilization, recording temperature, electrode positioning and developmental age. Furthermore, analysis of 3 days post fertilization (dpf) zebrafish embryos treated with known QT prolonging drugs such as terfenadine, verapamil and haloperidol led to reproducible detection of QT prolongation as previously shown for adult zebrafish. In addition, calculation of Z-factor scores revealed that the assay was sensitive and specific enough to detect large drug-induced changes in QTc intervals. Thus, the ECG recording system is a useful drug-screening tool to detect alteration to cardiac cycle components and secondary effects such as heart block and arrhythmias in zebrafish larvae before free feeding stage, and thus provides a suitable replacement for mammalian experimentation. PMID:23579446

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

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kwon, Hye-Joo, E-mail: hjkwon@pnu.edu.sa; Biology Department, Princess Nourah University, Riyadh 11671

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.more » 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.« less

Genome wide identification of Staufen2-bound mRNAs in embryonic rat brains.

PubMed

Maher-Laporte, Marjolaine; DesGroseillers, Luc

2010-05-01

Messenger ribonucleoprotein particles (mRNPs) are used to transport mRNAs along neuronal dendrites to their site of translation. Staufen2 is an mRNA-binding protein expressed in the cell bodies and cellular processes of different brain cells. It is notably involved in the transport of dendritic mRNAs along microtubules. Its knockdown expression was shown to change spine morphology and impair synaptic functions. However, the identity of Staufen2-bound mRNAs in brain cells is still completely unknown. As a mean to identify these mRNAs, we immunoprecipitated Staufen2-containing mRNPs from embryonic rat brains and used a genome wide approach to identify Staufen2-associated mRNAs. The genome wide approach identified 1780 mRNAs in Staufen2-containing mRNPs that code for proteins involved in cellular processes such as post-translational protein modifications, RNA metabolism, intracellular transport and translation. These results represent an additional and important step in the characterization of Staufen2- mediated neuronal functions in rat brains.

Zebrafish: A Versatile Animal Model for Fertility Research.

PubMed

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun; Goh, Bey Hing

2016-01-01

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.

Zebrafish: A Versatile Animal Model for Fertility Research

PubMed Central

Hoo, Jing Ying; Kumari, Yatinesh; Shaikh, Mohd Farooq; Hue, Seow Mun

2016-01-01

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. PMID:27556045

Temporally-Controlled Site-Specific Recombination in Zebrafish

PubMed Central

Hans, Stefan; Kaslin, Jan; Freudenreich, Dorian; Brand, Michael

2009-01-01

Conventional use of the site-specific recombinase Cre is a powerful technology in mouse, but almost absent in other vertebrate model organisms. In zebrafish, Cre-mediated recombination efficiency was previously very low. Here we show that using transposon-mediated transgenesis, Cre is in fact highly efficient in this organism. Furthermore, temporal control of recombination can be achieved by using the ligand-inducible CreERT2. Site-specific recombination only occurs upon administration of the drug tamoxifen (TAM) or its active metabolite, 4-hydroxy-tamoxifen (4-OHT). Cre-mediated recombination is detectable already 4 or 2 hours after administration of TAM or 4-OHT, demonstrating fast recombination kinetics. In addition, low doses of TAM allow mosaic labeling of single cells. Combined, our results show that conditional Cre/lox will be a valuable tool for both, embryonic and adult zebrafish studies. Furthermore, single copy insertion transgenesis of Cre/lox constructs suggest a strategy suitable also for other organisms. PMID:19247481

Evaluation in zebrafish model of the toxicity of rhodamine B-conjugated crotamine, a peptide potentially useful for diagnostics and therapeutics.

PubMed

Chan, Judy Yuet-Wa; Zhou, Hefeng; Kwan, Yiu Wa; Chan, Shun Wan; Radis-Baptista, Gandhi; Lee, Simon Ming-Yuen

2017-11-01

Crotamine is defensin-like cationic peptide from rattlesnake venom that possesses anticancer, antimicrobial, and antifungal properties. Despite these promising biological activities, toxicity is a major concern associated with the development of venom-derived peptides as therapeutic agents. In the present study, we used zebrafish as a system model to evaluate the toxicity of rhodamine B-conjugated (RhoB) crotamine derivative. The lethal toxic concentration of RhoB-crotamine was as low as 4 μM, which effectively kill zebrafish larvae in less than 10 min. With non-lethal concentrations (<1 μM), crotamine caused malformation in zebrafish embryos, delayed or completely halted hatching, adversely affected embryonic developmental programming, decreased the cardiac functions, and attenuated the swimming distance of zebrafish. The RhoB-crotamine translocated across vitelline membrane and accumulated in zebrafish yolk sac. These results demonstrate the sensitive responsivity of zebrafish to trial crotamine analogues for the development of novel therapeutic peptides with improved safety, bioavailability, and efficacy profiles. © 2017 Wiley Periodicals, Inc.

Disruption of Epithalamic Left-Right Asymmetry Increases Anxiety in Zebrafish.

PubMed

Facchin, Lucilla; Duboué, Erik R; Halpern, Marnie E

2015-12-02

Differences between the left and right sides of the brain are found throughout the animal kingdom, but the consequences of altered neural asymmetry are not well understood. In the zebrafish epithalamus, the parapineal is located on the left side of the brain where it influences development of the adjacent dorsal habenular (dHb) nucleus, causing the left and right dHb to differ in their organization, gene expression, and connectivity. Left-right (L-R) reversal of parapineal position and dHb asymmetry occurs spontaneously in a small percentage of the population, whereas the dHb develop symmetrically following experimental ablation of the parapineal. The habenular region was previously implicated in modulating fear in both mice and zebrafish, but the relevance of its L-R asymmetry is unclear. We now demonstrate that disrupting directionality of the zebrafish epithalamus causes reduced exploratory behavior and increased cortisol levels, indicative of enhanced anxiety. Accordingly, exposure to buspirone, an anxiolytic agent, significantly suppresses atypical behavior. Axonal projections from the parapineal to the dHb are more variable when it is located on the right side of the brain, revealing that L-R reversals do not necessarily represent a neuroanatomical mirror image. The results highlight the importance of directional asymmetry of the epithalamus in the regulation of stress responses in zebrafish. Copyright © 2015 the authors 0270-6474/15/3515847-13$15.00/0.

Exosome delivered anticancer drugs across the blood-brain barrier for brain cancer therapy in Danio rerio.

PubMed

Yang, Tianzhi; Martin, Paige; Fogarty, Brittany; Brown, Alison; Schurman, Kayla; Phipps, Roger; Yin, Viravuth P; Lockman, Paul; Bai, Shuhua

2015-06-01

The blood-brain barrier (BBB) essentially restricts therapeutic drugs from entering into the brain. This study tests the hypothesis that brain endothelial cell derived exosomes can deliver anticancer drug across the BBB for the treatment of brain cancer in a zebrafish (Danio rerio) model. Four types of exosomes were isolated from brain cell culture media and characterized by particle size, morphology, total protein, and transmembrane protein markers. Transport mechanism, cell uptake, and cytotoxicity of optimized exosome delivery system were tested. Brain distribution of exosome delivered anticancer drugs was evaluated using transgenic zebrafish TG (fli1: GFP) embryos and efficacies of optimized formations were examined in a xenotransplanted zebrafish model of brain cancer model. Four exosomes in 30-100 diameters showed different morphologies and exosomes derived from brain endothelial cells expressed more CD63 tetraspanins transmembrane proteins. Optimized exosomes increased the uptake of fluorescent marker via receptor mediated endocytosis and cytotoxicity of anticancer drugs in cancer cells. Images of the zebrafish showed exosome delivered anticancer drugs crossed the BBB and entered into the brain. In the brain cancer model, exosome delivered anticancer drugs significantly decreased fluorescent intensity of xenotransplanted cancer cells and tumor growth marker. Brain endothelial cell derived exosomes could be potentially used as a carrier for brain delivery of anticancer drug for the treatment of brain cancer.

Zebrafish Get Connected: Investigating Neurotransmission Targets and Alterations in Chemical Toxicity

PubMed Central

Horzmann, Katharine A.; Freeman, Jennifer L.

2016-01-01

Neurotransmission is the basis of neuronal communication and is critical for normal brain development, behavior, learning, and memory. Exposure to drugs and chemicals can alter neurotransmission, often through unknown pathways and mechanisms. The zebrafish (Danio rerio) model system is increasingly being used to study the brain and chemical neurotoxicity. In this review, the major neurotransmitter systems, including glutamate, GABA, dopamine, norepinephrine, serotonin, acetylcholine, histamine, and glutamate are surveyed and pathways of synthesis, transport, metabolism, and action are examined. Differences between human and zebrafish neurochemical pathways are highlighted. We also review techniques for evaluating neurological function, including the measurement of neurotransmitter levels, assessment of gene expression through transcriptomic analysis, and the recording of neurobehavior. Finally examples of chemical toxicity studies evaluating alterations in neurotransmitter systems in the zebrafish model are reviewed. PMID:28730152

Differential requirement for irf8 in formation of embryonic and adult macrophages in zebrafish

DOE PAGES

Shiau, Celia E.; Kaufman, Zoe; Meireles, Ana M.; ...

2015-01-23

Interferon regulatory factor 8 (Irf8) is critical for mammalian macrophage development and innate immunity, but its role in teleost myelopoiesis remains incompletely understood. Specifically, genetic tools to analyze the role of irf8 in zebrafish macrophage development at larval and adult stages are lacking. In this study, we generated irf8 null mutants in zebrafish using TALEN-mediated targeting. Our analysis defines different requirements for irf8 at different stages. irf8 is required for formation of all macrophages during primitive and transient definitive hematopoiesis, but not during adult-phase definitive hematopoiesis starting at 5-6 days postfertilization. At early stages, irf8 mutants have excess neutrophils andmore » excess cell death in pu.1-expressing myeloid cells. Macrophage fates were recovered in irf8 mutants after wildtype irf8 expression in neutrophil and macrophage lineages, suggesting that irf8 regulates macrophage specification and survival. In juvenile irf8 mutant fish, mature macrophages are present, but at numbers significantly reduced compared to wildtype, indicating an ongoing requirement for irf8 after embryogenesis. As development progresses, tissue macrophages become apparent in zebrafish irf8 mutants, with the possible exception of microglia. Our study defines distinct requirement for irf8 in myelopoiesis before and after transition to the adult hematopoietic system.« less

Toxic Effects of Silica Nanoparticles on Zebrafish Embryos and Larvae

PubMed Central

Shi, Huiqin; Tian, Linwei; Guo, Caixia; Huang, Peili; Zhou, Xianqing; Peng, Shuangqing; Sun, Zhiwei

2013-01-01

Silica nanoparticles (SiNPs) have been widely used in biomedical and biotechnological applications. Environmental exposure to nanomaterials is inevitable as they become part of our daily life. Therefore, it is necessary to investigate the possible toxic effects of SiNPs exposure. In this study, zebrafish embryos were treated with SiNPs (25, 50, 100, 200 µg/mL) during 4–96 hours post fertilization (hpf). Mortality, hatching rate, malformation and whole-embryo cellular death were detected. We also measured the larval behavior to analyze whether SiNPs had adverse effects on larvae locomotor activity. The results showed that as the exposure dosages increasing, the hatching rate of zebrafish embryos was decreased while the mortality and cell death were increased. Exposure to SiNPs caused embryonic malformations, including pericardial edema, yolk sac edema, tail and head malformation. The larval behavior testing showed that the total swimming distance was decreased in a dose-dependent manner. The lower dose (25 and 50 µg/mL SiNPs) produced substantial hyperactivity while the higher doses (100 and 200 µg/mL SiNPs) elicited remarkably hypoactivity in dark periods. In summary, our data indicated that SiNPs caused embryonic developmental toxicity, resulted in persistent effects on larval behavior. PMID:24058598

Identification of differentially expressed genes in the zebrafish hypothalamus - pituitary axis

PubMed Central

Toro, Sabrina; Wegner, Jeremy; Muller, Marc; Westerfield, Monte; Varga, Zoltan M.

2009-01-01

The vertebrate hypothalamic-pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in adenohypophysis ontogenesis, little is known about hypothalamic and neurohypophysial development or when the HP matures and becomes functional. To identify markers of the HP, we constructed subtractive cDNA libraries between adult zebrafish hypothalamus and pituitary. We identified previously published genes and ESTs and novel zebrafish genes, some of which were predicted by genomic database analysis. We also analyzed expression patterns of these genes and found that several are expressed in the embryonic and larval hypothalamus, neurohypophysis, and/or adenohypophysis. Expression at these stages makes these genes useful markers to study HP maturation and function. PMID:19166982

A Novel Model of Traumatic Brain Injury in Adult Zebrafish Demonstrates Response to Injury and Treatment Comparable with Mammalian Models.

PubMed

McCutcheon, Victoria; Park, Eugene; Liu, Elaine; Sobhebidari, Pooya; Tavakkoli, Jahan; Wen, Xiao-Yan; Baker, Andrew J

2017-04-01

Traumatic brain injury (TBI) is a leading cause of death and morbidity in industrialized countries with considerable associated health care costs. The cost and time associated with pre-clinical development of TBI therapeutics is lengthy and expensive with a poor track record of successful translation to the clinic. The zebrafish is an emerging model organism in research with unique technical and genomic strengths in the study of disease and development. Its high degree of genetic homology and cell signaling pathways relative to mammalian species and amenability to high and medium throughput assays has potential to accelerate the rate of therapeutic drug identification. Accordingly, we developed a novel closed-head model of TBI in adult zebrafish using a targeted, pulsed, high-intensity focused ultrasound (pHIFU) to induce mechanical injury of the brain. Western blot results indicated altered microtubule and neurofilament expression as well as increased expression of cleaved caspase-3 and beta APP (β-APP; p < 0.05). We used automated behavioral tracking software to evaluate locomotor deficits 24 and 48 h post-injury. Significant behavioral impairment included decreased swim distance and velocity (p < 0.05), as well as heightened anxiety and altered group social dynamics. Responses to injury were pHIFU dose-dependent and modifiable with MK-801, MDL-28170, or temperature modulation. Together, results indicate that the zebrafish exhibits responses to injury and intervention similar to mammalian TBI pathophysiology and suggest the potential for use to rapidly evaluate therapeutic compounds with high efficiency.

The zebrafish as a model for complex tissue regeneration

PubMed Central

Gemberling, Matthew; Bailey, Travis J.; Hyde, David R.; Poss, Kenneth D.

2013-01-01

For centuries, philosophers and scientists have been fascinated by the principles and implications of regeneration in lower vertebrate species. Two features have made zebrafish an informative model system for determining mechanisms of regenerative events. First, they are highly regenerative, able to regrow amputated fins, as well as a lesioned brain, retina, spinal cord, heart, and other tissues. Second, they are amenable to both forward and reverse genetic approaches, with a research toolset regularly updated by an expanding community of zebrafish researchers. Zebrafish studies have helped identify new mechanistic underpinnings of regeneration in multiple tissues, and in some cases have served as a guide for contemplating regenerative strategies in mammals. Here, we review the recent history of zebrafish as a genetic model system for understanding how and why tissue regeneration occurs. PMID:23927865

Behavioral effects of MDMA ('ecstasy') on adult zebrafish.

PubMed

Stewart, Adam; Riehl, Russell; Wong, Keith; Green, Jeremy; Cosgrove, Jessica; Vollmer, Karoly; Kyzar, Evan; Hart, Peter; Allain, Alexander; Cachat, Jonathan; Gaikwad, Siddharth; Hook, Molly; Rhymes, Kate; Newman, Alan; Utterback, Eli; Chang, Katie; Kalueff, Allan V

2011-06-01

3,4-methylenedioxymethamphetamine (MDMA, 'ecstasy') is a potent psychedelic drug inducing euphoria and hypersociability in humans, as well as hyperactivity and anxiety in rodents. Adult zebrafish (Danio rerio) have become a widely used species in neurobehavioral research. Here, we explore the effects of a wide range (0.25-120 mg/l) of acute MDMA doses on zebrafish behavior in the novel tank test. Although MDMA was inactive at lower doses (0.25-10 mg/l), higher doses reduced bottom swimming and immobility (40-120 mg/l) and impaired intrasession habituation (10-120 mg/l). MDMA also elevated brain c-fos expression, collectively confirming the usage of zebrafish models for screening of hallucinogenic compounds.

Establishment of oct4:gfp transgenic zebrafish line for monitoring cellular multipotency by GFP fluorescence.

PubMed

Kato, Hiroyuki; Abe, Kota; Yokota, Shinpei; Matsuno, Rinta; Mikekado, Tsuyoshi; Yokoi, Hayato; Suzuki, Tohru

2015-01-01

The establishment of induced pluripotent stem (iPS) cell technology in fish could facilitate the establishment of novel cryopreservation techniques for storing selected aquaculture strains as frozen cells. In order to apply iPS cell technology to fish, we established a transgenic zebrafish line, Tg(Tru.oct4:EGFP), using green fluorescent protein (GFP) expression under the control of the oct4 gene promoter as a marker to evaluate multipotency in iPS cell preparations. We used the oct4 promoter from fugu (Takifugu rubripes) due to the compact nature of the fugu genome and to facilitate future applications of this technology in marine fishes. During embryogenesis, maternal GFP fluorescence was observed at the cleavage stage and zygotic GFP expression was observed from the start of the shield stage until approximately 24 h after fertilization. gfp messenger RNA (mRNA) was expressed by whole embryonic cells at the shield stage, and then restricted to the caudal neural tube in the latter stages of embryogenesis. These observations showed that GFP fluorescence and the regulation of gfp mRNA expression by the exogenous fugu oct4 promoter are well suited for monitoring endogenous oct4 mRNA expression in embryos. Bisulfite sequencing revealed that the rate of CpG methylation in the transgenic oct4 promoter was high in adult cells (98%) and low in embryonic cells (37%). These findings suggest that, as with the endogenous oct4 promoter, demethylation and methylation both take place normally in the transgenic oct4 promoter during embryogenesis. The embryonic cells harvested at the shield stage formed embryonic body-like cellular aggregates and maintained GFP fluorescence for 6 d when cultured on Transwell-COL Permeable Supports or a feeder layer of adult fin cells. Loss of GFP fluorescence by cultured cells was correlated with cellular differentiation. We consider that the Tg(Tru.oct4:EGFP) zebrafish line established here is well suited for monitoring multipotency in

Early-life glucocorticoids programme behaviour and metabolism in adulthood in zebrafish

PubMed Central

Wilson, K S; Tucker, C S; Al-Dujaili, E A S; Holmes, M C; Hadoke, P W F; Kenyon, C J

2016-01-01

Glucocorticoids (GCs) in utero influence embryonic development with consequent programmed effects on adult physiology and pathophysiology and altered susceptibility to cardiovascular disease. However, in viviparous species, studies of these processes are compromised by secondary maternal influences. The zebrafish, being fertilised externally, avoids this problem and has been used here to investigate the effects of transient alterations in GC activity during early development. Embryonic fish were treated either with dexamethasone (a synthetic GC), an antisense GC receptor (GR) morpholino (GR Mo), or hypoxia for the first 120h post fertilisation (hpf); responses were measured during embryonic treatment or later, post treatment, in adults. All treatments reduced cortisol levels in embryonic fish to similar levels. However, morpholino- and hypoxia-treated embryos showed delayed physical development (slower hatching and straightening of head–trunk angle, shorter body length), less locomotor activity, reduced tactile responses and anxiogenic activity. In contrast, dexamethasone-treated embryos showed advanced development and thigmotaxis but no change in locomotor activity or tactile responses. Gene expression changes were consistent with increased (dexamethasone) and decreased (hypoxia, GR Mo) GC activity. In adults, stressed cortisol values were increased with dexamethasone and decreased by GR Mo and hypoxia pre-treatments. Other responses were similarly differentially affected. In three separate tests of behaviour, dexamethasone-programmed fish appeared ‘bolder’ than matched controls, whereas Mo and hypoxia pre-treated fish were unaffected or more reserved. Similarly, the dexamethasone group but not the Mo or hypoxia groups were heavier, longer and had a greater girth than controls. Hyperglycaemia and expression of GC responsive gene (pepck) were also increased in the dexamethasone group. We conclude that GC activity controls many aspects of early-life growth and

Lineage tracing of dlx1a/2a and dlx5a/6a expressing cells in the developing zebrafish brain.

PubMed

Solek, Cynthia M; Feng, Shengrui; Perin, Sofia; Weinschutz Mendes, Hellen; Ekker, Marc

2017-07-01

Lineage tracing of specific populations of progenitor cells provides crucial information about developmental programs. Four members of the Dlx homeobox gene family, Dlx1,2, 5 and 6, are involved in the specification of γ-aminobutyric acid (GABA)ergic neurons in the vertebrate forebrain. Orthologous genes in mammals and teleost show similarities in expression patterns and transcriptional regulation mechanisms. We have used lineage tracing to permanently label dlx-expressing cells in the zebrafish and have characterized the progeny of these cells in the larva and in the juvenile and adult brain. We have found that dlx1a/2a and dlx5a/6a expressing progenitors give rise, for the most part, to small populations of cells which constitute only a small proportion of GABAergic cells in the adult brain tissue. Moreover, some of the cells do not acquire a neuronal phenotype suggesting that, regardless of the time a cell expresses dlx genes in the brain, it can potentially give rise to cells other than neurons. In some instances, labeling larval dlx5a/6a-expressing cells, but not dlx1a/2a-expressing cells, results in massively expanding, widespread clonal expansion throughout the adult brain. Our data provide a detailed lineage analysis of the dlx1a/2a and dlx5a/6a expressing progenitors in the zebrafish brain and lays the foundation for further characterization of the role of these transcription factors beyond the specification of GABAergic neurons. Copyright © 2017 Elsevier Inc. All rights reserved.

Malformation of certain brain blood vessels caused by TCDD activation of Ahr2/Arnt1 signaling in developing zebrafish.

PubMed

Teraoka, Hiroki; Ogawa, Akira; Kubota, Akira; Stegeman, John J; Peterson, Richard E; Hiraga, Takeo

2010-08-15

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) causes various signs of toxicity in early life stages of vertebrates through activation of the aryl hydrocarbon receptor (AHR). The AHR also plays important roles in normal development in mice, and AHR(-/-) mice show abnormal development of vascular structures in various blood vessels. Our previous studies revealed that Ahr type 2 (Ahr2) activation by TCDD and beta-naphthoflavone (BNF) caused a significant decrease in blood flow in the dorsal midbrain of zebrafish embryos. Here we report effects of TCDD exposure on the morphology of some blood vessels in the head of developing zebrafish. TCDD caused concentration-dependent anatomical rearrangements in the shape of the prosencephalic artery in zebrafish larvae. In contrast, no major vascular defects were recognized in the trunk and tail regions following exposure to TCDD at least at the concentrations used. Essentially, the same observations were also confirmed in BNF-exposed larvae. Knock-down of either Ahr2 or Ahr nuclear translocator type 1 (Arnt1) by morpholino oligonucleotides (MOs) protected larvae against abnormal shape of the prosencephalic artery caused by TCDD and BNF. On the other hand, knock-down of Ahr2 or Arnt1 in vehicle-exposed zebrafish larvae had no clear effect on morphology of the prosencephalic artery or trunk vessels. Ascorbic acid, an antioxidant, protected against the TCDD-induced decrease in blood flow through the prosencephalic artery, but not the abnormal morphological changes in the shape of this artery. These results indicate that activation of Ahr2/Arnt1 pathway by TCDD and BNF affects the shape of certain blood vessels in the brain of developing zebrafish. (c) 2010 Elsevier B.V. All rights reserved.

Embryonic lethality is not sufficient to explain hourglass-like conservation of vertebrate embryos.

PubMed

Uchida, Yui; Uesaka, Masahiro; Yamamoto, Takayoshi; Takeda, Hiroyuki; Irie, Naoki

2018-01-01

Understanding the general trends in developmental changes during animal evolution, which are often associated with morphological diversification, has long been a central issue in evolutionary developmental biology. Recent comparative transcriptomic studies revealed that gene expression profiles of mid-embryonic period tend to be more evolutionarily conserved than those in earlier or later periods. While the hourglass-like divergence of developmental processes has been demonstrated in a variety of animal groups such as vertebrates, arthropods, and nematodes, the exact mechanism leading to this mid-embryonic conservation remains to be clarified. One possibility is that the mid-embryonic period (pharyngula period in vertebrates) is highly prone to embryonic lethality, and the resulting negative selections lead to evolutionary conservation of this phase. Here, we tested this "mid-embryonic lethality hypothesis" by measuring the rate of lethal phenotypes of three different species of vertebrate embryos subjected to two kinds of perturbations: transient perturbations and genetic mutations. By subjecting zebrafish ( Danio rerio ), African clawed frog ( Xenopus laevis ), and chicken ( Gallus gallus ) embryos to transient perturbations, namely heat shock and inhibitor treatments during three developmental periods [early (represented by blastula and gastrula), pharyngula, and late], we found that the early stages showed the highest rate of lethal phenotypes in all three species. This result was corroborated by perturbation with genetic mutations. By tracking the survival rate of wild-type embryos and embryos with genetic mutations induced by UV irradiation in zebrafish and African clawed frogs, we found that the highest decrease in survival rate was at the early stages particularly around gastrulation in both these species. In opposition to the "mid-embryonic lethality hypothesis," our results consistently showed that the stage with the highest lethality was not around the

Evaluation of the neurotoxic effects of chronic embryonic exposure with inorganic mercury on motor and anxiety-like responses in zebrafish (Danio rerio) larvae.

PubMed

Abu Bakar, Noraini; Mohd Sata, Nurul Syafida Asma'; Ramlan, Nurul Farhana; Wan Ibrahim, Wan Norhamidah; Zulkifli, Syaizwan Zahmir; Che Abdullah, Che Azurahanim; Ahmad, Syahida; Amal, Mohammad Noor Azmai

Chronic exposure to mercury (Hg) can lead to cumulative impairments in motor and cognitive functions including alteration in anxiety responses. Although several risk factors have been identified in recent year, little is known about the environmental factors that either due exposure toward low level of inorganic mercury that may led to the developmental disorders. The present study investigated the effects of embryonic exposure of mercury chloride on motor function and anxiety-like behavior. The embryo exposed to 6 different concentrations of HgCl 2 (7.5, 15, 30, 100, 125, 250nM) at 5hpf until hatching (72hpf) in a semi-static condition. The mortality rate increased in a dose dependent manner where the chronic embryonic exposure to 100nM decreased the number of tail coiling, heartbeat, and swimming activity. Aversive stimulus was used to examine the effects of 100nM interferes with the development of anxiety-related behavior. No elevation in both thigmotaxis and avoidance response of 6dpf larvae exposed with 100nM were found. Biochemical analysis showed HgCl 2 exposure affects proteins, lipids, carbohydrates and nucleic acids of the zebrafish larvae. These results showed that implication of HgCl 2 on locomotor and biochemical defects affects motor performance and anxiety-like responses. Yet, the potential underlying mechanisms these responses need to be further investigated which is crucial to prevent potential hazards on the developing organism due to neurotoxicant exposure. Copyright © 2016 Elsevier Inc. All rights reserved.

Purification of zebrafish erythrocytes as a means of identifying a novel regulator of haematopoiesis.

PubMed

Kulkeaw, Kasem; Inoue, Tomoko; Ishitani, Tohru; Nakanishi, Yoichi; Zon, Leonard I; Sugiyama, Daisuke

2018-02-01

Zebrafish embryos are useful to study haematopoietic gene function in vertebrates, although lack of antibodies to zebrafish proteins has limited the purification of specific cell populations. Here, we purified primitive zebrafish erythrocytes using 1, 5-bis{[2-(di-methylamino)ethyl]amino}-4, 8-dihydroxyanthracene-9, 10-dione (DRAQ5 TM ), a DNA-staining fluorescent dye. At 48-h post-fertilization, we sorted small-sized cells from embryos using forward scatter and found that they consisted of DRAQ5 high and DRAQ5 low populations. DRAQ5 high cells contained haemoglobin, lacked myeloperoxidase activity and expressed high levels of embryonic globin (hbae3 and hbbe1.1) mRNA, all characteristics of primitive erythrocytes. Following DRAQ5 TM analysis of gata1:dsRed transgenic embryos, we purified primitive DRAQ5 high dsRed+ erythrocytes from haematopoietic progenitor cells. Using this method, we identified docking protein 2 (Dok2) as functioning in differentiation of primitive erythrocytes. We conclude that DRAQ5 TM -based flow cytometry enables purification of primitive zebrafish erythrocytes. © 2017 John Wiley & Sons Ltd.

Behavioral and Molecular Analysis of Nicotine-Conditioned Place Preference in Zebrafish

PubMed Central

Kedikian, Ximena; Faillace, Maria Paula; Bernabeu, Ramón

2013-01-01

Studies using mice and rats have demonstrated that nicotine induces a conditioned place preference (CPP), with more effective results obtained by using biased procedures. Zebrafish have also been used as a model system to identify factors influencing nicotine-associated reward by using an unbiased design. Here, we report that zebrafish exhibited putative nicotine biased CPP to an initially aversive compartment (nicotine-paired group). A counterbalanced nicotine-exposed control group did not show a significant preference shift, providing evidence that the preference shift in the nicotine-paired group was not due to a reduction of aversion for this compartment. Zebrafish preference was corroborated by behavioral analysis of several indicators of drug preference, such as time spent in the drug-paired side, number of entries to the drug-paired side, and distance traveled. These results provided strong evidence that zebrafish may actually develop a preference for nicotine, although the drug was administrated in an aversive place for the fish, which was further supported by molecular studies. Reverse transcription-quantitative real-time PCR analysis depicted a significant increase in the expression of α7 and α6 but not α4 and β2 subunits of the nicotinic receptor in nicotine-paired zebrafish brains. In contrast, zebrafish brains from the counterbalanced nicotine group showed no significant changes. Moreover, CREB phosphorylation, an indicator of neural activity, accompanied the acquisition of nicotine-CPP. Our studies offered an incremental value to the drug addiction field, because they further describe behavioral features of CPP to nicotine in zebrafish. The results suggested that zebrafish exposed to nicotine in an unfriendly environment can develop a preference for that initially aversive place, which is likely due to the rewarding effect of nicotine. Therefore, this model can be used to screen exogenous and endogenous molecules involved in nicotine

Aspartame-fed zebrafish exhibit acute deaths with swimming defects and saccharin-fed zebrafish have elevation of cholesteryl ester transfer protein activity in hypercholesterolemia.

PubMed

Kim, Jae-Yong; Seo, Juyi; Cho, Kyung-Hyun

2011-11-01

Although many artificial sweeteners (AS) have safety issues, the AS have been widely used in industry. To determine the physiologic effect of AS in the presence of hyperlipidemia, zebrafish were fed aspartame or saccharin with a high-cholesterol diet (HCD). After 12 days, 30% of zebrafish, which consumed aspartame and HCD, died with exhibiting swimming defects. The aspartame group had 65% survivability, while the control and saccharin groups had 100% survivability. Under HCD, the saccharin-fed groups had the highest increase in the serum cholesterol level (599 mg/dL). Aspartame-fed group showed a remarkable increase in serum glucose (up to 125 mg/dL), which was 58% greater than the increase in the HCD alone group. The saccharin and HCD groups had the highest cholesteryl ester transfer protein (CETP) activity (52% CE-transfer), while the HCD alone group had 42% CE-transfer. Histologic analysis revealed that the aspartame and HCD groups showed more infiltration of inflammatory cells in the brain and liver sections. Conclusively, under presence of hyperlipidemia, aspartame-fed zebrafish exhibited acute swimming defects with an increase in brain inflammation. Saccharin-fed zebrafish had an increased atherogenic serum lipid profile with elevation of CETP activity. Copyright © 2011 Elsevier Ltd. All rights reserved.

Flat mount preparation for observation and analysis of zebrafish embryo specimens stained by whole mount in situ hybridization.

PubMed

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.

Flat Mount Preparation for Observation and Analysis of Zebrafish Embryo Specimens Stained by Whole Mount In situ Hybridization

PubMed Central

Cheng, Christina N.; Li, Yue; Marra, Amanda N.; Verdun, Valerie; Wingert, Rebecca A.

2014-01-01

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. PMID:25078510

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

PubMed

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.

Using Zebrafish to Test the Genetic Basis of Human Craniofacial Diseases.

PubMed

Machado, R Grecco; Eames, B Frank

2017-10-01

Genome-wide association studies (GWASs) opened an innovative and productive avenue to investigate the molecular basis of human craniofacial disease. However, GWASs identify candidate genes only; they do not prove that any particular one is the functional villain underlying disease or just an unlucky genomic bystander. Genetic manipulation of animal models is the best approach to reveal which genetic loci identified from human GWASs are functionally related to specific diseases. The purpose of this review is to discuss the potential of zebrafish to resolve which candidate genetic loci are mechanistic drivers of craniofacial diseases. Many anatomic, embryonic, and genetic features of craniofacial development are conserved among zebrafish and mammals, making zebrafish a good model of craniofacial diseases. Also, the ability to manipulate gene function in zebrafish was greatly expanded over the past 20 y, enabling systems such as Gateway Tol2 and CRISPR-Cas9 to test gain- and loss-of-function alleles identified from human GWASs in coding and noncoding regions of DNA. With the optimization of genetic editing methods, large numbers of candidate genes can be efficiently interrogated. Finding the functional villains that underlie diseases will permit new treatments and prevention strategies and will increase understanding of how gene pathways operate during normal development.

Differential expression of neuroligin genes in the nervous system of zebrafish.

PubMed

Davey, Crystal; Tallafuss, Alexandra; Washbourne, Philip

2010-02-01

The establishment and maturation of appropriate synaptic connections is crucial in the development of neuronal circuits. Cellular adhesion is believed to play a central role in this process. Neuroligins are neuronal cell adhesion molecules that are hypothesized to act in the initial formation and maturation of synaptic connections. In order to establish the zebrafish as a model to investigate the in vivo role of Neuroligin proteins in nervous system development, we identified the zebrafish orthologs of neuroligin family members and characterized their expression. Zebrafish possess seven neuroligin genes. Synteny analysis and sequence comparisons show that NLGN2, NLGN3, and NLGN4X are duplicated in zebrafish, but NLGN1 has a single zebrafish ortholog. All seven zebrafish neuroligins are expressed in complex patterns in the developing nervous system and in the adult brain. The spatial and temporal expression patterns of these genes suggest that they occupy a role in nervous system development and maintenance.

Differences in Acute Alcohol-Induced Behavioral Responses Among Zebrafish Populations

PubMed Central

Gerlai, Robert; Ahmad, Fahad; Prajapati, Sonal

2009-01-01

Background With the arsenal of genetic tools available for zebrafish, this species has been successfully used to investigate the genetic aspects of human diseases from developmental disorders to cancer. Interest in the behavior and brain function of zebrafish is also increasing as CNS disorders may be modeled and studied with this species. Alcoholism and alcohol abuse are among the most devastating and costliest diseases. However, the mechanisms of these diseases are not fully understood. Zebrafish has been proposed as a model organism to study such mechanisms. Characterization of alcohol’s effects on zebrafish is a necessary step in this research. Methods Here, we compare the effects of acute alcohol (EtOH) administration on the behavior of zebrafish from 4 distinct laboratory-bred populations using automated as well as observation based behavioral quantification methods. Results Alcohol treatment resulted in significant dose-dependent behavioral changes but the dose–response trajectories differed among zebrafish populations. Conclusions The results demonstrate for the first time a genetic component in alcohol responses in adult zebrafish and also show the feasibility of high throughput behavioral screening. We discuss the exploration and exploitation of the genetic differences found. PMID:18652595

Augmenter of Liver Regeneration (alr) Promotes Liver Outgrowth during Zebrafish Hepatogenesis

PubMed Central

Li, Yan; Farooq, Muhammad; Sheng, Donglai; Chandramouli, Chanchal; Lan, Tian; Mahajan, Nilesh K.; Kini, R. Manjunatha; Hong, Yunhan; Lisowsky, Thomas; Ge, Ruowen

2012-01-01

Augmenter of Liver Regeneration (ALR) is a sulfhydryl oxidase carrying out fundamental functions facilitating protein disulfide bond formation. In mammals, it also functions as a hepatotrophic growth factor that specifically stimulates hepatocyte proliferation and promotes liver regeneration after liver damage or partial hepatectomy. Whether ALR also plays a role during vertebrate hepatogenesis is unknown. In this work, we investigated the function of alr in liver organogenesis in zebrafish model. We showed that alr is expressed in liver throughout hepatogenesis. Knockdown of alr through morpholino antisense oligonucleotide (MO) leads to suppression of liver outgrowth while overexpression of alr promotes liver growth. The small-liver phenotype in alr morphants results from a reduction of hepatocyte proliferation without affecting apoptosis. When expressed in cultured cells, zebrafish Alr exists as dimer and is localized in mitochondria as well as cytosol but not in nucleus or secreted outside of the cell. Similar to mammalian ALR, zebrafish Alr is a flavin-linked sulfhydryl oxidase and mutation of the conserved cysteine in the CxxC motif abolishes its enzymatic activity. Interestingly, overexpression of either wild type Alr or enzyme-inactive AlrC131S mutant promoted liver growth and rescued the liver growth defect of alr morphants. Nevertheless, alr C131S is less efficacious in both functions. Meantime, high doses of alr MOs lead to widespread developmental defects and early embryonic death in an alr sequence-dependent manner. These results suggest that alr promotes zebrafish liver outgrowth using mechanisms that are dependent as well as independent of its sulfhydryl oxidase activity. This is the first demonstration of a developmental role of alr in vertebrate. It exemplifies that a low-level sulfhydryl oxidase activity of Alr is essential for embryonic development and cellular survival. The dose-dependent and partial suppression of alr expression through MO

Functional Investigation of a Non-coding Variant Associated with Adolescent Idiopathic Scoliosis in Zebrafish: Elevated Expression of the Ladybird Homeobox Gene Causes Body Axis Deformation

PubMed Central

Guo, Long; Yamashita, Hiroshi; Kou, Ikuyo; Takimoto, Aki; Meguro-Horike, Makiko; Horike, Shin-ichi; Sakuma, Tetsushi; Miura, Shigenori; Adachi, Taiji; Yamamoto, Takashi; Ikegawa, Shiro; Hiraki, Yuji; Shukunami, Chisa

2016-01-01

Previously, we identified an adolescent idiopathic scoliosis susceptibility locus near human ladybird homeobox 1 (LBX1) and FLJ41350 by a genome-wide association study. Here, we characterized the associated non-coding variant and investigated the function of these genes. A chromosome conformation capture assay revealed that the genome region with the most significantly associated single nucleotide polymorphism (rs11190870) physically interacted with the promoter region of LBX1-FLJ41350. The promoter in the direction of LBX1, combined with a 590-bp region including rs11190870, had higher transcriptional activity with the risk allele than that with the non-risk allele in HEK 293T cells. The ubiquitous overexpression of human LBX1 or either of the zebrafish lbx genes (lbx1a, lbx1b, and lbx2), but not FLJ41350, in zebrafish embryos caused body curvature followed by death prior to vertebral column formation. Such body axis deformation was not observed in transcription activator-like effector nucleases mediated knockout zebrafish of lbx1b or lbx2. Mosaic expression of lbx1b driven by the GATA2 minimal promoter and the lbx1b enhancer in zebrafish significantly alleviated the embryonic lethal phenotype to allow observation of the later onset of the spinal curvature with or without vertebral malformation. Deformation of the embryonic body axis by lbx1b overexpression was associated with defects in convergent extension, which is a component of the main axis-elongation machinery in gastrulating embryos. In embryos overexpressing lbx1b, wnt5b, a ligand of the non-canonical Wnt/planar cell polarity (PCP) pathway, was significantly downregulated. Injection of mRNA for wnt5b or RhoA, a key downstream effector of Wnt/PCP signaling, rescued the defective convergent extension phenotype and attenuated the lbx1b-induced curvature of the body axis. Thus, our study presents a novel pathological feature of LBX1 and its zebrafish homologs in body axis deformation at various stages of

Cell Cycle Control in the Early Embryonic Development of Aquatic Animal Species

PubMed Central

Siefert, Joseph C.; Clowdus, Emily A.; Sansam, Christopher L.

2016-01-01

The cell cycle is integrated with many aspects of embryonic development. Not only is proper control over the pace of cell proliferation important, but also the timing of cell cycle progression is coordinated with transcription, cell migration, and cell differentiation. Due to the ease with which the embryos of aquatic organisms can be observed and manipulated, they have been a popular choice for embryologists throughout history. In the cell cycle field, aquatic organisms have been extremely important because they have played a major role in the discovery and analysis of key regulators of the cell cycle. In particular, the frog Xenopus laevis has been instrumental for understanding how the basic embryonic cell cycle is regulated. More recently, the zebrafish has been used to understand how the cell cycle is remodeled during vertebrate development and how it is regulated during morphogenesis. This review describes how some of the unique strengths of aquatic species have been leveraged for cell cycle research and suggests how species such as Xenopus and zebrafish will continue to reveal the roles of the cell cycle in human biology and disease. PMID:26475527

Increased cell proliferation and neural activity by physostigmine in the telencephalon of adult zebrafish.

PubMed

Lee, Yunkyoung; Lee, Bongkyu; Jeong, Sumin; Park, Ji-Won; Han, Inn-Oc; Lee, Chang-Joong

2016-08-26

Physostigmine, an acetylcholinesterase inhibitor, is known to affect the brain function in various aspects. This study was conducted to test whether physostigmine affects cell proliferation in the telencephalon of zebrafish. BrdU-labeled cells was prominently observed in the ventral zone of the ventral telencephalon of zebrafish. The increased number of BrdU- and proliferating cell nuclear antigen-labeled cells were shown in zebrafish treated with 200μM physostigmine, which was inhibited by pretreatment with 200μM scopolamine. iNOS mRNA expression was increased in the brain of zebrafish treated with 200μM physostigmine. Consistently, aminoguanidine, an iNOS inhibitor, attenuated the increase in the number of BrdU-labeled cells by physostigmine treatment. Zebrafish also showed seizure-like locomotor activity characterized by a rapid and abrupt movement during a 30min treatment with 200μM physostigmine. Neural activity in response to an electrical stimulus was increased in the isolated telencephalon of zebrafish continuously perfused with 200μM physostigmine. None of the number of BrdU-labeled cells, neural activity, or locomotor activity was affected by treatment with 20μM physostigmine. These results suggest that 200μM physostigmine increased neural activity and induced cell proliferation via nitric oxide production in zebrafish. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

Post-Larval Developmental Trajectory of Zebrafish Fry is Altered by Exposure to T3 or T4 Analogues

EPA Science Inventory

The thyroid axis plays a key role in development. While the impacts of perturbing thyroid axis development and/or function are documented in embryonic and larval zebrafish, the effects on developmental milestones at later life stages are not well-delineated. To assess potential l...

Evolution of complexity in the zebrafish synapse proteome

PubMed Central

Bayés, Àlex; Collins, Mark O.; Reig-Viader, Rita; Gou, Gemma; Goulding, David; Izquierdo, Abril; Choudhary, Jyoti S.; Emes, Richard D.; Grant, Seth G. N.

2017-01-01

The proteome of human brain synapses is highly complex and is mutated in over 130 diseases. This complexity arose from two whole-genome duplications early in the vertebrate lineage. Zebrafish are used in modelling human diseases; however, its synapse proteome is uncharacterized, and whether the teleost-specific genome duplication (TSGD) influenced complexity is unknown. We report the characterization of the proteomes and ultrastructure of central synapses in zebrafish and analyse the importance of the TSGD. While the TSGD increases overall synapse proteome complexity, the postsynaptic density (PSD) proteome of zebrafish has lower complexity than mammals. A highly conserved set of ∼1,000 proteins is shared across vertebrates. PSD ultrastructural features are also conserved. Lineage-specific proteome differences indicate that vertebrate species evolved distinct synapse types and functions. The data sets are a resource for a wide range of studies and have important implications for the use of zebrafish in modelling human synaptic diseases. PMID:28252024

Response of avian embryonic brain to spatially segmented x-ray microbeams.

PubMed

Dilmanian, F A; Morris, G M; Le Duc, G; Huang, X; Ren, B; Bacarian, T; Allen, J C; Kalef-Ezra, J; Orion, I; Rosen, E M; Sandhu, T; Sathé, P; Wu, X Y; Zhong, Z; Shivaprasad, H L

2001-05-01

Duck embryo was studied as a model for assessing the effects of microbeam radiation therapy (MRT) on the human infant brain. Because of the high risk of radiation-induced disruption of the developmental process in the immature brain, conventional wide-beam radiotherapy of brain tumors is seldom carried out in infants under the age of three. Other types of treatment for pediatric brain tumors are frequently ineffective. Recent findings from studies in Grenoble on the brain of suckling rats indicate that MRT could be of benefit for the treatment of early childhood tumors. In our studies, duck embryos were irradiated at 3-4 days prior to hatching. Irradiation was carried out using a single exposure of synchrotron-generated X-rays, either in the form of parallel microplanar beams (microbeams), or as non-segmented broad beam. The individual microplanar beams had a width of 27 microm and height of 11 mm, and a center-to-center spacing of 100 microm. Doses to the exposed areas of embryo brain were 40, 80, 160 and 450 Gy (in-slice dose) for the microbeam, and 6, 12 and 18 Gy for the broad beam. The biological end point employed in the study was ataxia. This neurological symptom of radiation damage to the brain developed within 75 days of hatching. Histopathological analysis of brain tissue did not reveal any radiation induced lesions for microbeam doses of 40-160 Gy (in-slice), although some incidences of ataxia were observed in that dose group. However, severe brain lesions did occur in animals in the 450 Gy microbeam dose groups, and mild lesions in the 18 Gy broad beam dose group. These results indicate that embryonic duck brain has an appreciably higher tolerance to the microbeam modality, as compared to the broad beam modality. When the microbeam dose was normalized to the full volume of the irradiated tissue. i.e., the dose averaged over microbeams and the space between the microbeams, brain tolerance was estimated to be about three times higher to microbeam

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12

PubMed Central

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K.; Nielsen, G. Petur; Peterson, Randall T.; Drummond, Iain A.; Haber, Daniel A.

2014-01-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. PMID:24311731

Treatment with sodium benzoate leads to malformation of zebrafish larvae.

PubMed

Tsay, Huey-Jen; Wang, Yun-Hsin; Chen, Wei-Li; Huang, Mei-Yun; Chen, Yau-Hung

2007-01-01

Sodium benzoate (SB) is a commonly used food preservative and anti-microbial agent in many foods from soup to cereals. However, little is known about the SB-induced toxicity and teratogenicity during early embryonic development. Here, we used zebrafish as a model to test the toxicity and teratogenicity because of their transparent eggs; therefore, the organogenesis of zebrafish embryos is easy to observe. After low dosages of SB (1-1000 ppm) treatment, the zebrafish embryos exhibited a 100% survival rate. As the exposure dosages increased, the survival rates decreased. No embryos survived after treatment with 2000 ppm SB. The 50% lethal dose (LD(50)) of zebrafish is found to be in the range of 1400-1500 ppm. Gut abnormalities, malformation of pronephros, defective hatching gland and edema in pericardial sac were observed after treatment with SB. Compared to untreated littermates (vehicle-treated control), SB-treated embryos exhibited significantly reduced tactile sensitivity frequencies of touch-induced movement (vehicle-treated control: 27.60+/-1.98 v.s. 1000 ppm SB: 7.89+/-5.28; N=30). Subtle changes are easily observed by staining with specific monoclonal antibodies F59, Znp1 and alpha6F to detect morphology changes in muscle fibers, motor axons and pronephros, respectively. Our data showed that the treatment of SB led to misalignment of muscle fibers, motor neuron innervations, excess acetyl-choline receptor cluster and defective pronephric tubes. On the basis of these observations, we suggest that sodium benzoate is able to induce neurotoxicity and nephrotoxicity of zebrafish larvae.

Brain-wide mapping of neural activity controlling zebrafish exploratory locomotion

PubMed Central

Dunn, Timothy W; Mu, Yu; Narayan, Sujatha; Randlett, Owen; Naumann, Eva A; Yang, Chao-Tsung; Schier, Alexander F

2016-01-01

In the absence of salient sensory cues to guide behavior, animals must still execute sequences of motor actions in order to forage and explore. How such successive motor actions are coordinated to form global locomotion trajectories is unknown. We mapped the structure of larval zebrafish swim trajectories in homogeneous environments and found that trajectories were characterized by alternating sequences of repeated turns to the left and to the right. Using whole-brain light-sheet imaging, we identified activity relating to the behavior in specific neural populations that we termed the anterior rhombencephalic turning region (ARTR). ARTR perturbations biased swim direction and reduced the dependence of turn direction on turn history, indicating that the ARTR is part of a network generating the temporal correlations in turn direction. We also find suggestive evidence for ARTR mutual inhibition and ARTR projections to premotor neurons. Finally, simulations suggest the observed turn sequences may underlie efficient exploration of local environments. DOI: http://dx.doi.org/10.7554/eLife.12741.001 PMID:27003593

Identification of Chemical Vascular Disruptors During Development Using An Integrative Predictive Toxicity Model and Zebrafish and in Vitro Functional Angiogenesis Assays.

EPA Science Inventory

Identification of chemical vascular disruptors during development using an integrative predictive toxicity model and zebrafish and in vitro functional angiogenesis assays Chemically-induced vascular toxicity during embryonic development can result in a wide range of adverse pre...

Ion transport in the zebrafish kidney from a human disease angle: possibilities, considerations, and future perspectives.

PubMed

Kersten, Simone; Arjona, Francisco J

2017-01-01

Unique experimental advantages, such as its embryonic/larval transparency, high-throughput nature, and ease of genetic modification, underpin the rapid emergence of the zebrafish (Danio rerio) as a preeminent model in biomedical research. Particularly in the field of nephrology, the zebrafish provides a promising model for studying the physiological implications of human solute transport processes along consecutive nephron segments. However, although the zebrafish might be considered a valuable model for numerous renal ion transport diseases and functional studies of many channels and transporters, not all human renal electrolyte transport mechanisms and human diseases can be modeled in the zebrafish. With this review, we explore the ontogeny of zebrafish renal ion transport, its nephron structure and function, and thereby demonstrate the clinical translational value of this model. By critical assessment of genomic and amino acid conservation of human proteins involved in renal ion handling (channels, transporters, and claudins), kidney and nephron segment conservation, and renal electrolyte transport physiology in the zebrafish, we provide researchers and nephrologists with an indication of the possibilities and considerations of the zebrafish as a model for human renal ion transport. Combined with advanced techniques envisioned for the future, implementation of the zebrafish might expand beyond unraveling pathophysiological mechanisms that underlie distinct genetic or environmentally, i.e., pharmacological and lifestyle, induced renal transport deficits. Specifically, the ease of drug administration and the exploitation of improved genetic approaches might argue for the adoption of the zebrafish as a model for preclinical personalized medicine for distinct renal diseases and renal electrolyte transport proteins. Copyright © 2017 the American Physiological Society.

Stimulus-triggered enhancement of chilling tolerance in zebrafish embryos

PubMed Central

Szabó, Katalin; Budai, Csilla; Losonczi, Eszter; Bernáth, Gergely; Csenki-Bakos, Zsolt; Urbányi, Béla; Pribenszky, Csaba; Horváth, Ákos; Cserepes, Judit

2017-01-01

Background Cryopreservation of zebrafish embryos is still an unsolved problem despite market demand and massive efforts to preserve genetic variation among numerous existing lines. Chilled storage of embryos might be a step towards developing successful cryopreservation, but no methods to date have worked. Methods In the present study, we applied a novel strategy to improve the chilling tolerance of zebrafish embryos by introducing a preconditioning hydrostatic pressure treatment to the embryos. In our experiments, 26-somites and Prim-5 stage zebrafish embryos were chilled at 0°C for 24 hours after preconditioning. Embryo survival rate, ability to reach maturation and fertilizing capacity were tested. Results Our results indicate that applied preconditioning technology made it possible for the chilled embryos to develop normally until maturity, and to produce healthy offspring as normal, thus passing on their genetic material successfully. Treated embryos had a significantly higher survival and better developmental rate, moreover the treated group had a higher ratio of normal morphology during continued development. While all controls from chilled embryos died by 30 day-post-fertilization, the treated group reached maturity (~90–120 days) and were able to reproduce, resulting in offspring in expected quantity and quality. Conclusions Based on our results, we conclude that the preconditioning technology represents a significant improvement in zebrafish embryo chilling tolerance, thus enabling a long-time survival. Furthermore, as embryonic development is arrested during chilled storage this technology also provides a solution to synchronize or delay the development. PMID:28166301

Part II: morphological analysis of embryonic development following femtosecond laser manipulation

NASA Astrophysics Data System (ADS)

Kohli, V.; Elezzabi, A. Y.

2008-02-01

The zebrafish (Danio rerio) is an attractive model system that has received wide attention for its usefulness in the study of development and disease. This organism represents a closer analog to humans than the common invetebrates Drosophila melanogaster and Caenorhabditis elegans, making this species an ideal model for human health research. Non-invasive manipulation of the zebrafish has been challenging, owing to the outer proteinaceous membrane and multiple embryonic barriers. A novel tool capable of manipulating early cleavage stage embryonic cells would be important for future advancements in medial research and the aquaculture industry. Herein, we demonstrate the laser surgery of early cleavage stage (2-cell) blastomere cells using a range of average laser powers and beam dwell times. Since the novelty of this manipulation tool depends on its non-invasive application, we examined short- and long-term laser-induced developmental defects following embryonic surgery. Laser-manipulated embryos were reared to 2 and 7 days post-fertilization and compared to control embryos at the same developmental stages. Morphological analysis was performed using light microscopy and scanning electron microscopy. Developmental features that were examined included the antero- and dorsal-lateral whole body views of the larvae, the olfactory pit, dorsal, ventral and pectoral fins, notochord, pectoral fin buds, otic capsule, otic vesicle, neuromast patterning, and kinocilia of the olfactory pit rim and cristae of the lateral wall of the ear. Laser-manipulated embryos developed normally relative to the controls, with developmental patterning and morphology at 2 and 7 days indistinguishable from control larvae.

Movement maintains forebrain neurogenesis via peripheral neural feedback in larval zebrafish

PubMed Central

Hall, Zachary Jonas

2018-01-01

The postembryonic brain exhibits experience-dependent development, in which sensory experience guides normal brain growth. This neuroplasticity is thought to occur primarily through structural and functional changes in pre-existing neurons. Whether neurogenesis also mediates the effects of experience on brain growth is unclear. Here, we characterized the importance of motor experience on postembryonic neurogenesis in larval zebrafish. We found that movement maintains an expanded pool of forebrain neural precursors by promoting progenitor self-renewal over the production of neurons. Physical cues associated with swimming (bodily movement) increase neurogenesis and these cues appear to be conveyed by dorsal root ganglia (DRG) in the zebrafish body: DRG-deficient larvae exhibit attenuated neurogenic responses to movement and targeted photoactivation of DRG in immobilized larvae expands the pallial pool of proliferative cells. Our results demonstrate the importance of movement in neurogenic brain growth and reveal a fundamental sensorimotor association that may couple early motor and brain development. PMID:29528285

Multidimensional In Vivo Hazard Assessment Using Zebrafish

PubMed Central

Tanguay, Robert L.

2014-01-01

There are tens of thousands of man-made chemicals in the environment; the inherent safety of most of these chemicals is not known. Relevant biological platforms and new computational tools are needed to prioritize testing of chemicals with limited human health hazard information. We describe an experimental design for high-throughput characterization of multidimensional in vivo effects with the power to evaluate trends relating to commonly cited chemical predictors. We evaluated all 1060 unique U.S. EPA ToxCast phase 1 and 2 compounds using the embryonic zebrafish and found that 487 induced significant adverse biological responses. The utilization of 18 simultaneously measured endpoints means that the entire system serves as a robust biological sensor for chemical hazard. The experimental design enabled us to describe global patterns of variation across tested compounds, evaluate the concordance of the available in vitro and in vivo phase 1 data with this study, highlight specific mechanisms/value-added/novel biology related to notochord development, and demonstrate that the developmental zebrafish detects adverse responses that would be missed by less comprehensive testing strategies. PMID:24136191

Physiological roles of glucocorticoids during early embryonic development of the zebrafish (Danio rerio)

PubMed Central

Wilson, K S; Matrone, G; Livingstone, D E W; Al-Dujaili, E A S; Mullins, J J; Tucker, C S; Hadoke, P W F; Kenyon, C J; Denvir, M A

2013-01-01

While glucocorticoids (GCs) are known to be present in the zebrafish embryo, little is known about their physiological roles at this stage. We hypothesised that GCs play key roles in stress response, hatching and swim activity during early development. To test this, whole embryo cortisol (WEC) and corticosteroid-related genes were measured in embryos from 6 to 120 h post fertilisation (hpf) by enzyme linked immunosorbent assay (ELISA) and quantitative real-time polymerase chain reaction (qRT-PCR). Stress response was assessed by change in WEC following stirring, hypoxia or brief electrical impulses applied to the bathing water. The impact of pharmacological and molecular GC manipulation on the stress response, spontaneous hatching and swim activity at different stages of development was also assessed. WEC levels demonstrated a biphasic pattern during development with a decrease from 0 to 36 hpf followed by a progressive increase towards 120 hpf. This was accompanied by a significant and sustained increase in the expression of genes encoding cyp11b1 (GC biosynthesis), hsd11b2 (GC metabolism) and gr (GC receptor) from 48 to 120 hpf. Metyrapone (Met), an inhibitor of 11β-hydroxylase (encoded by cyp11b1), and cyp11b1 morpholino (Mo) knockdown significantly reduced basal and stress-induced WEC levels at 72 and 120 hpf but not at 24 hpf. Spontaneous hatching and swim activity were significantly affected by manipulation of GC action from approximately 48 hpf onwards. We have identified a number of key roles of GCs in zebrafish embryos contributing to adaptive physiological responses under adverse conditions. The ability to alter GC action in the zebrafish embryo also highlights its potential value for GC research. PMID:24167225

Early embryonic androgen exposure induces transgenerational epigenetic and metabolic changes.

PubMed

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.

Small heat shock proteins are necessary for heart migration and laterality determination in zebrafish

PubMed Central

Lahvic, Jamie L.; Ji, Yongchang; Marin, Paloma; Zuflacht, Jonah P.; Springel, Mark W.; Wosen, Jonathan E.; Davis, Leigh; Hutson, Lara D.; Amack, Jeffrey D.; Marvin, Martha J.

2013-01-01

Small heat shock proteins (sHsps) regulate cellular functions not only under stress, but also during normal development, when they are expressed in organ-specific patterns. Here we demonstrate that two small heat shock proteins expressed in embryonic zebrafish heart, hspb7 and hspb12, have roles in the development of left-right asymmetry. In zebrafish, laterality is determined by the motility of cilia in Kupffer’s vesicle (KV), where hspb7 is expressed; knockdown of hspb7 causes laterality defects by disrupting the motility of these cilia. In embryos with reduced hspb7, the axonemes of KV cilia have a 9+0 structure, while control embyros have a predominately 9+2 structure. Reduction of either hspb7 or hspb12 alters the expression pattern of genes that propagate the signals that establish left-right asymmetry: the nodal-related gene southpaw (spaw) in the lateral plate mesoderm, and its downstream targets pitx2, lefty1 and lefty2. Partial depletion of hspb7 causes concordant heart, brain and visceral laterality defects, indicating that loss of KV cilia motility leads causes coordinated but randomized laterality. Reducing hspb12 leads to similar alterations in the expression of downstream laterality genes, but at a lower penetrance. Simultaneous reduction of hspb7 and hspb12 randomizes heart, brain and visceral laterality, suggesting that these two genes have partially redundant functions in the establishment of left-right asymmetry. In addition, both hspb7 and hspb12 are expressed in the precardiac mesoderm and in the yolk syncytial layer, which supports the migration and fusion of mesodermal cardiac precursors. In embryos in which the reduction of hspb7 or hspb12 was limited to the yolk, migration defects predominated, suggesting that the yolk expression of these genes rather than heart expression is responsible for the migration defects. PMID:24140541

Satellite-like cells contribute to pax7-dependent skeletal muscle repair in adult zebrafish

PubMed Central

Berberoglu, Michael A.; Gallagher, Thomas L.; Morrow, Zachary T.; Talbot, Jared C.; Hromowyk, Kimberly J.; Tenente, Inês M.; Langenau, David M.; Amacher, Sharon L.

2017-01-01

Satellite cells, also known as muscle stem cells, are responsible for skeletal muscle growth and repair in mammals. Pax7 and Pax3 transcription factors are established satellite cell markers required for muscle development and regeneration, and there is great interest in identifying additional factors that regulate satellite cell proliferation, differentiation, and/or skeletal muscle regeneration. Due to the powerful regenerative capacity of many zebrafish tissues, even in adults, we are exploring the regenerative potential of adult zebrafish skeletal muscle. Here, we show that adult zebrafish skeletal muscle contains cells similar to mammalian satellite cells. Adult zebrafish satellite-like cells have dense heterochromatin, express Pax7 and Pax3, proliferate in response to injury, and show peak myogenic responses 4–5 days post-injury (dpi). Furthermore, using a pax7a-driven GFP reporter, we present evidence implicating satellite-like cells as a possible source of new muscle. In lieu of central nucleation, which distinguishes regenerating myofibers in mammals, we describe several characteristics that robustly identify newly-forming myofibers from surrounding fibers in injured adult zebrafish muscle. These characteristics include partially overlapping expression in satellite cells and regenerating myofibers of two RNA-binding proteins Rbfox2 and Rbfoxl1, known to regulate embryonic muscle development and function. Finally, by analyzing pax7a; pax7b double mutant zebrafish, we show that Pax7 is required for adult skeletal muscle repair, as it is in the mouse. PMID:28279710

Mind the fish: zebrafish as a model in cognitive social neuroscience

PubMed Central

Oliveira, Rui F.

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed. PMID:23964204

Mind the fish: zebrafish as a model in cognitive social neuroscience.

PubMed

Oliveira, Rui F

2013-01-01

Understanding how the brain implements social behavior on one hand, and how social processes feedback on the brain to promote fine-tuning of behavioral output according to changes in the social environment is a major challenge in contemporary neuroscience. A critical step to take this challenge successfully is finding the appropriate level of analysis when relating social to biological phenomena. Given the enormous complexity of both the neural networks of the brain and social systems, the use of a cognitive level of analysis (in an information processing perspective) is proposed here as an explanatory interface between brain and behavior. A conceptual framework for a cognitive approach to comparative social neuroscience is proposed, consisting of the following steps to be taken across different species with varying social systems: (1) identification of the functional building blocks of social skills; (2) identification of the cognitive mechanisms underlying the previously identified social skills; and (3) mapping these information processing mechanisms onto the brain. Teleost fish are presented here as a group of choice to develop this approach, given the diversity of social systems present in closely related species that allows for planned phylogenetic comparisons, and the availability of neurogenetic tools that allows the visualization and manipulation of selected neural circuits in model species such as the zebrafish. Finally, the state-of-the art of zebrafish social cognition and of the tools available to map social cognitive abilities to neural circuits in zebrafish are reviewed.

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

PubMed

Chatterjee, Aniruddha; Lagisz, Malgorzata; Rodger, Euan J; Zhen, Li; Stockwell, Peter A; Duncan, Elizabeth J; Horsfield, Julia A; Jeyakani, Justin; Mathavan, Sinnakaruppan; Ozaki, Yuichi; Nakagawa, Shinichi

2016-09-30

The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time. Copyright © 2016 Elsevier B.V. All rights reserved.

High-throughput identification of small molecules that affect human embryonic vascular development

PubMed Central

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R.; Honório, Inês; de Vries, Margreet R.; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H. A.; Pereira, Carlos F.; Mercader, Nadia; Ferreira, Lino

2017-01-01

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature. PMID:28348206

High-throughput identification of small molecules that affect human embryonic vascular development.

PubMed

Vazão, Helena; Rosa, Susana; Barata, Tânia; Costa, Ricardo; Pitrez, Patrícia R; Honório, Inês; de Vries, Margreet R; Papatsenko, Dimitri; Benedito, Rui; Saris, Daniel; Khademhosseini, Ali; Quax, Paul H A; Pereira, Carlos F; Mercader, Nadia; Fernandes, Hugo; Ferreira, Lino

2017-04-11

Birth defects, which are in part caused by exposure to environmental chemicals and pharmaceutical drugs, affect 1 in every 33 babies born in the United States each year. The current standard to screen drugs that affect embryonic development is based on prenatal animal testing; however, this approach yields low-throughput and limited mechanistic information regarding the biological pathways and potential adverse consequences in humans. To develop a screening platform for molecules that affect human embryonic development based on endothelial cells (ECs) derived from human pluripotent stem cells, we differentiated human pluripotent stem cells into embryonic ECs and induced their maturation under arterial flow conditions. These cells were then used to screen compounds that specifically affect embryonic vasculature. Using this platform, we have identified two compounds that have higher inhibitory effect in embryonic than postnatal ECs. One of them was fluphenazine (an antipsychotic), which inhibits calmodulin kinase II. The other compound was pyrrolopyrimidine (an antiinflammatory agent), which inhibits vascular endothelial growth factor receptor 2 (VEGFR2), decreases EC viability, induces an inflammatory response, and disrupts preformed vascular networks. The vascular effect of the pyrrolopyrimidine was further validated in prenatal vs. adult mouse ECs and in embryonic and adult zebrafish. We developed a platform based on human pluripotent stem cell-derived ECs for drug screening, which may open new avenues of research for the study and modulation of embryonic vasculature.

Prolactin-dependent modulation of organogenesis in the vertebrate: Recent discoveries in zebrafish.

PubMed

Nguyen, Nhu; Stellwag, Edmund J; Zhu, Yong

2008-11-01

The scientific literature is replete with evidence of the multifarious functions of the prolactin (PRL)/growth hormone (GH) superfamily in adult vertebrates. However, little information is available on the roles of PRL and related hormones prior to the adult stage of development. A limited number of studies suggest that GH functions to stimulate glucose transport and protein synthesis in mouse blastocytes and may be involved during mammalian embryogenesis. In contrast, the evidence for a role of PRL during vertebrate embryogenesis is limited and controversial. Genes encoding GH/PRL hormones and their respective receptors are actively transcribed and translated in various animal models at different time points, particularly during tissue remodeling. We have addressed the potential function of GH/PRL hormones during embryonic development in zebrafish by the temporary inhibition of in vivo PRL translation. This treatment caused multiple morphological defects consistent with a role of PRL in embryonic-stage organogenesis. The affected organs and tissues are known targets of PRL activity in fish and homologous structures in mammalian species. Traditionally, the GH/PRL hormones are viewed as classical endocrine hormones, mediating functions through the circulatory system. More recent evidence points to cytokine-like actions of these hormones through either an autocrine or a paracrine mechanism. In some situations they could mimic actions of developmentally regulated genes as suggested by experiments in multiple organisms. In this review, we present similarities and disparities between zebrafish and mammalian models in relation to PRL and PRLR activity. We conclude that the zebrafish could serve as a suitable alternative to the rodent model to study PRL functions in development, especially in relation to organogenesis.

FishFace: interactive atlas of zebrafish craniofacial development at cellular resolution

PubMed Central

2013-01-01

Background The vertebrate craniofacial skeleton may exhibit anatomical complexity and diversity, but its genesis and evolution can be understood through careful dissection of developmental programs at cellular resolution. Resources are lacking that include introductory overviews of skeletal anatomy coupled with descriptions of craniofacial development at cellular resolution. In addition to providing analytical guidelines for other studies, such an atlas would suggest cellular mechanisms underlying development. Description We present the Fish Face Atlas, an online, 3D-interactive atlas of craniofacial development in the zebrafish Danio rerio. Alizarin red-stained skulls scanned by fluorescent optical projection tomography and segmented into individual elements provide a resource for understanding the 3D structure of the zebrafish craniofacial skeleton. These data provide the user an anatomical entry point to confocal images of Alizarin red-stained zebrafish with transgenically-labelled pharyngeal arch ectomesenchyme, chondrocytes, and osteoblasts, which illustrate the appearance, morphogenesis, and growth of the mandibular and hyoid cartilages and bones, as viewed in live, anesthetized zebrafish during embryonic and larval development. Confocal image stacks at high magnification during the same stages provide cellular detail and suggest developmental and evolutionary hypotheses. Conclusion The FishFace Atlas is a novel learning tool for understanding craniofacial skeletal development, and can serve as a reference for a variety of studies, including comparative and mutational analyses. PMID:23714426

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

PubMed Central

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

2016-01-01

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. PMID:27468180

A forward chemical screen in zebrafish identifies a retinoic acid derivative with receptor specificity.

PubMed

Das, Bhaskar C; McCartin, Kellie; Liu, Ting-Chun; Peterson, Randall T; Evans, Todd

2010-04-02

Retinoids regulate key developmental pathways throughout life, and have potential uses for differentiation therapy. It should be possible to identify novel retinoids by coupling new chemical reactions with screens using the zebrafish embryonic model. We synthesized novel retinoid analogues and derivatives by amide coupling, obtaining 80-92% yields. A small library of these compounds was screened for bioactivity in living zebrafish embryos. We found that several structurally related compounds significantly affect development. Distinct phenotypes are generated depending on time of exposure, and we characterize one compound (BT10) that produces specific cardiovascular defects when added 1 day post fertilization. When compared to retinoic acid (ATRA), BT10 shows similar but not identical changes in the expression pattern of embryonic genes that are known targets of the retinoid pathway. Reporter assays determined that BT10 interacts with all three RAR receptor sub-types, but has no activity for RXR receptors, at all concentrations tested. Our screen has identified a novel retinoid with specificity for retinoid receptors. This lead compound may be useful for manipulating components of retinoid signaling networks, and may be further derivatized for enhanced activity.

Induction of Social Behavior in Zebrafish: Live Versus Computer Animated Fish as Stimuli

PubMed Central

Qin, Meiying; Wong, Albert; Seguin, Diane

2014-01-01

Abstract The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish. PMID:24575942

Induction of social behavior in zebrafish: live versus computer animated fish as stimuli.

PubMed

Qin, Meiying; Wong, Albert; Seguin, Diane; Gerlai, Robert

2014-06-01

The zebrafish offers an excellent compromise between system complexity and practical simplicity and has been suggested as a translational research tool for the analysis of human brain disorders associated with abnormalities of social behavior. Unlike laboratory rodents zebrafish are diurnal, thus visual cues may be easily utilized in the analysis of their behavior and brain function. Visual cues, including the sight of conspecifics, have been employed to induce social behavior in zebrafish. However, the method of presentation of these cues and the question of whether computer animated images versus live stimulus fish have differential effects have not been systematically analyzed. Here, we compare the effects of five stimulus presentation types: live conspecifics in the experimental tank or outside the tank, playback of video-recorded live conspecifics, computer animated images of conspecifics presented by two software applications, the previously employed General Fish Animator, and a new application Zebrafish Presenter. We report that all stimuli were equally effective and induced a robust social response (shoaling) manifesting as reduced distance between stimulus and experimental fish. We conclude that presentation of live stimulus fish, or 3D images, is not required and 2D computer animated images are sufficient to induce robust and consistent social behavioral responses in zebrafish.

Toxicity evaluation of β-diketone antibiotics on the development of embryo-larval zebrafish (Danio rerio).

PubMed

Wang, Huili; Che, Baoguang; Duan, Ailian; Mao, Jingwen; Dahlgren, Randy A; Zhang, Minghua; Zhang, Hongqin; Zeng, Aibing; Wang, Xuedong

2014-10-01

This study evaluated the effects of β-diketone antibiotics (DKAs) on the development of embryo-larval zebrafish (Danio rerio). When exposure to DKAs, developmental malformations, such as hatching delay, curved body axis, pericardial edema, uninflated swim bladder and yolk sac edema, were observed at 120 h postfertilization (hpf). The estimated 120 hpf nominal concentrations of no observed effect concentration and lowest observed effect concentration for DKAs were 18.75 and 37.50 mg/L, respectively, suggesting that DKAs have much lower toxicity than other persistent pollutants. Following DKA exposure, embryonic heart rates were significantly reduced as compared to the controls at 48 and 60 hpf. The peak bending motion frequency appeared 1 h earlier than in control embryos. The 2.34 and 9.38-mg/L treatment groups had a higher basal swim rate than control groups at 120 hpf in both light and light-to-dark photoperiod experiments. The occurrence of high speed swim rates was enhanced approximately threefold to sevenfold in the 2.34 and 9.38 mg/L treatments compared to the control. Glutathione (GSH) concentrations in the 2.34 and 9.38-mg/L treatments were significantly higher than the control at 72 hpf, suggesting that GSH production was induced at the end of the hatching period. When exposed to DKAs, zebrafish superoxide dismutase enzyme (SOD) activities were significantly inhibited in the early embryonic period, demonstrating that the clearing ability in zebrafish was lower than the generation rate of free radicals. In summary, the combined DKAs were developmentally toxic to zebrafish in their early life stages and had the ability to impair individual behaviors that are of great importance in the assessment of their ecological fitness. Copyright © 2013 Wiley Periodicals, Inc., a Wiley company.

The Morphogenesis of Cranial Sutures in Zebrafish

PubMed Central

Topczewska, Jolanta M.; Shoela, Ramy A.; Tomaszewski, Joanna P.; Mirmira, Rupa B.; Gosain, Arun K.

2016-01-01

Using morphological, histological, and TEM analyses of the cranium, we provide a detailed description of bone and suture growth in zebrafish. Based on expression patterns and localization, we identified osteoblasts at different degrees of maturation. Our data confirm that, unlike in humans, zebrafish cranial sutures maintain lifelong patency to sustain skull growth. The cranial vault develops in a coordinated manner resulting in a structure that protects the brain. The zebrafish cranial roof parallels that of higher vertebrates and contains five major bones: one pair of frontal bones, one pair of parietal bones, and the supraoccipital bone. Parietal and frontal bones are formed by intramembranous ossification within a layer of mesenchyme positioned between the dermal mesenchyme and meninges surrounding the brain. The supraoccipital bone has an endochondral origin. Cranial bones are separated by connective tissue with a distinctive architecture of osteogenic cells and collagen fibrils. Here we show RNA in situ hybridization for col1a1a, col2a1a, col10a1, bglap/osteocalcin, fgfr1a, fgfr1b, fgfr2, fgfr3, foxq1, twist2, twist3, runx2a, runx2b, sp7/osterix, and spp1/ osteopontin, indicating that the expression of genes involved in suture development in mammals is preserved in zebrafish. We also present methods for examining the cranium and its sutures, which permit the study of the mechanisms involved in suture patency as well as their pathological obliteration. The model we develop has implications for the study of human disorders, including craniosynostosis, which affects 1 in 2,500 live births. PMID:27829009

Zebrafish on a chip: a novel platform for real-time monitoring of drug-induced developmental toxicity.

PubMed

Li, Yinbao; Yang, Fan; Chen, Zuanguang; Shi, Lijuan; Zhang, Beibei; Pan, Jianbin; Li, Xinchun; Sun, Duanping; Yang, Hongzhi

2014-01-01

Pharmaceutical safety testing requires a cheap, fast and highly efficient platform for real-time evaluation of drug toxicity and secondary effects. In this study, we have developed a microfluidic system for phenotype-based evaluation of toxic and teratogenic effects of drugs using zebrafish (Danio rerio) embryos and larvae as the model organism. The microfluidic chip is composed of two independent functional units, enabling the assessment of zebrafish embryos and larvae. Each unit consists of a fluidic concentration gradient generator and a row of seven culture chambers to accommodate zebrafish. To test the accuracy of this new chip platform, we examined the toxicity and teratogenicity of an anti-asthmatic agent-aminophylline (Apl) on 210 embryos and 210 larvae (10 individuals per chamber). The effect of Apl on zebrafish embryonic development was quantitatively assessed by recording a series of physiological indicators such as heart rate, survival rate, body length and hatch rate. Most importantly, a new index called clonic convulsion rate, combined with mortality was used to evaluate the toxicities of Apl on zebrafish larvae. We found that Apl can induce deformity and cardiovascular toxicity in both zebrafish embryos and larvae. This microdevice is a multiplexed testing apparatus that allows for the examination of indexes beyond toxicity and teratogenicity at the sub-organ and cellular levels and provides a potentially cost-effective and rapid pharmaceutical safety assessment tool.

Brain Transcriptomic Response to Social Eavesdropping in Zebrafish (Danio rerio)

PubMed Central

Oliveira, Rui F.

2015-01-01

Public information is widely available at low cost to animals living in social groups. For instance, bystanders may eavesdrop on signaling interactions between conspecifics and use it to adapt their subsequent behavior towards the observed individuals. This social eavesdropping ability is expected to require specialized mechanisms such as social attention, which selects social information available for learning. To begin exploring the genetic basis of social eavesdropping, we used a previously established attention paradigm in the lab to study the brain gene expression profile of male zebrafish (Danio rerio) in relation to the attention they paid towards conspecifics involved or not involved in agonistic interactions. Microarray gene chips were used to characterize their brain transcriptomes based on differential expression of single genes and gene sets. These analyses were complemented by promoter region-based techniques. Using data from both approaches, we further drafted protein interaction networks. Our results suggest that attentiveness towards conspecifics, whether interacting or not, activates pathways linked to neuronal plasticity and memory formation. The network analyses suggested that fos and jun are key players on this response, and that npas4a, nr4a1 and egr4 may also play an important role. Furthermore, specifically observing fighting interactions further triggered pathways associated to a change in the alertness status (dnajb5) and to other genes related to memory formation (btg2, npas4b), which suggests that the acquisition of eavesdropped information about social relationships activates specific processes on top of those already activated just by observing conspecifics. PMID:26713440

Zebrafish models for the functional genomics of neurogenetic disorders.

PubMed

Kabashi, Edor; Brustein, Edna; Champagne, Nathalie; Drapeau, Pierre

2011-03-01

In this review, we consider recent work using zebrafish to validate and study the functional consequences of mutations of human genes implicated in a broad range of degenerative and developmental disorders of the brain and spinal cord. Also we present technical considerations for those wishing to study their own genes of interest by taking advantage of this easily manipulated and clinically relevant model organism. Zebrafish permit mutational analyses of genetic function (gain or loss of function) and the rapid validation of human variants as pathological mutations. In particular, neural degeneration can be characterized at genetic, cellular, functional, and behavioral levels. Zebrafish have been used to knock down or express mutations in zebrafish homologs of human genes and to directly express human genes bearing mutations related to neurodegenerative disorders such as spinal muscular atrophy, ataxia, hereditary spastic paraplegia, amyotrophic lateral sclerosis (ALS), epilepsy, Huntington's disease, Parkinson's disease, fronto-temporal dementia, and Alzheimer's disease. More recently, we have been using zebrafish to validate mutations of synaptic genes discovered by large-scale genomic approaches in developmental disorders such as autism, schizophrenia, and non-syndromic mental retardation. Advances in zebrafish genetics such as multigenic analyses and chemical genetics now offer a unique potential for disease research. Thus, zebrafish hold much promise for advancing the functional genomics of human diseases, the understanding of the genetics and cell biology of degenerative and developmental disorders, and the discovery of therapeutics. This article is part of a Special Issue entitled Zebrafish Models of Neurological Diseases. Copyright © 2010 Elsevier B.V. All rights reserved.

Pharmacological analyses of learning and memory in zebrafish (Danio rerio).

PubMed

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.

Intrinsic Properties of Larval Zebrafish Neurons in Ethanol

PubMed Central

Ikeda, Hiromi; Delargy, Alison H.; Yokogawa, Tohei; Urban, Jason M.; Burgess, Harold A.; Ono, Fumihito

2013-01-01

The behavioral effects of ethanol have been studied in multiple animal models including zebrafish. Locomotion of zebrafish larvae is resistant to high concentrations of ethanol in bath solution. This resistance has been attributed to a lower systemic concentration of ethanol in zebrafish when compared with bath solution, although the mechanism to maintain such a steep gradient is unclear. Here we examined whether the intrinsic properties of neurons play roles in this resistance. In order to minimize the contribution of metabolism and diffusional barriers, larvae were hemisected and the anterior half immersed in a range of ethanol concentrations thereby ensuring the free access of bath ethanol to the brain. The response to vibrational stimuli of three types of reticulospinal neurons: Mauthner neurons, vestibulospinal neurons, and MiD3 neurons were examined using an intracellular calcium indicator. The intracellular [Ca2+] response in MiD3 neurons decreased in 100 mM ethanol, while Mauthner neurons and vestibulospinal neurons required >300 mM ethanol to elicit similar effects. The ethanol effect in Mauthner neurons was reversible following removal of ethanol. Interestingly, activities of MiD3 neurons displayed spontaneous recovery in 300 mM ethanol, suggestive of acute tolerance. Finally, we examined with mechanical vibration the startle response of free-swimming larvae in 300 mM ethanol. Ethanol treatment abolished long latency startle responses, suggesting a functional change in neural processing. These data support the hypothesis that individual neurons in larval zebrafish brains have distinct patterns of response to ethanol dictated by specific molecular targets. PMID:23658822

Embryotoxicity of poorly soluble nanoparticles at various stages of Zebrafish development

NASA Astrophysics Data System (ADS)

Morgaleva, Tamara; Morgalev, Yuri; Gosteva, Irina; Morgalev, Sergey; Nesterenya, Daria

2017-11-01

The biological effects of the poorly soluble nanoparticles (NPs) of different chemical nature and structural characteristics were evaluated. It was established that the Zebrafish test response to contamination of aqueous medium with nickel NPs (nNi), platinum (nPt), zinc oxide (nZnO) and cerium oxide (nCeO2) depends on the physicochemical properties of the NPs and embryo development stage. The concentrations of NPs not causing disruptions in embryonic development of Zebrafish were determined. The smallest impact on embryogenesis was exerted by nCeO2: coagulation of a small number of embryos was observed only at C = 20.0 mg/L. The same effect was observed when exposed to lower concentrations of nPt (C = 5.0 mg/L) and nNi (C = 0.1 mg/L). The greatest number of coagulated embryos was observed when grown in the DS nZnO: 37.5% of embryos died at the DS concentration of C = 0.1 mg/L. Zebrafish cultivation in the DS with low concentrations (C ≤ LC10) of nNi and nZnO caused distortions in the development of embryos: development of scoliosis, malformation of somites, inhibited mobility.

Using visual lateralization to model learning and memory in zebrafish larvae

PubMed Central

Andersson, Madelene Åberg; Ek, Fredrik; Olsson, Roger

2015-01-01

Impaired learning and memory are common symptoms of neurodegenerative and neuropsychiatric diseases. Present, there are several behavioural test employed to assess cognitive functions in animal models, including the frequently used novel object recognition (NOR) test. However, although atypical functional brain lateralization has been associated with neuropsychiatric conditions, spanning from schizophrenia to autism, few animal models are available to study this phenomenon in learning and memory deficits. Here we present a visual lateralization NOR model (VLNOR) in zebrafish larvae as an assay that combines brain lateralization and NOR. In zebrafish larvae, learning and memory are generally assessed by habituation, sensitization, or conditioning paradigms, which are all representatives of nondeclarative memory. The VLNOR is the first model for zebrafish larvae that studies a memory similar to the declarative memory described for mammals. We demonstrate that VLNOR can be used to study memory formation, storage, and recall of novel objects, both short and long term, in 10-day-old zebrafish. Furthermore we show that the VLNOR model can be used to study chemical modulation of memory formation and maintenance using dizocilpine (MK-801), a frequently used non-competitive antagonist of the NMDA receptor, used to test putative antipsychotics in animal models. PMID:25727677

rbm47, a novel RNA binding protein, regulates zebrafish head development.

PubMed

Guan, Rui; El-Rass, Suzan; Spillane, David; Lam, Simon; Wang, Yuodong; Wu, Jing; Chen, Zhuchu; Wang, Anan; Jia, Zhengping; Keating, Armand; Hu, Jim; Wen, Xiao-Yan

2013-12-01

Vertebrate trunk induction requires inhibition of bone morphogenetic protein (BMP) signaling, whereas vertebrate head induction requires concerted inhibition of both Wnt and BMP signaling. RNA binding proteins play diverse roles in embryonic development and their roles in vertebrate head development remain to be elucidated. We first characterized the human RBM47 as an RNA binding protein that specifically binds RNA but not single-stranded DNA. Next, we knocked down rbm47 gene function in zebrafish using morpholinos targeting the start codon and exon-1/intron-1 splice junction. Down-regulation of rbm47 resulted in headless and small head phenotypes, which can be rescued by a wnt8a blocking morpholino. To further reveal the mechanism of rbm47's role in head development, microarrays were performed to screen genes differentially expressed in normal and knockdown embryos. epcam and a2ml were identified as the most significantly up- and down-regulated genes, respectively. The microarrays also confirmed up-regulation of several genes involved in head development, including gsk3a, otx2, and chordin, which are important regulators of Wnt signaling. Altogether, our findings reveal that Rbm47 is a novel RNA-binding protein critical for head formation and embryonic patterning during zebrafish embryogenesis which may act through a Wnt8a signaling pathway. Copyright © 2013 Wiley Periodicals, Inc.

Multilayer mounting for long-term light sheet microscopy of zebrafish.

PubMed

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-02-27

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology.

A zebrafish model of chordoma initiated by notochord-driven expression of HRASV12.

PubMed

Burger, Alexa; Vasilyev, Aleksandr; Tomar, Ritu; Selig, Martin K; Nielsen, G Petur; Peterson, Randall T; Drummond, Iain A; Haber, Daniel A

2014-07-01

Chordoma is a malignant tumor thought to arise from remnants of the embryonic notochord, with its origin in the bones of the axial skeleton. Surgical resection is the standard treatment, usually in combination with radiation therapy, but neither chemotherapeutic nor targeted therapeutic approaches have demonstrated success. No animal model and only few chordoma cell lines are available for preclinical drug testing, and, although no druggable genetic drivers have been identified, activation of EGFR and downstream AKT-PI3K pathways have been described. Here, we report a zebrafish model of chordoma, based on stable transgene-driven expression of HRASV12 in notochord cells during development. Extensive intra-notochordal tumor formation is evident within days of transgene expression, ultimately leading to larval death. The zebrafish tumors share characteristics of human chordoma as demonstrated by immunohistochemistry and electron microscopy. The mTORC1 inhibitor rapamycin, which has some demonstrated activity in a chordoma cell line, delays the onset of tumor formation in our zebrafish model, and improves survival of tumor-bearing fish. Consequently, the HRASV12-driven zebrafish model of chordoma could enable high-throughput screening of potential therapeutic agents for the treatment of this refractory cancer. © 2014. Published by The Company of Biologists Ltd.

Seeing the whole picture: A comprehensive imaging approach to functional mapping of circuits in behaving zebrafish.

PubMed

Feierstein, C E; Portugues, R; Orger, M B

2015-06-18

In recent years, the zebrafish has emerged as an appealing model system to tackle questions relating to the neural circuit basis of behavior. This can be attributed not just to the growing use of genetically tractable model organisms, but also in large part to the rapid advances in optical techniques for neuroscience, which are ideally suited for application to the small, transparent brain of the larval fish. Many characteristic features of vertebrate brains, from gross anatomy down to particular circuit motifs and cell-types, as well as conserved behaviors, can be found in zebrafish even just a few days post fertilization, and, at this early stage, the physical size of the brain makes it possible to analyze neural activity in a comprehensive fashion. In a recent study, we used a systematic and unbiased imaging method to record the pattern of activity dynamics throughout the whole brain of larval zebrafish during a simple visual behavior, the optokinetic response (OKR). This approach revealed the broadly distributed network of neurons that were active during the behavior and provided insights into the fine-scale functional architecture in the brain, inter-individual variability, and the spatial distribution of behaviorally relevant signals. Combined with mapping anatomical and functional connectivity, targeted electrophysiological recordings, and genetic labeling of specific populations, this comprehensive approach in zebrafish provides an unparalleled opportunity to study complete circuits in a behaving vertebrate animal. Copyright © 2014. Published by Elsevier Ltd.

Aryl hydrocarbon receptor 2 mediates the toxicity of Paclobutrazol on the digestive system of zebrafish embryos.

PubMed

Wang, Wen-Der; Chen, Guan-Ting; Hsu, Hwei-Jan; Wu, Chang-Yi

2015-02-01

Paclobutrazol (PBZ), a trazole-containing fungicide and plant growth retardant, has been widely used for over 30 years to regulate plant growth and promote early fruit setting. Long-term usage of PBZ in agriculture and natural environments has resulted in residual PBZ in the soil and water. Chronic exposure to waterborne PBZ can cause various physiological effects in fish, including hepatic steatosis, antioxidant activity, and disruption of spermatogenesis. We have previously shown that PBZ also affects the rates of zebrafish embryonic survival and hatching, and causes developmental failure of the head skeleton and eyes; here, we further show that PBZ has embryonic toxic effects on digestive organs of zebrafish, and describe the underlying mechanisms. PBZ treatment of embryos resulted in dose-dependent morphological and functional abnormalities of the digestive organs. Real-time RT-PCR and in situ hybridization were used to show that PBZ strongly induces cyp1a1 expression in the digestive system, and slightly induces ahr2 expression in zebrafish embryos. Knockdown of ahr2 with morpholino oligonucleotides prevents PBZ toxicity. Thus, the toxic effect of PBZ on digestive organs is mediated by AhR2, as was previously reported for retene and TCDD. These findings have implications for understanding the potential toxicity of PBZ during embryogenesis, and thus the potential impact of fungicides on public health and the environment. Copyright © 2014 Elsevier B.V. All rights reserved.

Screening for angiogenic inhibitors in zebrafish to evaluate a predictive model for developmental vascular toxicity.

PubMed

Tal, Tamara; Kilty, Claire; Smith, Andrew; LaLone, Carlie; Kennedy, Brendán; Tennant, Alan; McCollum, Catherine W; Bondesson, Maria; Knudsen, Thomas; Padilla, Stephanie; Kleinstreuer, Nicole

2017-06-01

Chemically-induced vascular toxicity during embryonic development may cause a wide range of adverse effects. To identify putative vascular disrupting chemicals (pVDCs), a predictive pVDC signature was constructed from 124 U.S. EPA ToxCast high-throughput screening (HTS) assays and used to rank 1060 chemicals for their potential to disrupt vascular development. Thirty-seven compounds were selected for targeted testing in transgenic Tg(kdrl:EGFP) and Tg(fli1:EGFP) zebrafish embryos to identify chemicals that impair developmental angiogenesis. We hypothesized that zebrafish angiogenesis toxicity data would correlate with human cell-based and cell-free in vitro HTS ToxCast data. Univariate statistical associations used to filter HTS data based on correlations with zebrafish angiogenic inhibition in vivo revealed 132 total significant associations, 33 of which were already captured in the pVDC signature, and 689 non-significant assay associations. Correlated assays were enriched in cytokine and extracellular matrix pathways. Taken together, the findings indicate the utility of zebrafish assays to evaluate an HTS-based predictive toxicity signature and also provide an experimental basis for expansion of the pVDC signature with novel HTS assays. Published by Elsevier Inc.

The Ndst Gene Family in Zebrafish: Role of Ndst1b in Pharyngeal Arch Formation

PubMed Central

Haitina, Tatjana; Habicher, Judith; Ledin, Johan; Kjellén, Lena

2015-01-01

Heparan sulfate (HS) proteoglycans are ubiquitous components of the extracellular matrix and plasma membrane of metazoans. The sulfation pattern of the HS glycosaminoglycan chain is characteristic for each tissue and changes during development. The glucosaminyl N-deacetylase/N-sulfotransferase (NDST) enzymes catalyze N-deacetylation and N-sulfation during HS biosynthesis and have a key role in designing the sulfation pattern. We here report on the presence of five NDST genes in zebrafish. Zebrafish ndst1a, ndst1b, ndst2a and ndst2b represent duplicated mammalian orthologues of NDST1 and NDST2 that arose through teleost specific genome duplication. Interestingly, the single zebrafish orthologue ndst3, is equally similar to tetrapod Ndst3 and Ndst4. It is likely that a local duplication in the common ancestor of lobe-finned fish and tetrapods gave rise to these two genes. All zebrafish Ndst genes showed distinct but partially overlapping expression patterns during embryonic development. Morpholino knockdown of ndst1b resulted in delayed development, craniofacial cartilage abnormalities, shortened body and pectoral fin length, resembling some of the features of the Ndst1 mouse knockout. PMID:25767878

The role of Sox6 in zebrafish muscle fiber type specification.

PubMed

Jackson, Harriet E; Ono, Yosuke; Wang, Xingang; Elworthy, Stone; Cunliffe, Vincent T; Ingham, Philip W

2015-01-01

The transcription factor Sox6 has been implicated in regulating muscle fiber type-specific gene expression in mammals. In zebrafish, loss of function of the transcription factor Prdm1a results in a slow to fast-twitch fiber type transformation presaged by ectopic expression of sox6 in slow-twitch progenitors. Morpholino-mediated Sox6 knockdown can suppress this transformation but causes ectopic expression of only one of three slow-twitch specific genes assayed. Here, we use gain and loss of function analysis to analyse further the role of Sox6 in zebrafish muscle fiber type specification. The GAL4 binary misexpression system was used to express Sox6 ectopically in zebrafish embryos. Cis-regulatory elements were characterized using transgenic fish. Zinc finger nuclease mediated targeted mutagenesis was used to analyse the effects of loss of Sox6 function in embryonic, larval and adult zebrafish. Zebrafish transgenic for the GCaMP3 Calcium reporter were used to assay Ca2+ transients in wild-type and mutant muscle fibres. Ectopic Sox6 expression is sufficient to downregulate slow-twitch specific gene expression in zebrafish embryos. Cis-regulatory elements upstream of the slow myosin heavy chain 1 (smyhc1) and slow troponin c (tnnc1b) genes contain putative Sox6 binding sites required for repression of the former but not the latter. Embryos homozygous for sox6 null alleles expressed tnnc1b throughout the fast-twitch muscle whereas other slow-specific muscle genes, including smyhc1, were expressed ectopically in only a subset of fast-twitch fibers. Ca2+ transients in sox6 mutant fast-twitch fibers were intermediate in their speed and amplitude between those of wild-type slow- and fast-twitch fibers. sox6 homozygotes survived to adulthood and exhibited continued misexpression of tnnc1b as well as smaller slow-twitch fibers. They also exhibited a striking curvature of the spine. The Sox6 transcription factor is a key regulator of fast-twitch muscle fiber differentiation

Optical manipulation for optogenetics: otoliths manipulation in zebrafish (Conference Presentation)

NASA Astrophysics Data System (ADS)

Favre-Bulle, Itia A.; Scott, Ethan; Rubinsztein-Dunlop, Halina

2016-03-01

Otoliths play an important role in Zebrafish in terms of hearing and sense of balance. Many studies have been conducted to understand its structure and function, however the encoding of its movement in the brain remains unknown. Here we developed a noninvasive system capable of manipulating the otolith using optical trapping while we image its behavioral response and brain activity. We'll also present our tools for behavioral response detection and brain activity mapping. Acceleration is sensed through movements of the otoliths in the inner ear. Because experimental manipulations involve movements, electrophysiology and fluorescence microscopy are difficult. As a result, the neural codes underlying acceleration sensation are poorly understood. We have developed a technique for optically trapping otoliths, allowing us to simulate acceleration in stationary larval zebrafish. By applying forces to the otoliths, we can elicit behavioral responses consistent with compensation for perceived acceleration. Since the animal is stationary, we can use calcium imaging in these animals' brains to identify the functional circuits responsible for mediating responses to acceleration in natural settings.

Dechorionation of Zebrafish Embryos on Day 1 Post Fertilization Alters Response to an Acute Chemical Challenge at 6 Days Post Fertilization

EPA Science Inventory

Dechorionation is a method used to enable image acquisition in embryonic and larval zebrafish studies. As it is assumed that dechorionation has no long-term effects on fish embryo development, it is important to determine if that assumption is correct. The present study explored ...

Early Embryonic Androgen Exposure Induces Transgenerational Epigenetic and Metabolic Changes

PubMed Central

Xu, Ning; Chua, Angela K.; Jiang, Hong; Liu, Ning-Ai

2014-01-01

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. PMID:24992182

Neuronal expression of fibroblast growth factor receptors in zebrafish.

PubMed

Rohs, Patricia; Ebert, Alicia M; Zuba, Ania; McFarlane, Sarah

2013-12-01

Fibroblast growth factor (FGF) signaling is important for a host of developmental processes such as proliferation, differentiation, tissue patterning, and morphogenesis. In vertebrates, FGFs signal through a family of four fibroblast growth factor receptors (FGFR 1-4), one of which is duplicated in zebrafish (FGFR1). Here we report the mRNA expression of the five known zebrafish fibroblast growth factor receptors at five developmental time points (24, 36, 48, 60, and 72h postfertilization), focusing on expression within the central nervous system. We show that the receptors have distinct and dynamic expression in the developing zebrafish brain, eye, inner ear, lateral line, and pharynx. In many cases, the expression patterns are similar to those of homologous FGFRs in mouse, chicken, amphibians, and other teleosts. Copyright © 2013 Elsevier B.V. All rights reserved.

Live imaging reveals a conserved role of fatty acid β-oxidation in early lymphatic development in zebrafish.

PubMed

Zecchin, Annalisa; Wong, Brian W; Tembuyser, Bieke; Souffreau, Joris; Van Nuffelen, An; Wyns, Sabine; Vinckier, Stefan; Carmeliet, Peter; Dewerchin, Mieke

2018-06-18

During embryonic development, lymphatic endothelial cells (LECs) differentiate from venous endothelial cells (VECs), a process that is tightly regulated by several genetic signals. While the aquatic zebrafish model is regularly used for studying lymphangiogenesis and offers the unique advantage of time-lapse video-imaging of lymphatic development, some aspects of lymphatic development in this model differ from those in the mouse. It therefore remained to be determined whether fatty acid β-oxidation (FAO), which we showed to regulate lymphatic formation in the mouse, also co-determines lymphatic development in this aquatic model. Here, we took advantage of the power of the zebrafish embryo model to visualize the earliest steps of lymphatic development through time-lapse video-imaging. By targeting zebrafish isoforms of carnitine palmitoyltransferase 1a (cpt1a), a rate controlling enzyme of FAO, with multiple morpholinos, we demonstrate that reducing CPT1A levels and FAO flux during zebrafish development impairs lymphangiogenic secondary sprouting, the initiation of lymphatic development in the zebrafish trunk, and the formation of the first lymphatic structures. These findings not only show evolutionary conservation of the importance of FAO for lymphatic development, but also suggest a role for FAO in co-regulating the process of VEC-to-LEC differentiation in zebrafish in vivo. Copyright © 2018 Elsevier Inc. All rights reserved.

Distinct myocardial lineages break atrial symmetry during cardiogenesis in zebrafish

PubMed Central

Stone, Oliver; Arnaout, Rima; Guenther, Stefan; Ahuja, Suchit; Uribe, Verónica; Vanhollebeke, Benoit; Stainier, Didier YR

2018-01-01

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry. PMID:29762122

Multilayer Mounting for Long-term Light Sheet Microscopy of Zebrafish

PubMed Central

Weber, Michael; Mickoleit, Michaela; Huisken, Jan

2014-01-01

Light sheet microscopy is the ideal imaging technique to study zebrafish embryonic development. Due to minimal photo-toxicity and bleaching, it is particularly suited for long-term time-lapse imaging over many hours up to several days. However, an appropriate sample mounting strategy is needed that offers both confinement and normal development of the sample. Multilayer mounting, a new embedding technique using low-concentration agarose in optically clear tubes, now overcomes this limitation and unleashes the full potential of light sheet microscopy for real-time developmental biology. PMID:24637614

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration

NASA Astrophysics Data System (ADS)

Gibbs, Holly C.; Dodson, Colin R.; Bai, Yuqiang; Lekven, Arne C.; Yeh, Alvin T.

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

Combined lineage mapping and gene expression profiling of embryonic brain patterning using ultrashort pulse microscopy and image registration.

PubMed

Gibbs, Holly C; Dodson, Colin R; Bai, Yuqiang; Lekven, Arne C; Yeh, Alvin T

2014-12-01

During embryogenesis, presumptive brain compartments are patterned by dynamic networks of gene expression. The spatiotemporal dynamics of these networks, however, have not been characterized with sufficient resolution for us to understand the regulatory logic resulting in morphogenetic cellular behaviors that give the brain its shape. We have developed a new, integrated approach using ultrashort pulse microscopy [a high-resolution, two-photon fluorescence (2PF)-optical coherence microscopy (OCM) platform using 10-fs pulses] and image registration to study brain patterning and morphogenesis in zebrafish embryos. As a demonstration, we used time-lapse 2PF to capture midbrain-hindbrain boundary morphogenesis and a wnt1 lineage map from embryos during brain segmentation. We then performed in situ hybridization to deposit NBT/BCIP, where wnt1 remained actively expressed, and reimaged the embryos with combined 2PF-OCM. When we merged these datasets using morphological landmark registration, we found that the mechanism of boundary formation differs along the dorsoventral axis. Dorsally, boundary sharpening is dominated by changes in gene expression, while ventrally, sharpening may be accomplished by lineage sorting. We conclude that the integrated visualization of lineage reporter and gene expression domains simultaneously with brain morphology will be useful for understanding how changes in gene expression give rise to proper brain compartmentalization and structure.

The 8-oxoguanine DNA glycosylase 1 (ogg1) decreases the vulnerability of the developing brain to DNA damage.

PubMed

Gu, Aihua; Ji, Guixiang; Yan, Lifeng; Zhou, Yong

2013-12-01

The developing brain is particularly vulnerable to oxidative DNA damage, which may be the cause of most major congenital mental anomalies. The repair enzyme ogg1 initiates the highly conserved base-excision repair pathway. However, its function in the embryonic brain is largely unknown. This study is the first to validate the function of ogg1 during brain development using zebrafish embryos. Ogg1 was found to be highly expressed in the brain throughout early embryonic development, with particularly enrichment observed in the midbrain. The lack of ogg1 causes severe brain defects including changes in brain volume and integrity, destruction of the midbrain-hindbrain boundary, and balance and motor impairment, while overexpression of ogg1 can partially rescue these defects. Multiple cellular and molecular events were involved in the manifestation of brain defects due primarily to the lack of ogg1. These included (1) increased apoptosis; (2) decreased proliferation; and (3) aberrant axon distribution and extension from the inner surface towards the outer layers. The results of a microarray analysis showed that the expression of genes involved in cell cycle checkpoint, apoptosis, and neurogenesis were significantly changed in response to ogg1 knockdown. Cmyb was the key downstream gene that responses to DNA damage caused by ogg1 deficiency. Notably, the recruitment of ogg1 mRNA can alleviate the effects on the brain due to neural DNA damage. In summary, we introduce here that ogg1 is fundamentally required for protecting the developing brain, which may be helpful in understanding the aetiology of congenital brain deficits. Copyright © 2013 Elsevier B.V. All rights reserved.

MicroXRF tomographic visualization of zinc and iron in the zebrafish embryo at the onset of the hatching period

DOE Office of Scientific and Technical Information (OSTI.GOV)

Bourassa, Daisy; Gleber, Sophie-Charlotte; Vogt, Stefan

2016-01-01

Transition metals such as zinc, copper, and iron play key roles in cellular proliferation, cell differentiation, growth, and development. Over the past decade, advances in synchrotron X-ray fluorescence instrumentation presented new opportunities for the three-dimensional mapping of trace metal distributions within intact specimens. Taking advantage of microXRF tomography, we visualized the 3D distribution of zinc and iron in a zebrafish embryo at the onset of the hatching period. The reconstructed volumetric data revealed distinct differences in the elemental distributions, with zinc predominantly localized to the yolk and yolk extension, and iron to various regions of the brain as well asmore » the myotome extending along the dorsal side of the embryo. The data set complements an earlier tomographic study of an embryo at the pharyngula stage (24 hpf), thus offering new insights into the trace metal distribution at key stages of embryonic development.« less

Zebrafish Collagen Type I: Molecular and Biochemical Characterization of the Major Structural Protein in Bone and Skin

PubMed Central

Gistelinck, C.; Gioia, R.; Gagliardi, A.; Tonelli, F.; Marchese, L.; Bianchi, L.; Landi, C.; Bini, L.; Huysseune, A.; Witten, P. E.; Staes, A.; Gevaert, K.; De Rocker, N.; Menten, B.; Malfait, F.; Leikin, S.; Carra, S.; Tenni, R.; Rossi, A.; De Paepe, A.; Coucke, P.; Willaert, A.; Forlino, A.

2016-01-01

Over the last years the zebrafish imposed itself as a powerful model to study skeletal diseases, but a limit to its use is the poor characterization of collagen type I, the most abundant protein in bone and skin. In tetrapods collagen type I is a trimer mainly composed of two α1 chains and one α2 chain, encoded by COL1A1 and COL1A2 genes, respectively. In contrast, in zebrafish three type I collagen genes exist, col1a1a, col1a1b and col1a2 coding for α1(I), α3(I) and α2(I) chains. During embryonic and larval development the three collagen type I genes showed a similar spatio-temporal expression pattern, indicating their co-regulation and interdependence at these stages. In both embryonic and adult tissues, the presence of the three α(I) chains was demonstrated, although in embryos α1(I) was present in two distinct glycosylated states, suggesting a developmental-specific collagen composition. Even though in adult bone, skin and scales equal amounts of α1(I), α3(I) and α2(I) chains are present, the presented data suggest a tissue-specific stoichiometry and/or post-translational modification status for collagen type I. In conclusion, this data will be useful to properly interpret results and insights gained from zebrafish models of skeletal diseases. PMID:26876635

Afferent Connectivity of the Zebrafish Habenulae

PubMed Central

Turner, Katherine J.; Hawkins, Thomas A.; Yáñez, Julián; Anadón, Ramón; Wilson, Stephen W.; Folgueira, Mónica

2016-01-01

The habenulae are bilateral nuclei located in the dorsal diencephalon that are conserved across vertebrates. Here we describe the main afferents to the habenulae in larval and adult zebrafish. We observe afferents from the subpallium, nucleus rostrolateralis, posterior tuberculum, posterior hypothalamic lobe, median raphe; we also see asymmetric afferents from olfactory bulb to the right habenula, and from the parapineal to the left habenula. In addition, we find afferents from a ventrolateral telencephalic nucleus that neurochemical and hodological data identify as the ventral entopeduncular nucleus (vENT), confirming and extending observations of Amo et al. (2014). Fate map and marker studies suggest that vENT originates from the diencephalic prethalamic eminence and extends into the lateral telencephalon from 48 to 120 hour post-fertilization (hpf). No afferents to the habenula were observed from the dorsal entopeduncular nucleus (dENT). Consequently, we confirm that the vENT (and not the dENT) should be considered as the entopeduncular nucleus “proper” in zebrafish. Furthermore, comparison with data in other vertebrates suggests that the vENT is a conserved basal ganglia nucleus, being homologous to the entopeduncular nucleus of mammals (internal segment of the globus pallidus of primates) by both embryonic origin and projections, as previously suggested by Amo et al. (2014). PMID:27199671

Developmental toxicity of 2,4-dichlorophenoxyacetic acid in zebrafish embryos.

PubMed

Li, Kang; Wu, Jia-Qi; Jiang, Ling-Ling; Shen, Li-Zhen; Li, Jian-Ying; He, Zhi-Heng; Wei, Ping; Lv, Zhuo; He, Ming-Fang

2017-03-01

2,4-Dichlorophenoxyacetic acid (2,4-D) is widely used in agriculture as herbicide/pesticide, plant growth regulator and fruit preservative agent. It progressively accumulates in the environment including surface water, air and soil. It could be detected in human food and urine, which poses great risk to the living organisms. In the present study, we investigated the developmental toxicity of 2,4-D on zebrafish (Danio rerio) embryo. 2,4-D exposure significantly decreased both the survival rate (LC 50  = 46.71 mg/L) and hatching rate (IC 50  = 46.26 mg/L) of zebrafish embryos. The most common developmental defect in 2,4-D treated embryos was pericardial edema. 2,4-D (25 mg/L) upregulated marker genes of cardiac development (vmhc, amhc, hand2, vegf, and gata1) and downregulated marker genes of oxidative stress (cat and gpx1a). Whole mount in situ hybridization confirmed the vmhc and amhc upregulation by 2,4-D treatment. LC/MS/MS showed that the bioaccumulation of 2,4-D in zebrafish embryos were increased in a time-dependent manner after 25 mg/L of 2,4-D treatment. Taken together, our study investigated the toxic effects of 2,4-D on zebrafish embryonic development and its potential molecular mechanisms, gave evidence for the full understanding of 2,4-D toxicity on living organisms and shed light on its environmental impact. Copyright © 2016 Elsevier Ltd. All rights reserved.

A Single-Cell Roadmap of Lineage Bifurcation in Human ESC Models of Embryonic Brain Development.

PubMed

Yao, Zizhen; Mich, John K; Ku, Sherman; Menon, Vilas; Krostag, Anne-Rachel; Martinez, Refugio A; Furchtgott, Leon; Mulholland, Heather; Bort, Susan; Fuqua, Margaret A; Gregor, Ben W; Hodge, Rebecca D; Jayabalu, Anu; May, Ryan C; Melton, Samuel; Nelson, Angelique M; Ngo, N Kiet; Shapovalova, Nadiya V; Shehata, Soraya I; Smith, Michael W; Tait, Leah J; Thompson, Carol L; Thomsen, Elliot R; Ye, Chaoyang; Glass, Ian A; Kaykas, Ajamete; Yao, Shuyuan; Phillips, John W; Grimley, Joshua S; Levi, Boaz P; Wang, Yanling; Ramanathan, Sharad

2017-01-05

During human brain development, multiple signaling pathways generate diverse cell types with varied regional identities. Here, we integrate single-cell RNA sequencing and clonal analyses to reveal lineage trees and molecular signals underlying early forebrain and mid/hindbrain cell differentiation from human embryonic stem cells (hESCs). Clustering single-cell transcriptomic data identified 41 distinct populations of progenitor, neuronal, and non-neural cells across our differentiation time course. Comparisons with primary mouse and human gene expression data demonstrated rostral and caudal progenitor and neuronal identities from early brain development. Bayesian analyses inferred a unified cell-type lineage tree that bifurcates between cortical and mid/hindbrain cell types. Two methods of clonal analyses confirmed these findings and further revealed the importance of Wnt/β-catenin signaling in controlling this lineage decision. Together, these findings provide a rich transcriptome-based lineage map for studying human brain development and modeling developmental disorders. Copyright © 2017 Elsevier Inc. All rights reserved.

The effects of cobalt on the development, oxidative stress, and apoptosis in zebrafish embryos.

PubMed

Cai, Guiquan; Zhu, Junfeng; Shen, Chao; Cui, Yimin; Du, Jiulin; Chen, Xiaodong

2012-12-01

Metal-on-metal hip arthroplasty has been performed with increasing frequency throughout the world, particularly in younger and more active patients, including women of childbearing age. The potential toxicity of cobalt exposure on fetus is concerned since cobalt ions generated by metal-on-metal bearings can traverse the placenta and be detected in fetal blood and amniotic fluid. This study examined the effects of cobalt exposure on early embryonic development and the mechanisms underlying its toxicity. Zebrafish embryos were exposed to a range of cobalt concentrations (0-100 mg/L) between 1 and 144 h postfertilization. The survival and early development of embryos were not significantly affected by cobalt at concentrations <100 μg/L. However, embryos exposed to higher concentrations (>100 μg/L) displayed reduced survival rates and abnormal development, including delayed hatching, aberrant morphology, retarded growth, and bradycardia. Furthermore, this study examined oxidative stress and apoptosis in embryos exposed to cobalt at concentrations of 0-500 μg/L. Lipid peroxidation levels were increased in cobalt-treated embryos at concentrations of 100 and 500 μg/L. The mRNA levels of catalase, superoxide dismutase 2, p53, caspase-3, and caspase-9 genes were upregulated in a dose-dependent manner. Terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling assays also revealed abnormal apoptotic signals in the brain, trunk, and tail when treated with 500 μg/L cobalt. These data suggest that oxidative stress and apoptosis are associated with cobalt toxicity in zebrafish embryos.

Pax2.1 is required for the development of thyroid follicles in zebrafish.

PubMed

Wendl, Thomas; Lun, Klaus; Mione, Marina; Favor, Jack; Brand, Michael; Wilson, Stephen W; Rohr, Klaus B

2002-08-01

The thyroid gland is an organ primarily composed of endoderm-derived follicular cells. Although disturbed embryonic development of the thyroid gland leads to congenital hypothyroidism in humans and mammals, the underlying principles of thyroid organogenesis are largely unknown. In this study, we introduce zebrafish as a model to investigate the molecular and genetic mechanisms that control thyroid development. Marker gene expression suggests that the molecular pathways of early thyroid development are essentially conserved between fish and mammals. However during larval stages, we find both conserved and divergent features of development compared with mammals. A major difference is that in fish, we find evidence for hormone production not only in thyroid follicular cells, but also in an anterior non-follicular group of cells. We show that pax2.1 and pax8, members of the zebrafish pax2/5/8 paralogue group, are expressed in the thyroid primordium. Whereas in mice, only Pax8 has a function during thyroid development, analysis of the zebrafish pax2.1 mutant no isthmus (noi(-/-)) demonstrates that pax2.1 has a role comparable with mouse Pax8 in differentiation of the thyroid follicular cells. Early steps of thyroid development are normal in noi(-/-), but later expression of molecular markers is lost and the formation of follicles fails. Interestingly, the anterior non-follicular site of thyroid hormone production is not affected in noi(-/-). Thus, in zebrafish, some remaining thyroid hormone synthesis takes place independent of the pathway leading to thyroid follicle formation. We suggest that the noi(-/-) mutant serves as a new zebrafish model for hypothyroidism.

Expression of thyroid hormone transporters and deiodinases at the brain barriers in the embryonic chicken: Insights into the regulation of thyroid hormone availability during neurodevelopment.

PubMed

Van Herck, Stijn L J; Delbaere, Joke; Bourgeois, Nele M A; McAllan, Bronwyn M; Richardson, Samantha J; Darras, Veerle M

2015-04-01

Thyroid hormones (THs) are key regulators in the development of the vertebrate brain. Therefore, TH access to the developing brain needs to be strictly regulated. The brain barriers separate the central nervous system from the rest of the body and impose specific transport mechanisms on the exchange of molecules between the general circulation and the nervous system. As such they form ideal structures for regulating TH exchange between the blood and the brain. To investigate the mechanism by which the developing brain regulates TH availability, we investigated the ontogenetic expression profiles of TH transporters, deiodinases and the TH distributor protein transthyretin (TTR) at the brain barriers during embryonic and early postnatal development using the chicken as a model. In situ hybridisation revealed expression of the TH transporters monocarboxylate transporter 8 (MCT8) and 10 (MCT10), organic anion transporting polypeptide 1C1 (OATP1C1) and L-type amino acid transporter 1 (LAT1) and the inactivating type 3 deiodinase (D3) in the choroid plexus which forms the blood-cerebrospinal fluid barrier. This was confirmed by quantitative PCR which additionally indicated strongly increasing expression of TTR as well as detectable expression of the activating type 2 deiodinase (D2) and the (in)activating type 1 deiodinase (D1). In the brain capillaries forming the blood-brain barrier in situ hybridisation showed exclusive expression of LAT1 and D2. The combined presence of LAT1 and D2 in brain capillaries suggests that the blood-brain barrier forms the main route for receptor-active T3 uptake into the embryonic chicken brain. Expression of multiple transporters, deiodinases and TTR in the choroid plexus indicates that the blood-cerebrospinal fluid barrier is also important in regulating early TH availability. The impact of these barrier systems can be deduced from the clear difference in T3 and T4 levels as well as the T3/T4 ratio between the developing brain and the

Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108.

PubMed

Wang, Ruhung; N Meredith, Alicea; Lee, Michael; Deutsch, Dakota; Miadzvedskaya, Lizaveta; Braun, Elizabeth; Pantano, Paul; Harper, Stacey; Draper, Rockford

2016-08-01

Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity.

Toxicity assessment and bioaccumulation in zebrafish embryos exposed to carbon nanotubes suspended in Pluronic® F-108

PubMed Central

Wang, Ruhung; Meredith, Alicea N.; Lee, Michael; Deutsch, Dakota; Miadzvedskaya, Lizaveta; Braun, Elizabeth; Pantano, Paul; Harper, Stacey; Draper, Rockford

2015-01-01

Carbon nanotubes (CNTs) are often suspended in Pluronic® surfactants by sonication, which may confound toxicity studies because sonication of surfactants can create degradation products that are toxic to mammalian cells. Here, we present a toxicity assessment of Pluronic® F-108 with and without suspended CNTs using embryonic zebrafish as an in vivo model. Pluronic® sonolytic degradation products were toxic to zebrafish embryos just as they were to mammalian cells. When the toxic Pluronic® fragments were removed, there was little effect of pristine multi-walled CNTs (pMWNTs), carboxylated MWNTs (cMWNTs) or pristine single-walled carbon nanotubes (pSWNTs) on embryo viability and development, even at high concentrations. A gel electrophoretic method coupled with Raman imaging was developed to measure the bioaccumulation of CNTs by zebrafish embryos, and dose-dependent uptake of CNTs was observed. These data indicate that embryos accumulate pMWNTs, cMWNTs and pSWNTs yet there is very little embryo toxicity. PMID:26559437

Pan-neuronal calcium imaging with cellular resolution in freely swimming zebrafish.

PubMed

Kim, Dal Hyung; Kim, Jungsoo; Marques, João C; Grama, Abhinav; Hildebrand, David G C; Gu, Wenchao; Li, Jennifer M; Robson, Drew N

2017-11-01

Calcium imaging with cellular resolution typically requires an animal to be tethered under a microscope, which substantially restricts the range of behaviors that can be studied. To expand the behavioral repertoire amenable to imaging, we have developed a tracking microscope that enables whole-brain calcium imaging with cellular resolution in freely swimming larval zebrafish. This microscope uses infrared imaging to track a target animal in a behavior arena. On the basis of the predicted trajectory of the animal, we applied optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We combined this motion-cancellation system with differential illumination focal filtering, a variant of HiLo microscopy, which enabled us to image the brain of a freely swimming larval zebrafish for more than an hour. This work expands the repertoire of natural behaviors that can be studied with cellular-resolution calcium imaging to potentially include spatial navigation, social behavior, feeding and reward.

Distribution, pharmacokinetics and primary metabolism model of tramadol in zebrafish.

PubMed

Zhuo, Huiqin; Jin, Hongwei; Peng, Huifang; Huang, Heqing

2016-12-01

The current study aimed to develop a rapid, robust and adequately sensitive method for simultaneous determination of the concentration of tramadol and its active metabolites in zebrafish. The pharmacokinetic and elimination pattern of tramadol and its major phase I metabolites following oral or intramuscular administration in zebrafish tissues was achieved using electrospray ionization‑quadrupole‑time of flight/mass spectrometry (ESI‑Q‑TOF/MS) and gas chromatography/mass spectrometry (GC‑MS). Following administration, the metabolisms were detected in the brain, eyes, muscle and gill tissues within 1 h. Two tramadol metabolites, O‑ and N‑desmethyltramadol, were detected in brain tissue, with N‑desmethyltramadol detected at a higher level. Following GC‑MS detection the curve indicated an initial rapid phase, corresponding to the detection of the tramadol within 1 min, and reached peak value in the brain at 5 min. Faster drug clearance was detected in low‑dose groups, and concentration had dropped around the to initial level (1.11 µg) at 20 min, but was detectable for up to 3 h. However, it took 80 min to fall back to the initial value (1.73 µg) in the high‑dose groups, and tramadol was detectable for up to 4 h. This study developed and validated a simple and high throughput analytical procedure to determine the distribution and pharmacokinetic profiles of tramadol, and its primary metabolites in tissues of zebrafish.

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

PubMed

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.

A Brain-wide Circuit Model of Heat-Evoked Swimming Behavior in Larval Zebrafish.

PubMed

Haesemeyer, Martin; Robson, Drew N; Li, Jennifer M; Schier, Alexander F; Engert, Florian

2018-05-16

Thermosensation provides crucial information, but how temperature representation is transformed from sensation to behavior is poorly understood. Here, we report a preparation that allows control of heat delivery to zebrafish larvae while monitoring motor output and imaging whole-brain calcium signals, thereby uncovering algorithmic and computational rules that couple dynamics of heat modulation, neural activity and swimming behavior. This approach identifies a critical step in the transformation of temperature representation between the sensory trigeminal ganglia and the hindbrain: A simple sustained trigeminal stimulus representation is transformed into a representation of absolute temperature as well as temperature changes in the hindbrain that explains the observed motor output. An activity constrained dynamic circuit model captures the most prominent aspects of these sensori-motor transformations and predicts both behavior and neural activity in response to novel heat stimuli. These findings provide the first algorithmic description of heat processing from sensory input to behavioral output. Copyright © 2018 Elsevier Inc. All rights reserved.

An integrative analysis of ethanol tolerance and withdrawal in zebrafish (Danio rerio)

PubMed Central

Tran, Steven; Chatterjee, Diptendu; Gerlai, Robert

2014-01-01

The zebrafish is emerging as a popular animal model for alcohol (ethanol or EtOH) addiction due to its simplicity and practical advantages. Two phenomena associated with ethanol addiction are the development of tolerance and withdrawal. Using a multi-level approach in the current study, we characterize ethanol tolerance and withdrawal in zebrafish. We first investigate the temporal trajectory of ethanol concentration in the zebrafish brain in response to an acute exposure and during withdrawal. We report that ethanol concentrations approach a steady state within 60 minutes of exposure to 0.50% and 1.00% v/v ethanol and rapidly decline and return to zero within 60 minutes following withdrawal from chronic ethanol exposure (0.50% v/v). We characterize the changes associated with ethanol tolerance and withdrawal in zebrafish by focusing on 3 domains relevant to ethanol addiction: motor patterns, physiological responses (i.e. cortisol levels), and neurochemical alterations. The use of multiple domains of investigation allowed an in-depth analysis of ethanol induced changes in zebrafish. PMID:24598276

MANF regulates dopaminergic neuron development in larval zebrafish.

PubMed

Chen, Y-C; Sundvik, M; Rozov, S; Priyadarshini, M; Panula, P

2012-10-15

Mesencephalic astrocyte derived neurotrophic factor (MANF) is recognized as a dopaminergic neurotrophic factor, which can protect dopaminergic neurons from neurotoxic damage. However, little is known about the function of MANF during the vertebrate development. Here, we report that MANF expression is widespread during embryonic development and in adult organs analyzed by qPCR and in situ hybridization in zebrafish. Knockdown of MANF expression with antisense splice-blocking morpholino oligonucleotides resulted in no apparent abnormal phenotype. Nevertheless, the dopamine level of MANF morphants was lower than that of the wild type larvae, the expression levels of the two tyrosine hydroxylase gene transcripts were decreased and a decrease in neuron number in certain groups of th1 and th2 cells in the diencephalon region in MANF morphants was observed. These defects were rescued by injection of exogenous manf mRNA. Strikingly, manf mRNA could partly restore the decrease of th1 positive cells in Nr4a2-deficient larvae. These results suggest that MANF is involved in the regulation of the development of dopaminergic system in zebrafish. Copyright © 2012 Elsevier Inc. All rights reserved.

Thyroid Hormone Regulates the Expression of the Sonic Hedgehog Signaling Pathway in the Embryonic and Adult Mammalian Brain

PubMed Central

Desouza, Lynette A.; Sathanoori, Malini; Kapoor, Richa; Rajadhyaksha, Neha; Gonzalez, Luis E.; Kottmann, Andreas H.; Tole, Shubha

2011-01-01

Thyroid hormone is important for development and plasticity in the immature and adult mammalian brain. Several thyroid hormone-responsive genes are regulated during specific developmental time windows, with relatively few influenced across the lifespan. We provide novel evidence that thyroid hormone regulates expression of the key developmental morphogen sonic hedgehog (Shh), and its coreceptors patched (Ptc) and smoothened (Smo), in the early embryonic and adult forebrain. Maternal hypo- and hyperthyroidism bidirectionally influenced Shh mRNA in embryonic forebrain signaling centers at stages before fetal thyroid hormone synthesis. Further, Smo and Ptc expression were significantly decreased in the forebrain of embryos derived from hypothyroid dams. Adult-onset thyroid hormone perturbations also regulated expression of the Shh pathway bidirectionally, with a significant induction of Shh, Ptc, and Smo after hyperthyroidism and a decline in Smo expression in the hypothyroid brain. Short-term T3 administration resulted in a significant induction of cortical Shh mRNA expression and also enhanced reporter gene expression in Shh+/LacZ mice. Further, acute T3 treatment of cortical neuronal cultures resulted in a rapid and significant increase in Shh mRNA, suggesting direct effects. Chromatin immunoprecipitation assays performed on adult neocortex indicated enhanced histone acetylation at the Shh promoter after acute T3 administration, providing further support that Shh is a thyroid hormone-responsive gene. Our results indicate that maternal and adult-onset perturbations of euthyroid status cause robust and region-specific changes in the Shh pathway in the embryonic and adult forebrain, implicating Shh as a possible mechanistic link for specific neurodevelopmental effects of thyroid hormone. PMID:21363934

Thyroid hormone regulates the expression of the sonic hedgehog signaling pathway in the embryonic and adult Mammalian brain.

PubMed

Desouza, Lynette A; Sathanoori, Malini; Kapoor, Richa; Rajadhyaksha, Neha; Gonzalez, Luis E; Kottmann, Andreas H; Tole, Shubha; Vaidya, Vidita A

2011-05-01

Thyroid hormone is important for development and plasticity in the immature and adult mammalian brain. Several thyroid hormone-responsive genes are regulated during specific developmental time windows, with relatively few influenced across the lifespan. We provide novel evidence that thyroid hormone regulates expression of the key developmental morphogen sonic hedgehog (Shh), and its coreceptors patched (Ptc) and smoothened (Smo), in the early embryonic and adult forebrain. Maternal hypo- and hyperthyroidism bidirectionally influenced Shh mRNA in embryonic forebrain signaling centers at stages before fetal thyroid hormone synthesis. Further, Smo and Ptc expression were significantly decreased in the forebrain of embryos derived from hypothyroid dams. Adult-onset thyroid hormone perturbations also regulated expression of the Shh pathway bidirectionally, with a significant induction of Shh, Ptc, and Smo after hyperthyroidism and a decline in Smo expression in the hypothyroid brain. Short-term T₃ administration resulted in a significant induction of cortical Shh mRNA expression and also enhanced reporter gene expression in Shh(+/LacZ) mice. Further, acute T₃ treatment of cortical neuronal cultures resulted in a rapid and significant increase in Shh mRNA, suggesting direct effects. Chromatin immunoprecipitation assays performed on adult neocortex indicated enhanced histone acetylation at the Shh promoter after acute T₃ administration, providing further support that Shh is a thyroid hormone-responsive gene. Our results indicate that maternal and adult-onset perturbations of euthyroid status cause robust and region-specific changes in the Shh pathway in the embryonic and adult forebrain, implicating Shh as a possible mechanistic link for specific neurodevelopmental effects of thyroid hormone.

Toxicity Evaluation of Pig Slurry Using Luminescent Bacteria and Zebrafish

PubMed Central

Chen, Wenyan; Cai, Qiang; Zhao, Yuan; Zheng, Guojuan; Liang, Yuting

2014-01-01

Biogas slurry has become a serious pollution problem and anaerobic digestion is widely applied to pig manure treatment for environmental protection and energy recovery. To evaluate environmental risk of the emission of biogas slurry, luminescent bacteria (Vibrio fischeri), larvae and embryos of zebrafish (Danio rerio) were used to detect the acute and development toxicity of digested and post-treated slurry. Then the ability of treatment process was evaluated. The results showed that digested slurry displayed strong toxicity to both zebrafish and luminescent bacteria, while the EC50 for luminescent bacteria and the LC50 for larvae were only 6.81% (v/v) and 1.95% (v/v) respectively, and embryonic development was inhibited at just 1% (v/v). Slurry still maintained a high level of toxicity although it had been treated by membrane bioreactor (MBR), while the LC50 of larvae was 75.23% (v/v) and there was a little effect on the development of embryos and V. fischeri; the results also revealed that the zebrafish larvae are more sensitive than embryos and luminescent bacteria to pig slurry. Finally, we also found the toxicity removal rate was higher than 90% after the treatment of MBR according to toxicity tests. In conclusion, further treatment should be used in pig slurry disposal or reused of final effluent. PMID:24995598

Trimethyltin chloride inhibits neuronal cell differentiation in zebrafish embryo neurodevelopment.

PubMed

Kim, Jin; Kim, C-Yoon; Song, Juha; Oh, Hanseul; Kim, Cheol-Hee; Park, Jae-Hak

2016-01-01

Trimethyltin chloride (TMT) is a neurotoxicant widely present in the aquatic environment, primarily from effluents of the plastic industry. It is known to cause acute neuronal death in the limbic-cerebellar system, particularly in the hippocampus. However, relatively few studies have estimated the effects of TMT toxicity on neurodevelopment. In this study, we confirmed the dose-dependent effects of TMT on neurodevelopmental stages through analysis of morphological changes and fluorescence assays using HuC-GFP and olig2-dsRed transgenic zebrafish embryos. In addition, we analyzed the expression of genes and proteins related to neurodevelopment. Exposure of embryos to TMT for 4 days post fertilization (dpf) elicited a concentration-related decrease in body length and increase in axial malformation. TMT affected the fluorescent CNS structure by decreasing pattern of HuC-GFP and olig2-dsRed transgenic zebrafish. In addition, it significantly modulated the expression patterns of Sonic hedgehog a (Shha), Neurogenin1 (Ngn1), Embryonic lethal abnormal vision like protein 3 (Elavl3), and Glial fibrillary acidic protein (Gfap). The overexpression of Shha and Ngn1, and downregulation of Elavl3 and Gfap, indicate repression of proneural cell differentiation. Our study demonstrates that TMT inhibits specific neurodevelopmental stages in zebrafish embryos and suggests a possible mechanism for the toxicity of TMT in vertebrate neurodevelopment. Copyright © 2015 Elsevier Inc. All rights reserved.

Dopamine receptor antagonism disrupts social preference in zebrafish, a strain comparison study

PubMed Central

Scerbina, Tanya; Chatterjee, Diptendu; Gerlai, Robert

2012-01-01

Zebrafish form shoals in nature and in the laboratory. The sight of conspecifics has been found reinforcing in zebrafish learning tasks. However, the mechanisms of shoaling, and those of its reinforcing properties, are not known. The dopaminergic system has been implicated in reward among other functions and it is also engaged by drugs of abuse as shown in a variety of vertebrates including zebrafish. The ontogenetic changes in dopamine levels and, to a lesser degree, in serotonin levels, have been found to accompany the maturation of shoaling in zebrafish. Thus, we hypothesized that the dopaminergic system may contribute to shoaling in zebrafish. To test this we employed a D1-receptor antagonist and quantified behavioral responses of our subjects using a social preference (shoaling) paradigm. We found significant reduction of social preference induced by the D1-R antagonist, SCH23390, in the AB strain of zebrafish, an alteration that was not accompanied by changes in motor function or vision. We also detected D1-R antagonist induced changes in the level of dopamine, DOPAC, serotonin and 5HIAA, respectively, in the brain of AB zebrafish as quantified by HPLC with electrochemical detection. We found the antagonist induced behavioral changes to be absent and the levels of these neurochemicals to be lower in another zebrafish population, SF, demonstrating naturally occurring genetic variability in these traits. We conclude that this variability may be utilized to unravel the mechanisms of social behavior in zebrafish, a line of research that may be extended to other vertebrates including our own species. PMID:22491827

Modulation of p53 and met expression by Krüppel-like factor 8 regulates zebrafish cerebellar development.

PubMed

Tsai, Ming-Yuan; Lu, Yu-Fen; Liu, Yu-Hsiu; Lien, Huang-Wei; Huang, Chang-Jen; Wu, Jen-Leih; Hwang, Sheng-Ping L

2015-09-01

Krüppel-like factor 8 (Klf8) is a zinc-finger transcription factor implicated in cell proliferation, and cancer cell survival and invasion; however, little is known about its role in normal embryonic development. Here, we show that Klf8 is required for normal cerebellar development in zebrafish embryos. Morpholino knockdown of klf8 resulted in abnormal cerebellar primordium morphology and the induction of p53 in the brain region at 24 hours post-fertilization (hpf). Both p53-dependent reduction of cell proliferation and augmentation of apoptosis were observed in the cerebellar anlage of 24 hpf-klf8 morphants. In klf8 morphants, expression of ptf1a in the ventricular zone was decreased from 48 to 72 hpf; on the other hand, expression of atohla in the upper rhombic lip was unaffected. Consistent with this finding, Purkinje cell development was perturbed and granule cell number was reduced in 72 hpf-klf8 morphants; co-injection of p53 MO(sp) or klf8 mRNA substantially rescued development of cerebellar Purkinje cells in klf8 morphants. Hepatocyte growth factor/Met signaling is known to regulate cerebellar development in zebrafish and mouse. We observed decreased met expression in the tectum and rhombomere 1 of 24 hpf-klf8 morphants, which was largely rescued by co-injection with klf8 mRNA. Moreover, co-injection of met mRNA substantially rescued formation of Purkinje cells in klf8 morphants at 72 hpf. Together, these results demonstrate that Klf8 modulates expression of p53 and met to maintain ptf1a-expressing neuronal progenitors, which are required for the appropriate development of cerebellar Purkinje and granule cells in zebrafish embryos. © 2014 Wiley Periodicals, Inc.

The Mammalian “Obesogen” Tributyltin Targets Hepatic Triglyceride Accumulation and the Transcriptional Regulation of Lipid Metabolism in the Liver and Brain of Zebrafish

PubMed Central

Lyssimachou, Angeliki; Santos, Joana G.; André, Ana; Soares, Joana; Lima, Daniela; Guimarães, Laura; Almeida, C. Marisa R.; Teixeira, Catarina; Castro, L. Filipe C.; Santos, Miguel M.

2015-01-01

Recent findings indicate that different Endocrine Disrupting Chemicals (EDCs) interfere with lipid metabolic pathways in mammals and promote fat accumulation, a previously unknown site of action for these compounds. The antifoulant and environmental pollutant tributyltin (TBT), which causes imposex in gastropod snails, induces an “obesogenic” phenotype in mammals, through the activation of the nuclear receptors retinoid X receptor (RXR) and peroxisome proliferator-activated receptor gamma (PPARγ). In teleosts, the effects of TBT on the lipid metabolism are poorly understood, particularly following exposure to low, environmental concentrations. In this context, the present work shows that exposure of zebrafish to 10 and 50 ng/L of TBT (as Sn) from pre-hatch to 9 months of age alters the body weight, condition factor, hepatosomatic index and hepatic triglycerides in a gender and dose related manner. Furthermore, TBT modulated the transcription of key lipid regulating factors and enzymes involved in adipogenesis, lipogenesis, glucocorticoid metabolism, growth and development in the brain and liver of exposed fish, revealing sexual dimorphic effects in the latter. Overall, the present study shows that the model mammalian obesogen TBT interferes with triglyceride accumulation and the transcriptional regulation of lipid metabolism in zebrafish and indentifies the brain lipogenic transcription profile of fish as a new target of this compound. PMID:26633012

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

PubMed

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.

The search for evolutionary developmental origins of aging in zebrafish: a novel intersection of developmental and senescence biology in the zebrafish model system.

PubMed

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

Wnt-regulated dynamics of positional information in zebrafish somitogenesis

PubMed Central

Bajard, Lola; Morelli, Luis G.; Ares, Saúl; Pécréaux, Jacques; Jülicher, Frank; Oates, Andrew C.

2014-01-01

How signaling gradients supply positional information in a field of moving cells is an unsolved question in patterning and morphogenesis. Here, we ask how a Wnt signaling gradient regulates the dynamics of a wavefront of cellular change in a flow of cells during somitogenesis. Using time-controlled perturbations of Wnt signaling in the zebrafish embryo, we changed segment length without altering the rate of somite formation or embryonic elongation. This result implies specific Wnt regulation of the wavefront velocity. The observed Wnt signaling gradient dynamics and timing of downstream events support a model for wavefront regulation in which cell flow plays a dominant role in transporting positional information. PMID:24595291

A zebrafish transgenic model of Ewing's sarcoma reveals conserved mediators of EWS-FLI1 tumorigenesis.

PubMed

Leacock, Stefanie W; Basse, Audrey N; Chandler, Garvin L; Kirk, Anne M; Rakheja, Dinesh; Amatruda, James F

2012-01-01

Ewing's sarcoma, a malignant bone tumor of children and young adults, is a member of the small-round-blue-cell tumor family. Ewing's sarcoma family tumors (ESFTs), which include peripheral primitive neuroectodermal tumors (PNETs), are characterized by chromosomal translocations that generate fusions between the EWS gene and ETS-family transcription factors, most commonly FLI1. The EWS-FLI1 fusion oncoprotein represents an attractive therapeutic target for treatment of Ewing's sarcoma. The cell of origin of ESFT and the molecular mechanisms by which EWS-FLI1 mediates tumorigenesis remain unknown, and few animal models of Ewing's sarcoma exist. Here, we report the use of zebrafish as a vertebrate model of EWS-FLI1 function and tumorigenesis. Mosaic expression of the human EWS-FLI1 fusion protein in zebrafish caused the development of tumors with histology strongly resembling that of human Ewing's sarcoma. The incidence of tumors increased in a p53 mutant background, suggesting that the p53 pathway suppresses EWS-FLI1-driven tumorigenesis. Gene expression profiling of the zebrafish tumors defined a set of genes that might be regulated by EWS-FLI1, including the zebrafish ortholog of a crucial EWS-FLI1 target gene in humans. Stable zebrafish transgenic lines expressing EWS-FLI1 under the control of the heat-shock promoter exhibit altered embryonic development and defective convergence and extension, suggesting that EWS-FLI1 interacts with conserved developmental pathways. These results indicate that functional targets of EWS-FLI1 that mediate tumorigenesis are conserved from zebrafish to human and provide a novel context in which to study the function of this fusion oncogene.

The zebrafish as a model system to study cardiovascular development.

PubMed

Stainier, D Y; Fishman, M C

1994-01-01

The zebrafish, Brachydanio rerio, is rapidly becoming a system of choice for vertebrate developmental biologists. It presents unique embryological attributes and is amenable to saturation style mutagenesis, a powerful approach that, in invertebrates, has already led to the identification of a large number of key developmental genes. Since fertilization is external, the zebrafish embryo develops in the dish and is thus accessible for continued observation and manipulation at all stages of development. Furthermore, because the embryo is transparent, the developing heart and vessels can be resolved at the single-cell level. A large number of mutations that affect the development of cardiovascular form and function have recently been isolated from large-scale genetic screens for zygotic embryonic lethals. Our further understanding of the development of the cardiovascular system is important not only because of the high incidence, and familial inheritance, of congenital abnormalities, but also because it should lead to novel, differentiation-based strategies for the analysis and therapy of the diseased state. Copyright © 1994. Published by Elsevier Inc.

Targeting autophagy to modulate cell survival: a comparative analysis in cancer, normal and embryonic cells.

PubMed

Divac Rankov, Aleksandra; Ljujić, Mila; Petrić, Marija; Radojković, Dragica; Pešić, Milica; Dinić, Jelena

2017-11-01

Autophagy is linked to multiple cancer-related signaling pathways, and represents a defense mechanism for cancer cells under therapeutic stress. The crosstalk between apoptosis and autophagy is essential for both tumorigenesis and embryonic development. We studied the influence of autophagy on cell survival in pro-apoptotic conditions induced by anticancer drugs in three model systems: human cancer cells (NCI-H460, COR-L23 and U87), human normal cells (HaCaT and MRC-5) and zebrafish embryos (Danio rerio). Autophagy induction with AZD2014 and tamoxifen antagonized the pro-apoptotic effect of chemotherapeutics doxorubicin and cisplatin in cell lines, while autophagy inhibition by wortmannin and chloroquine synergized the action of both anticancer agents. This effect was further verified by assessing cleaved caspase-3 and PARP-1 levels. Autophagy inhibitors significantly increased both apoptotic markers when applied in combination with doxorubicin while autophagy inducers had the opposite effect. In a similar manner, autophagy induction in zebrafish embryos prevented cisplatin-induced apoptosis in the tail region while autophagy inhibition increased cell death in the tail and retina of cisplatin-treated animals. Autophagy modulation with direct inhibitors of the PI3kinase/Akt/mTOR pathway (AZD2014 and wortmannin) triggered the cellular response to anticancer drugs more effectively in NCI-H460 and zebrafish embryonic models compared to HaCaT suggesting that these modulators are selective towards rapidly proliferating cells. Therefore, evaluating the autophagic properties of chemotherapeutics could help determine more accurately the fate of different cell types under treatment. Our study underlines the importance of testing autophagic activity of potential anticancer agents in a comparative approach to develop more rational anticancer therapeutic strategies.

Lethal Dysregulation of Energy Metabolism During Embryonic Vitamin E Deficiency

PubMed Central

McDougall, Melissa; Choi, Jaewoo; Kim, Hye-Kyeong; Bobe, Gerd; Stevens, J. Frederik; Cadenas, Enrique; Tanguay, Robert; Traber, Maret G.

2017-01-01

Vitamin E (α-tocopherol, VitE) was discovered in 1922 for its role in preventing embryonic mortality. We investigated the underlying mechanisms causing lethality using targeted metabolomics analyses of zebrafish VitE-deficient embryos over five days of development, which coincided with their increased morbidity and mortality. VitE deficiency resulted in peroxidation of docosahexaenoic acid (DHA), depleting DHA-containing phospholipids, especially phosphatidylcholine, which also caused choline depletion. This increased lipid peroxidation also increased NADPH oxidation, which depleted glucose by shunting it to the pentose phosphate pathway. VitE deficiency was associated with mitochondrial dysfunction with concomitant impairment of energy homeostasis. The observed morbidity and mortality outcomes could be attenuated, but not fully reversed, by glucose injection into VitE-deficient embryos at developmental day one. Thus, embryonic VitE deficiency in vertebrates leads to a metabolic reprogramming that adversely affects methyl donor status and cellular energy homeostasis with lethal outcomes. PMID:28095320

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

PubMed

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.

Lipidomics and H218O labeling techniques reveal increased remodeling of DHA-containing membrane phospholipids associated with abnormal locomotor responses in α-tocopherol deficient zebrafish (danio rerio) embryos

PubMed Central

McDougall, Melissa Q.; Choi, Jaewoo; Stevens, Jan F.; Truong, Lisa; Tanguay, Robert L.; Traber, Maret G.

2016-01-01

We hypothesized that vitamin E (α-tocopherol) is required by the developing embryonic brain to prevent depletion of highly polyunsaturated fatty acids, especially docosahexaenoic acid (DHA, 22:6), the loss of which we predicted would underlie abnormal morphological and behavioral outcomes. Therefore, we fed adult 5D zebrafish (Danio rerio) defined diets without (E−) or with added α-tocopherol (E+, 500 mg RRR-α-tocopheryl acetate/kg diet) for a minimum of 80 days, and then spawned them to obtain E− and E+ embryos. The E− compared with E+ embryos were 82% less responsive (p<0.01) to a light/dark stimulus at 96 h post-fertilization (hpf), demonstrating impaired locomotor behavior, even in the absence of gross morphological defects. Evaluation of phospholipid (PL) and lysophospholipid (lyso-PL) composition using untargeted lipidomics in E− compared with E+ embryos at 24, 48, 72, and 120 hpf showed that four PLs and three lyso-PLs containing docosahexaenoic acid (DHA), including lysophosphatidylcholine (LPC 22:6, required for transport of DHA into the brain, p<0.001), were at lower concentrations in E− at all time-points. Additionally, H218O labeling experiments revealed enhanced turnover of LPC 22:6 (p<0.001) and three other DHA-containing PLs in the E− compared with the E+ embryos, suggesting that increased membrane remodeling is a result of PL depletion. Together, these data indicate that α-tocopherol deficiency in the zebrafish embryo causes the specific depletion and increased turnover of DHA-containing PL and lyso-PLs, which may compromise DHA delivery to the brain and thereby contribute to the functional impairments observed in E− embryos. PMID:26774753

Light directs zebrafish period2 expression via conserved D and E boxes.

PubMed

Vatine, Gad; Vallone, Daniela; Appelbaum, Lior; Mracek, Philipp; Ben-Moshe, Zohar; Lahiri, Kajori; Gothilf, Yoav; Foulkes, Nicholas S

2009-10-01

For most species, light represents the principal environmental signal for entraining the endogenous circadian clock. The zebrafish is a fascinating vertebrate model for studying this process since unlike mammals, direct exposure of most of its tissues to light leads to local clock entrainment. Importantly, light induces the expression of a set of genes including certain clock genes in most zebrafish cell types in vivo and in vitro. However, the mechanism linking light to gene expression remains poorly understood. To elucidate this key mechanism, here we focus on how light regulates transcription of the zebrafish period2 (per2) gene. Using transgenic fish and stably transfected cell line-based assays, we define a Light Responsive Module (LRM) within the per2 promoter. The LRM lies proximal to the transcription start site and is both necessary and sufficient for light-driven gene expression and also for a light-dependent circadian clock regulation. Curiously, the LRM sequence is strongly conserved in other vertebrate per2 genes, even in species lacking directly light-sensitive peripheral clocks. Furthermore, we reveal that the human LRM can substitute for the zebrafish LRM to confer light-regulated transcription in zebrafish cells. The LRM contains E- and D-box elements that are critical for its function. While the E-box directs circadian clock regulation by mediating BMAL/CLOCK activity, the D-box confers light-driven expression. The zebrafish homolog of the thyrotroph embryonic factor binds efficiently to the LRM D-box and transactivates expression. We demonstrate that tef mRNA levels are light inducible and that knock-down of tef expression attenuates light-driven transcription from the per2 promoter in vivo. Together, our results support a model where a light-dependent crosstalk between E- and D-box binding factors is a central determinant of per2 expression. These findings extend the general understanding of the mechanism whereby the clock is entrained by light

Altered gene expression in the brain and ovaries of zebrafish (Danio rerio) exposed to the aromatase inhibitor fadrozole: microarray analysis and hypothesis generation.

PubMed

Villeneuve, L; Wang, Rong-Lin; Bencic, David C; Biales, Adam D; Martinović, Dalma; Lazorchak, James M; Toth, Gregory; Ankley, Gerald T

2009-08-01

As part of a research effort examining system-wide responses of the hypothalamic-pituitary-gonadal (HPG) axis in fish to endocrine-active chemicals (EACs) with different modes of action, zebrafish (Danio rerio) were exposed to 25 or 100 microg/L of the aromatase inhibitor fadrozole for 24, 48, or 96 h. Global transcriptional response in brain and ovarian tissue of fish exposed to 25 microg/L of fadrozole was compared to that in control fish using a commercially available, 22,000-gene oligonucleotide microarray. Transcripts altered in brain were functionally linked to differentiation, development, DNA replication, and cell cycle. Additionally, multiple genes associated with the one-carbon pool by folate pathway (KEGG 00670) were significantly up-regulated. Transcripts altered in ovary were functionally linked to cell-cell adhesion, extracellular matrix, vasculogenesis, and development. Promoter motif analysis identified GATA-binding factor 2, Ikaros 2, alcohol dehydrogenase gene regulator 1, myoblast-determining factor, and several heat shock factors as being associated with coexpressed gene clusters that were differentially expressed following exposure to fadrozole. Based on the transcriptional changes observed, it was hypothesized that fadrozole elicits neurodegenerative stress in brain tissue and that fish cope with this stress through proliferation of radial glial cells. Additionally, it was hypothesized that changes of gene expression in the ovary of fadrozole-exposed zebrafish reflect disruption of oocyte maturation and ovulation because of impaired vitellogenesis. These hypotheses and others derived from the microarray results provide a foundation for future studies aimed at understanding responses of the HPG axis to EACs and other chemical stressors.

Embryonic blood-cerebrospinal fluid barrier formation and function

PubMed Central

Bueno, David; Parvas, Maryam; Hermelo, Ismaïl; Garcia-Fernàndez, Jordi

2014-01-01

During embryonic development and adult life, brain cavities and ventricles are filled with cerebrospinal fluid (CSF). CSF has attracted interest as an active signaling medium that regulates brain development, homeostasis and disease. CSF is a complex protein-rich fluid containing growth factors and signaling molecules that regulate multiple cell functions in the central nervous system (CNS). The composition and substance concentrations of CSF are tightly controlled. In recent years, it has been demonstrated that embryonic CSF (eCSF) has a key function as a fluid pathway for delivering diffusible signals to the developing brain, thus contributing to the proliferation, differentiation and survival of neural progenitor cells, and to the expansion and patterning of the brain. From fetal stages through to adult life, CSF is primarily produced by the choroid plexus. The development and functional activities of the choroid plexus and other blood–brain barrier (BBB) systems in adults and fetuses have been extensively analyzed. However, eCSF production and control of its homeostasis in embryos, from the closure of the anterior neuropore when the brain cavities become physiologically sealed, to the formation of the functional fetal choroid plexus, has not been studied in as much depth and remains open to debate. This review brings together the existing literature, some of which is based on experiments conducted by our research group, concerning the formation and function of a temporary embryonic blood–CSF barrier in the context of the crucial roles played by the molecules in eCSF. PMID:25389383

Cardio-respirometry disruption in zebrafish (Danio rerio) embryos exposed to hydraulic fracturing flowback and produced water.

PubMed

Folkerts, Erik J; Blewett, Tamzin A; He, Yuhe; Goss, Greg G

2017-12-01

Hydraulic fracturing to extract oil and natural gas reserves is an increasing practice in many international energy sectors. Hydraulic fracturing flowback and produced water (FPW) is a hyper saline wastewater returned to the surface from a fractured well containing chemical species present in the initial fracturing fluid, geogenic contaminants, and potentially newly synthesized chemicals formed in the fracturing well environment. However, information on FPW toxicological mechanisms of action remain largely unknown. Both cardiotoxic and respirometric responses were explored in zebrafish (Danio rerio) embryos after either an acute sediment-free (FPW-SF) or raw/sediment containing (FPW-S) fraction exposure of 24 and 48 h at 2.5% and 5% dilutions. A 48 h exposure to either FPW fraction in 24-72 h post fertilization zebrafish embryos significantly increased occurrences of pericardial edema, yolk-sac edema, and tail/spine curvature. In contrast, larval heart rates significantly decreased after FPW fraction exposures. FPW-S, but not FPW-SF, at 2.5% doses significantly reduced embryonic respiration/metabolic rates (MO 2 ), while for 5% FPW, both fractions reduced MO 2 . Expression of select cardiac genes were also significantly altered in each FPW exposure group, implicating a cardiovascular system compromise as the potential cause for reduced embryonic MO 2 . Collectively, these results support our hypothesis that organics are major contributors to cardiac and respiratory responses to FPW exposure in zebrafish embryos. Our study is the first to investigate cardiac and respiratory sub-lethal effects of FPW exposure, demonstrating that FPW effects extend beyond initial osmotic stressors and verifies the use of respirometry as a potential marker for FPW exposure. Copyright © 2017 Elsevier Ltd. All rights reserved.

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

PubMed

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.

Emotions and motivated behavior converge on an amygdala-like structure in the zebrafish

PubMed Central

von Trotha, Jakob William; Vernier, Philippe; Bally-Cuif, Laure

2014-01-01

The brain reward circuitry plays a key role in emotional and motivational behaviors, and its dysfunction underlies neuropsychiatric disorders such as schizophrenia, depression and drug addiction. Here, we characterized the neuronal activity pattern induced by acute amphetamine administration and during drug-seeking behavior in the zebrafish, and demonstrate the existence of conserved underlying brain circuitry. Combining quantitative analyses of cfos expression with neuronal subtype-specific markers at single-cell resolution, we show that acute d-amphetamine administration leads to both increased neuronal activation and the recruitment of neurons in the medial (Dm) and the lateral (Dl) domains of the adult zebrafish pallium, which contain homologous structures to the mammalian amygdala and hippocampus, respectively. Calbindin-positive and glutamatergic neurons are recruited in Dm, and glutamatergic and γ-aminobutyric acid (GABAergic) neurons in Dl. The drug-activated neurons in Dm and Dl are born at juvenile stage rather than in the embryo or during adulthood. Furthermore, the same territory in Dm is activated during both drug-seeking approach and light avoidance behavior, while these behaviors do not elicit activation in Dl. These data identify the pallial territories involved in acute psychostimulant response and reward formation in the adult zebrafish. They further suggest an evolutionarily conserved function of amygdala-like structures in positive emotions and motivated behavior in zebrafish and mammals. PMID:25145867

C2orf71a/pcare1 is important for photoreceptor outer segment morphogenesis and visual function in zebrafish.

PubMed

Corral-Serrano, Julio C; Messchaert, Muriël; Dona, Margo; Peters, Theo A; Kamminga, Leonie M; van Wijk, Erwin; Collin, Rob W J

2018-06-26

Mutations in C2orf71 are causative for autosomal recessive retinitis pigmentosa and occasionally cone-rod dystrophy. We have recently discovered that the protein encoded by this gene is important for modulation of the ciliary membrane through the recruitment of an actin assembly module, and have therefore renamed the gene to PCARE (photoreceptor cilium actin regulator). Here, we report on the identification of two copies of the c2orf71/pcare gene in zebrafish, pcare1 and pcare2. To study the role of the gene most similar to human PCARE, pcare1, we have generated a stable pcare1 mutant zebrafish model (designated pcare1 rmc100/rmc100 ) in which the coding sequence was disrupted using CRISPR/Cas9 technology. Retinas of both embryonic (5 dpf) and adult (6 mpf) pcare1 rmc100/rmc100 zebrafish display a clear disorganization of photoreceptor outer segments, resembling the phenotype observed in Pcare -/- mice. Optokinetic response and visual motor response measurements indicated visual impairment in pcare1 rmc100/rmc100 zebrafish larvae at 5 dpf. In addition, electroretinogram measurements showed decreased b-wave amplitudes in pcare1 rmc100/rmc100 zebrafish as compared to age- and strain-matched wild-type larvae, indicating a defect in the transretinal current. Altogether, our data show that lack of pcare1 causes a retinal phenotype in zebrafish and indicate that the function of the PCARE gene is conserved across species.

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

PubMed

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.

Gene expression changes in a zebrafish model of drug dependency suggest conservation of neuro-adaptation pathways.

PubMed

Kily, Layla J M; Cowe, Yuka C M; Hussain, Osman; Patel, Salma; McElwaine, Suzanne; Cotter, Finbarr E; Brennan, Caroline H

2008-05-01

Addiction is a complex psychiatric disorder considered to be a disease of the brain's natural reward reinforcement system. Repeated stimulation of the 'reward' pathway leads to adaptive changes in gene expression and synaptic organization that reinforce drug taking and underlie long-term changes in behaviour. The primitive nature of reward reinforcement pathways and the near universal ability of abused drugs to target the same system allow drug-associated reward and reinforcement to be studied in non-mammalian species. Zebrafish have proved to be a valuable model system for the study of vertebrate development and disease. Here we demonstrate that adult zebrafish show a dose-dependent acute conditioned place preference (CPP) reinforcement response to ethanol or nicotine. Repeated exposure of adult zebrafish to either nicotine or ethanol leads to a robust CPP response that persists following 3 weeks of abstinence and in the face of adverse stimuli, a behavioural indicator of the establishment of dependence. Microarray analysis using whole brain samples from drug-treated and control zebrafish identified 1362 genes that show a significant change in expression between control and treated individuals. Of these genes, 153 are common to both ethanol- and nicotine-treated animals. These genes include members of pathways and processes implicated in drug dependence in mammalian models, revealing conservation of neuro-adaptation pathways between zebrafish and mammals.

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

PubMed

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.

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

PubMed Central

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

Graph theoretical model of a sensorimotor connectome in zebrafish.

PubMed

Stobb, Michael; Peterson, Joshua M; Mazzag, Borbala; Gahtan, Ethan

2012-01-01

Mapping the detailed connectivity patterns (connectomes) of neural circuits is a central goal of neuroscience. The best quantitative approach to analyzing connectome data is still unclear but graph theory has been used with success. We present a graph theoretical model of the posterior lateral line sensorimotor pathway in zebrafish. The model includes 2,616 neurons and 167,114 synaptic connections. Model neurons represent known cell types in zebrafish larvae, and connections were set stochastically following rules based on biological literature. Thus, our model is a uniquely detailed computational representation of a vertebrate connectome. The connectome has low overall connection density, with 2.45% of all possible connections, a value within the physiological range. We used graph theoretical tools to compare the zebrafish connectome graph to small-world, random and structured random graphs of the same size. For each type of graph, 100 randomly generated instantiations were considered. Degree distribution (the number of connections per neuron) varied more in the zebrafish graph than in same size graphs with less biological detail. There was high local clustering and a short average path length between nodes, implying a small-world structure similar to other neural connectomes and complex networks. The graph was found not to be scale-free, in agreement with some other neural connectomes. An experimental lesion was performed that targeted three model brain neurons, including the Mauthner neuron, known to control fast escape turns. The lesion decreased the number of short paths between sensory and motor neurons analogous to the behavioral effects of the same lesion in zebrafish. This model is expandable and can be used to organize and interpret a growing database of information on the zebrafish connectome.

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

PubMed

Glisic, Branka; Mihaljevic, Ivan; Popovic, Marta; Zaja, Roko; Loncar, Jovica; Fent, Karl; Kovacevic, Radmila; Smital, Tvrtko

2015-01-01

Glutathione-S-transferases (GSTs) are one of the key enzymes that mediate phase II of cellular detoxification. The aim of our study was a comprehensive characterization of GSTs in zebrafish (Danio rerio) as an important vertebrate model species frequently used in environmental research. A detailed phylogenetic analysis of GST superfamily revealed 27 zebrafish gst genes. Further insights into the orthology relationships between human and zebrafish GSTs/Gsts were obtained by the conserved synteny analysis. Expression of gst genes in six tissues (liver, kidney, gills, intestine, brain and gonads) of adult male and female zebrafish was determined using qRT-PCR. Functional characterization was performed on 9 cytosolic Gst enzymes after overexpression in E. coli and subsequent protein purification. Enzyme kinetics was measured for GSH and a series of model substrates. Our data revealed ubiquitously high expression of gstp, gstm (except in liver), gstr1, mgst3a and mgst3b, high expression of gsto2 in gills and ovaries, gsta in intestine and testes, gstt1a in liver, and gstz1 in liver, kidney and brain. All zebrafish Gsts catalyzed the conjugation of GSH to model GST substrates 1-chloro-2,4-dinitrobenzene (CDNB) and monochlorobimane (MCB), apart from Gsto2 and Gstz1 that catalyzed GSH conjugation to dehydroascorbate (DHA) and dichloroacetic acid (DCA), respectively. Affinity toward CDNB varied from 0.28 mM (Gstp2) to 3.69 mM (Gstm3), while affinity toward MCB was in the range of 5 μM (Gstt1a) to 250 μM (Gstp1). Affinity toward GSH varied from 0.27 mM (Gstz1) to 4.45 mM (Gstt1a). Turnover number for CDNB varied from 5.25s(-1) (Gstt1a) to 112s(-1) (Gstp2). Only Gst Pi enzymes utilized ethacrynic acid (ETA). We suggest that Gstp1, Gstp2, Gstt1a, Gstz1, Gstr1, Mgst3a and Mgst3b have important role in the biotransformation of xenobiotics, while Gst Alpha, Mu, Pi, Zeta and Rho classes are involved in the crucial physiological processes. In summary, this study provides the

A novel TRIM family member, Trim69, regulates zebrafish development through p53-mediated apoptosis.

PubMed

Han, Ruiqin; Zhao, Qing; Zong, Shudong; Miao, Shiying; Song, Wei; Wang, Linfang

2016-05-01

Trim69 contains the hallmark domains of a tripartite motif (TRIM) protein, including a Ring-finger domain, B-box domain, and coiled-coil domain. Trim69 is structurally and evolutionarily conserved in zebrafish, mouse, rat, human, and chimpanzee. The role of this protein is unclear, however, so we investigated its function in zebrafish development. Trim69 is extensively expressed in zebrafish adults and developing embryos-particularly in the testis, brain, ovary, and heart-and its expression decreases in a time- and stage-dependent manner. Loss of trim69 in zebrafish induces apoptosis and activates apoptosis-related processes; indeed, the tp53 pathway was up-regulated in response to the knockdown. Expression of human trim69 rescued the apoptotic phenotype, while overexpression of trim69 does not increase cellular apoptosis. Taken together, our results suggest that trim69 participates in tp53-mediated apoptosis during zebrafish development. Mol. Reprod. Dev. 83: 442-454, 2016. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Mitragynine Attenuates Withdrawal Syndrome in Morphine-Withdrawn Zebrafish

PubMed Central

Khor, Beng-Siang; Amar Jamil, Mohd Fadzly; Adenan, Mohamad Ilham; Chong Shu-Chien, Alexander

2011-01-01

A major obstacle in treating drug addiction is the severity of opiate withdrawal syndrome, which can lead to unwanted relapse. Mitragynine is the major alkaloid compound found in leaves of Mitragyna speciosa, a plant widely used by opiate addicts to mitigate the harshness of drug withdrawal. A series of experiments was conducted to investigate the effect of mitragynine on anxiety behavior, cortisol level and expression of stress pathway related genes in zebrafish undergoing morphine withdrawal phase. Adult zebrafish were subjected to two weeks chronic morphine exposure at 1.5 mg/L, followed by withdrawal for 24 hours prior to tests. Using the novel tank diving tests, we first showed that morphine-withdrawn zebrafish display anxiety-related swimming behaviors such as decreased exploratory behavior and increased erratic movement. Morphine withdrawal also elevated whole-body cortisol levels, which confirms the phenotypic stress-like behaviors. Exposing morphine-withdrawn fish to mitragynine however attenuates majority of the stress-related swimming behaviors and concomitantly lower whole-body cortisol level. Using real-time PCR gene expression analysis, we also showed that mitragynine reduces the mRNA expression of corticotropin releasing factor receptors and prodynorphin in zebrafish brain during morphine withdrawal phase, revealing for the first time a possible link between mitragynine's ability to attenuate anxiety during opiate withdrawal with the stress-related corticotropin pathway. PMID:22205946

Automated processing of zebrafish imaging data: a survey.

PubMed

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.

Automated Processing of Zebrafish Imaging Data: A Survey

PubMed Central

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

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

PubMed

Rong, Xiaozhi; Chen, Chen; Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

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.

R-Spondin 3 Regulates Dorsoventral and Anteroposterior Patterning by Antagonizing Wnt/β-Catenin Signaling in Zebrafish Embryos

PubMed Central

Zhou, Pin; Zhou, Yumei; Li, Yun; Lu, Ling; Liu, Yunzhang; Zhou, Jianfeng; Duan, Cunming

2014-01-01

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. PMID:24918770

Histone deacetylase 1 is required for the development of the zebrafish inner ear

PubMed Central

He, Yingzi; Tang, Dongmei; Li, Wenyan; Chai, Renjie; Li, Huawei

2016-01-01

Histone deacetylase 1 (HDAC1) has been reported to be important for multiple aspects of normal embryonic development, but little is known about its function in the development of mechanosensory organs. Here, we first confirmed that HDAC1 is expressed in the developing otic vesicles of zebrafish by whole-mount in situ hybridization. Knockdown of HDAC1 using antisense morpholino oligonucleotides in zebrafish embryos induced smaller otic vesicles, abnormal otoliths, malformed or absent semicircular canals, and fewer sensory hair cells. HDAC1 loss of function also caused attenuated expression of a subset of key genes required for otic vesicle formation during development. Morpholino-mediated knockdown of HDAC1 resulted in decreased expression of members of the Fgf family in the otic vesicles, suggesting that HDAC1 is involved in the development of the inner ear through regulation of Fgf signaling pathways. Taken together, our results indicate that HDAC1 plays an important role in otic vesicle formation. PMID:26832938

Novel degenerative and developmental defects in a zebrafish model of mucolipidosis type IV

PubMed Central

Li, Huiqing; Pei, Wuhong; Vergarajauregui, Sivia; Zerfas, Patricia M.; Raben, Nina; Burgess, Shawn M.; Puertollano, Rosa

2017-01-01

Abstract Mucolipidosis type IV (MLIV) is a lysosomal storage disease characterized by neurologic and ophthalmologic abnormalities. There is currently no effective treatment. MLIV is caused by mutations in MCOLN1, a lysosomal cation channel from the transient receptor potential (TRP) family. In this study, we used genome editing to knockout the two mcoln1 genes present in Danio rerio (zebrafish). Our model successfully reproduced the retinal and neuromuscular defects observed in MLIV patients, indicating that this model is suitable for studying the disease pathogenesis. Importantly, our model revealed novel insights into the origins and progression of the MLIV pathology, including the contribution of autophagosome accumulation to muscle dystrophy and the role of mcoln1 in embryonic development, hair cell viability and cellular maintenance. The generation of a MLIV model in zebrafish is particularly relevant given the suitability of this organism for large-scale in vivo drug screening, thus providing unprecedented opportunities for therapeutic discovery. PMID:28449103