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PubMed1999

Effects of triphenyltin acetate on survival, hatching success, and liver ultrastructure of early life stages of zebrafish (Danio rerio).

In order to evaluate the toxicity of the fungicide triphenyltin acetate (TPTA) on hatching, survival, morphology, and histology of early life stages of zebrafish (Danio rerio), newly fertilized eggs were exposed to concentrations of 0.5, 5, 25, 75, and 100 microg/liter TPTA for 96 h at 28+/-0.5 degrees C. Embryos and larvae were kept under constant observation for up to 7 days and studied with respect to mortality and teratogenic effects as well as histological and cytological alterations in the liver as endpoints of sublethal toxicity of TPTA. Exposure to TPTA caused dose- and time-related effects with respect to all parameters investigated: (1) Hatching was delayed at concentrations >/=0.5 microg/liter TPTA; (2) mortality increased at >/=25 microg/liter TA after 96 h exposure, with TPTA toxicity being higher in larval than in embryonic stages; (3) from >/=25 microg/liter, larvae exhibited skeletal malformation, retarded yolk sac resorption, and edema in the heart and yolk sac regions; and (4) histo- and cytopathological alterations of larval liver included changes in nuclei and mitochondria as well as glycogen depletion from >/=0.5 microg/liter TPTA. The study thus demonstrates not only an elevated sensitivity of zebrafish embryos to TPTA in stages prior to hatching, but also the importance of continuous observation over extended periods after termination of the actual exposure for a comprehensive evaluation of the toxicity of chemical compounds.

Animals↗
PubMed2003

Novel regulatory sequence -82/-62 functions as a key element to drive the somite-specificity of zebrafish myf-5.

Myf-5, a transcription factor that controls muscle differentiation, is expressed in somites during early embryogenesis. However, gene regulation of myf-5 is poorly understood and detailed functional analysis of the regulatory cis-elements is needed. In zebrafish, the myf-5 upstream sequence from -82 to -62 (-82/-62) was fused with a basal promoter and transferred to fertilized zebrafish eggs. The -82/-62 cassette drove green fluorescent protein (GFP) reporter gene expression specifically in the somites. Moreover, GFP signals were detected exclusively in the somites of 28-hpf embryos derived from eggs injected with pCMV-5x(-82/-62), which contained five copies of the -82/-62 cassette inserted within cytomegalovirus promoter/enhancer. Thus, the -82/-62 cassette, conserved in mouse myf-5, functions to drive somite-specific expression and to repress nonspecific expression during the early development of zebrafish embryos. Mutated sequence analysis of -82/-62 cassette showed that the -70/-62 sequence was the key element for controlling myf-5 specificity. The putative CCAAT-like box, located at -66/-62, could not direct somite-specific expression. A DNA-protein complex was specifically formed between the -70/-62 probe and embryonic nuclear extracts. We conclude that the -70/-62 motif is essential for controlling somite-specific expression and the CCAAT-like box is essential for activating gene transcription.

Animals↗
PubMed2006

Stereospecificity and PAX6 function direct Hoxd4 neural enhancer activity along the antero-posterior axis.

The antero-posterior (AP) and dorso-ventral (DV) patterning of the neural tube is controlled in part by HOX and PAX transcription factors, respectively. We have reported on a neural enhancer of Hoxd4 that directs expression in the CNS with the correct anterior border in the hindbrain. Comparison to the orthologous enhancer of zebrafish revealed seven conserved footprints including an obligatory retinoic acid response element (RARE), and adjacent sites D, E and F. Whereas enhancer function in the embryonic CNS is destroyed by separation of the RARE from sites D-E-F by a half turn of DNA, it is rescued by one full turn, suggesting stereospecific constraints between DNA-bound retinoid receptors and the factor(s) recognizing sites D-E-F. Alterations in the DV trajectory of the Hoxd4 anterior expression border following mutation of site D or E implicated transcriptional regulators active across the DV axis. We show that PAX6 specifically binds sites D and E in vitro, and use chromatin immunoprecipitation to demonstrate recruitment of PAX6 to the Hoxd4 neural enhancer in mouse embryos. Hoxd4 expression throughout the CNS is reduced in Pax6 mutant Sey(Neu) animals on embryonic day 8. Additionally, stage-matched zebrafish embryos having decreased pax6a and/or pax6b activity display malformed rhombomere boundaries and an anteriorized hoxd4a expression border. These results reveal an evolutionarily conserved role for Pax6 in AP-restricted expression of vertebrate Hoxd4 orthologs.

Animals↗
PubMed2001

Zebrafish mdk2, a novel secreted midkine, participates in posterior neurogenesis.

Patterning the neural plate in vertebrates depends on complex interactions between a variety of secreted growth factors. Here we describe a novel secreted factor in zebrafish, named mdk2, related to the midkine family of heparin-binding growth factors that is involved in posterior neural development. mdk2 is expressed shortly after the onset of gastrulation in the presumptive neural plate cells of the epiblast, and this expression is enhanced by exogenous retinoic acid. Ectopic expression of mdk2 enhances neural crest cell fates at the lateral edges of the caudal neural plate, concomitant with a repression of anterior structures and mesendodermal and ectodermal markers. Reciprocally, ectopic expression of a dominant negative mdk2 results in severe deficiencies of structures posterior to the midbrain-hindbrain boundary, with negligible effects on anterior structures. In these embryos, the expression of hindbrain and neural crest markers is strongly reduced, and the formation of posterior primary moto- and sensory neurons is blocked. Analyses in mutant zebrafish embryos shows that expression of mdk2 is independent of FGF8 and nodal-related-1 signaling, but is under negative control of BMP signaling. These data support the hypothesis that mdk2 participates in posterior neural development in zebrafish.

Amino Acid Sequence↗
PubMed1995

Transparent things: cell fates and cell movements during early embryogenesis of zebrafish.

Development of an animal embryo involves the coordination of cell divisions, a variety of inductive interactions and extensive cellular rearrangements. One of the biggest challenges in developmental biology is to explain the relationships between these processes and the mechanisms that regulate them. Teleost embryos provide an ideal subject for the study of these issues. Their optical lucidity combined with modern techniques for the marking and observation of individual living cells allow high resolution investigations of specific morphogenetic movements and the construction of detailed fate maps. In this review we describe the patterns of cell divisions, cellular movements and other morphogenetic events during zebrafish early development and discuss how these events relate to the formation of restricted lineages.

Animals↗
PubMed1998

The zebrafish organizer.

Signals from the organizer play a crucial role in patterning the vertebrate embryo. Recent molecular analysis of zebrafish mutations has established an essential role for BMP2 and chordin in organizer function and has identified one-eyed pinhead as a novel EGF-like gene involved in prechordal plate and endoderm formation. In addition, embryological studies have suggested that the zebrafish organizer is induced by extraembryonic cues and have defined two novel organizing centers that pattern the nervous system along the anterior-posterior axes.

Animals↗
PubMed1996

Specific localization of zebrafish hsp90 alpha mRNA to myoD-expressing cells suggests a role for hsp90 alpha during normal muscle development.

Members of the eukaryotic hsp90 family function as important molecular chaperones in the assembly, folding and activation of a select group of cellular signalling molecules and transcription factors. Several of the molecules with which hsp90 interacts, such as the bHLH transcription factor myoD, are known to be important regulators of developmental events in vertebrates. However, little information is available in support of any specific role for hsp90 in developing embryos in vivo. In this study, we provide the first in vivo evidence that the hsp90 alpha gene may play a role in the process of myogenesis. We show that constitutive hsp90 alpha mRNA in zebrafish embryos is restricted primarily to a subset of cells within the somites and pectoral fin buds which also express myoD. Furthermore, expression of the hsp90 alpha gene is down-regulated along with myoD in differentiated muscles of the trunk at a time when levels of mRNA encoding the muscle structural protein alpha-tropomyosin remain high. No hsp90 alpha mRNA is detectable within the CNS at control temperatures. In contrast, heat shock-induced expression of the hsp90 alpha gene occurs throughout the embryo at all stages of development examined. The expression patterns strongly suggest that the hsp90 alpha gene plays a specific role in the normal process of myogenesis in addition to providing protection to all cells of the embryo during periods of environmental stress.

Animals↗
PubMed2006

Critical role of Brg1 member of the SWI/SNF chromatin remodeling complex during neurogenesis and neural crest induction in zebrafish.

Brg1 is a member of the SWI/SNF chromatin-remodeling complex, and in some organisms Brg1 has been shown to interact with beta-catenin and positively control the TCF/LEF transcription factor that is located downstream of the Wnt signal transduction pathway. During development, TCF/LEF activity is critical during neurogenesis and head induction. In zebrafish, Brg1-deficient embryos exhibit retinal cell differentiation and eye defects; however, the role of Brg1 in neurogenesis and neural crest cell induction remains elusive. We used zebrafish deficient in Brg1 (yng) or Brg1 specific-morpholino oligonucleotide-mediated knockdown to analyze the embryonic requirements of Brg1. Our results indicate that reduction in Brg1 expression leads to the expansion of the forebrain-specific transcription factor, six3, and marked reduction in expression of the mid/hind-brain boundary and hind-brain genes, engrailed2 and krox20, respectively. At 12 hpf, the expression of neural crest specifiers are severely affected in Brg1-morpholino-injected embryos. These results suggest that Brg1 is involved in neural crest induction, which is critical for the development of neurons, glia, pigment cells, and craniofacial structures. Brg1 is a maternal factor, and brg1-deficient embryos bearing the yng mutation derived from heterozygote intercrosses exhibit lesser effects on neural crest-specific gene expression, but show defects in neurogenesis and neural crest cell differentiation. This is exhibited by the aberrant brain patterning, a reduction in the sensory neurons, and craniofacial defects. These results further elucidate the critical role for Brg1 in neurogenesis, neural crest induction, and differentiation.

Adaptor Proteins, Signal Transducing↗
PubMed2003

Hematologic and serum biochemical values for zebrafish (Danio rerio).

The zebrafish (Danio rerio) has proven an excellent model for study of vertebrate development and genetics. Mutagenesis studies have produced many blood mutants with defects ranging from hematopoiesis to coagulation. The overwhelming majority of zebrafish studies have focused on development and mutational effects in embryos, whereas effects in mature zebrafish have gone largely unexplored. We believe that zebrafish will prove a valuable model for study of aging and age-related diseases, and we have sought to characterize some of the basic features of mature zebrafish. Accordingly, blood was collected from adult zebrafish and was analyzed to determine reference hematologic and biochemical parameters. White blood cell differential counts indicated predominantly lymphocytes, with mean proportion of 82.95%. Total red blood cell counts averaged 3.02 x 10(6) cells/microl. Except for increases in alanine transaminase (ALT), amylase, and phosphorus values, serum biochemical analytes were within the range of reported values for mammals and other species of fish. Accurate analysis of the many zebrafish mutants generated requires determination of normal characteristics of zebrafish. We believe results such as these will help define normal adult zebrafish, which have a tremendous potential for use in the study of human disease and aging.

Animals↗
PubMed2002

Pattern formation: swimming in retinoic acid.

The metabolite retinoic acid has been implicated as a key player during anterior-posterior patterning in vertebrate embryos. Recent studies in zebrafish extend this model by demonstrating the influence of retinoic acid on endoderm regionalization.

Animals↗
PubMed2002

Zebrafish DeltaNp63 is a direct target of Bmp signaling and encodes a transcriptional repressor blocking neural specification in the ventral ectoderm.

Bone morphogenetic proteins (Bmps) promote ventral specification in both the mesoderm and the ectoderm of vertebrate embryos. Here we identify zebrafish DeltaNp63, encoding an isoform of the p53-related protein p63, as an ectoderm-specific direct transcriptional target of Bmp signaling. DeltaNp63 itself acts as a transcriptional repressor required for ventral specification in the ectoderm of gastrulating embryos. Loss of DeltaNp63 function leads to reduced nonneural ectoderm followed by defects in epidermal development during skin and fin bud formation. In contrast, forced DeltaNp63 expression blocks neural development and promotes nonneural development, even in the absence of Bmp signaling. Together, DeltaNp63 fulfills the criteria to be the neural repressor postulated by the "neural default model."

Animals↗
PubMed2006

Real-time PCR analysis of cytochrome P450 aromatase expression in zebrafish: gene specific tissue distribution, sex differences, developmental programming, and estrogen regulation.

In teleost fish, the predominant brain form of cytochrome P450 aromatase (P450aromB) is a neural marker of estrogen effect, and an entry point for studying the role of hormonal and environmental estrogens on neurodevelopment and neuroplasticity. As part of a project using zebrafish to investigate these issues, we developed and validated a rapid, sensitive, and reproducible real-time polymerase chain reaction (PCR) assay for quantifying and comparing P450aromB and P450aromA expression in unfertilized eggs, embryos/larvae, and dissected tissues of adult fish. Results confirm that P450aromB and -A predominate in brain and ovary, respectively, and further show that the degree of overlapping expression (ratio, B:A) is 100:1 in brain, 1:50 in ovary, 1:1 in eye, and 2:1 in testis. Sex differences were observed in eye only (female>male). When compared to whole ovaries, unfertilized eggs had similar levels of P450aromA but enrichment of P450aromB, which suggests preferential synthesis or accumulation in mature oocytes. Both of the maternally derived aromatase isoforms were rapidly degraded post-fertilization, but the onset of embryonic P450aromB expression (5 hpf) was much earlier than P450aromA (48 hpf), and reached higher maximum levels (e.g., 10-fold at 72 hpf). Consistent with earlier reports, P450aromB but not -A was estrogen-inducible, but the estrogen response system in embryos was far more robust than in adults (>100- vs. <4-fold maximal induction, respectively). Application of this real-time PCR assay to measurement of P450aromB and -A in zebrafish embryos has utility for routine screening of chemicals and environmental samples for estrogen-like bioactivity and neural effects.

Animals↗
PubMed2005

Analysis of thrombocyte development in CD41-GFP transgenic zebrafish.

Thrombocytes are the nucleated equivalent of platelets in nonmammalian vertebrates such as the zebrafish, Danio rerio. We have cloned zebrafish CD41 cDNA (alpha(IIb), glycoprotein IIb [GPIIb]) and its promoter and have generated transgenic zebrafish lines with green fluorescent protein (GFP)-tagged thrombocytes. CD41 mRNA transcripts appeared 42 hours after fertilization (hpf) by reverse-transcriptase-polymerase chain reaction (RT-PCR) and at 48 hpf in circulating hematopoietic cells. Flow sorting of thrombocytes from the mesonephros of adult CD41-GFP zebrafish showed a GFP(high) subset, which had the morphologic appearance of mature thrombocytes, and a GFP(low) subset with an immature appearance, suggesting that they may be thrombocyte precursors. Confocal laser microscopy of embryos 40 and 48 hpf also showed a nonmobile population of GFP+ cells in a discrete area between the dorsal aorta and caudal vein. Production of circulating thrombocytes was inhibited by the injection of antisense morpholinos for the stem-cell transcription factor scl and c-mpl, the receptor for thrombopoietin. The nonmobile pool of GFP+ cells was abolished by scl knockdown and partially inhibited by c-mpl knockdown. These studies have shown that it is possible to identify thrombocytes, thrombocyte precursors, and, possibly, early hematopoietic stem cells in zebrafish embryos and track their proliferation and maturation.

Animals↗
PubMed2006

Zebrafish cypher is important for somite formation and heart development.

Mammalian CYPHER (Oracle, KIA0613), a member of the PDZ-LIM family of proteins (Enigma/LMP-1, ENH, ZASP/Cypher, RIL, ALP, and CLP-36), has been associated with cardiac and muscular myopathies. Targeted deletion of Cypher in mice is neonatal lethal possibly caused by myopathies. To further investigate the role of cypher in development, we have cloned the zebrafish orthologue. We present here the gene, domain structure, and expression pattern of zebrafish cypher during development. Cypher was not present as a maternal mRNA and was absent during early development. Cypher mRNA was first detected at the 3-somite stage in adaxial somites, and as somites matured, cypher expression gradually enveloped the whole somite. Later, cypher expression was also found in the heart, in head and jaw musculature, and in the brain. We further identified 13 alternative spliced forms of cypher from zebrafish heart and skeletal muscle tissue, among them a very short form containing the PDZ domain but lacking the ZM (ZASP-like) motif and the LIM domains. Targeted gene knock-down experiments using cypher antisense morpholinos led to severe defects, including truncation of the embryo, deformation of somites, dilatation of the pericardium, and thinning of the ventricular wall. The phenotype could be rescued by a cypher form, which contains the PDZ domain and the ZM motif, but lacks all three LIM domains. These findings indicate that a PDZ domain protein is important for normal somite formation and in normal heart development. Treatment of zebrafish embryos with cyclopamine, which disrupts hedgehog signaling, abolished cypher expression in 9 somite and 15-somite stage embryos. Taken together, our data suggest that cypher may play a role downstream of sonic hedgehog, in a late stage of somite development, when slow muscle fibers differentiate and migrate from the adaxial cells.

Alternative Splicing↗
PubMed2002

Green fluorescent protein expression in germ-line transmitted transgenic zebrafish under a stratified epithelial promoter from keratin8.

A zebrafish cDNA encoding a novel keratin protein was characterized and named keratin8, or krt8. krt8 expression was initiated at 4.5 hr postfertilization, immediately after the time of zygotic genome activation. The expression is limited to a single layer of envelope cells on the surface of embryos and, in later stages, it also appears in the innermost epithelial layer of the anterior- and posteriormost portions of the digestive tract. In adult, its expression was limited to the surface layer of stratified epithelial tissues, including skin epidermis and epithelia of mouth, pharynx, esophagus, and rectum but not in the gastral and intestinal epithelia. By using a 2.2-kb promoter from krt8, several stable green fluorescent protein (gfp) transgenic zebrafish lines were established. All of these transgenic lines displayed GFP expression in tissues mentioned above except for the rectum; therefore, the pattern of transgenic GFP expression is essentially identical to that of the endogenous krt8 mRNAs. krt8-GFP fusion protein was also expressed in zebrafish embryos under a ubiquitous promoter, and the fusion protein was capable of assembling into intermediate filaments only in the epithelia that normally expressed krt8 mRNAs, indicating the specificity of keratin assembly in vivo.

Amino Acid Sequence↗
PubMed1999

Characterization of zebrafish smad1, smad2 and smad5: the amino-terminus of smad1 and smad5 is required for specific function in the embryo.

Members of the TGFbeta superfamily of signalling molecules play important roles in mesendoderm induction and dorsoventral patterning of the vertebrate embryo. We cloned three intracellular mediators of TGFbeta signalling, smad1, 2 and 5, from the zebrafish. The three smad genes are expressed ubiquitously at the onset of gastrulation. The pattern of expression becomes progressively restricted during somitogenesis suggesting that at later stages not only the distribution of the TGFbeta signal but also that of the intracellular smad signal transducer determine the regionally restricted effects of TGFbeta signalling. Forced expression of smad1 leads to an expansion of blood cells resembling the phenotype of moderately ventralized zebrafish mutants. In contrast to Smad1, neither Smad2 nor Smad5 caused a detectable effect when expressed as full-length molecules suggesting that these latter two Smads are more dependent on activation by the cognate TGFbeta ligands. N-terminal truncated Smad2 dorsalized embryos, in agreement with a role downstream of dorsalizing TGFbeta members such as Nodals. In contrast to the C-terminal MH2 domain of Smad2, the C-terminal region of Smad1 and Smad5 lead to pleiotropic effects in embryos giving rize to both dorsalized and ventralized characteristics in injected embryos. Analysis of truncated zebrafish Smad1 in Xenopus embryos supports the notion that the C-terminal domain of smad1 is both a hypomorph and antimorph which can act as activator or inhibitor depending on the region of expression in the embryo. These results indicate a specific function of the MH1 domain of Smad1 and 5 for activity of the molecules.

Amino Acid Sequence↗
PubMed2005

Epithelial Ca(2+) channel expression and Ca(2+) uptake in developing zebrafish.

The purpose of the present work was to study the possible role of the epithelial Ca(2+) channel (ECaC) in the Ca(2+) uptake mechanism in developing zebrafish (Danio rerio). With rapid amplification of cDNA ends, full-length cDNA encoding the ECaC of zebrafish (zECaC) was cloned and sequenced. The cloned zECaC was 2,578 bp in length and encoded a protein of 709 amino acids that showed up to 73% identity with previously described vertebrate ECaCs. The zECaC was found to be expressed in all tissues examined and began to be expressed in the skin covering the yolk sac of embryos at 24 h postfertilization (hpf). zECaC-expressing cells expanded to cover the skin of the entire yolk sac after embryonic development and began to occur in the gill filaments at 96 hpf, and thereafter zECaC-expressing cells rapidly increased in both gills and yolk sac skin. Corresponding to ECaC expression profile, the Ca(2+) influx and content began to increase at 36-72 hpf. Incubating zebrafish embryos in low-Ca(2+) (0.02 mM) freshwater caused upregulation of the whole body Ca(2+) influx and zECaC expression in both gills and skin. Colocalization of zECaC mRNA and the Na(+)-K(+)-ATPase alpha-subunit (a marker for mitochondria-rich cells) indicated that only a portion of the mitochondria-rich cells expressed zECaC mRNA. These results suggest that the zECaC plays a key role in Ca(2+) absorption in developing zebrafish.

Amino Acid Sequence↗