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Cytokeratin 8 is a suitable epidermal marker during zebrafish development.

We have cloned and characterized the zebrafish (Danio rerio) homologous cytokeratin 8 (zf-K8) cDNA. This cytokeratin belongs to the gene family of intermediate filaments and it is a component of the cytoskeleton of epithelial cells. Gene expression analysis during embryonic development and at adult stages presented here revealed that zf-K8 mRNA is inherited maternally and that it is present in the oocyte, the zygote and in the cleavage stage embryo. After mid blastula transition this gene is expressed in all surface cells, notably in those of the enveloping layer (EVL) and of the periderm, as well as in a subpopulation of the deep cells (DEL) presumed to be intestinal progenitors. During later embryonic stage zf-K8 mRNA is strongly expressed in the developing pectoral fin. In adult zebrafish, the zf-K8 gene is not only expressed in simple epithelia such as the colorectal intestine, but also, in contrast to other vertebrates, it is present in stratified skin and differentiated fins. These observations suggest that the zf-K8 gene is an appropriate epidermal marker during zebrafish ontogenesis.

Amino Acid Sequence↗

Effects of embryonic exposure to ethanol on zebrafish visual function.

Across a variety of species, including humans, it has been shown that embryos exposed to ethanol display eye abnormalities as well as deficiencies in visual physiology and behavior. The purpose of this study was to examine the effects of embryonic exposure to ethanol on visual function in zebrafish. Visual function was assessed physiologically, via electroretinogram (ERG) recordings, and behaviorally, by measuring visual acuity with the optomotor response. Zebrafish larvae were exposed to 1.5% ethanol at various times during development, including the period of maximal eye development. The results show that ethanol effects on visual function were most pronounced when exposure occurred during eye development. ERG recordings from ethanol-exposed larvae differed from normal subjects both in shape of the response waveform and in visual thresholds under both light and dark adaptation; the differences were more pronounced under lower levels of adaptation. Also, ethanol-exposed larvae displayed lower visual acuity as determined from the optomotor response. These results indicate embryonic ethanol exposure affects visual function particularly when exposure occurs during eye development. In addition, these findings illustrate the usefulness of the zebrafish as a viable animal model for studying Fetal Alcohol Syndrome (FAS).

Alcohol-Induced Disorders, Nervous System↗

Cloning and embryonic expression patterns of the zebrafish Runt domain genes, runxa and runxb.

We isolated zebrafish homologues of the Runt-related transcription factor gene family (Runx family), runxa and runxb, and analyzed their developmental expression patterns. The deduced amino acid sequence of Runxa was highly homologous to that of AML1 (also called CBFA2, PEBP2alphaB or Runx1), a critical regulator of mammalian hematopoiesis expressed in cells of the hematopoietic lineage as well as other tissues. During zebrafish development, the runxa gene was not expressed in hematopoietic tissues but in the olfactory placodes and cells attached to the otic vesicles. We identified three kinds of runxb transcripts, which encoded two types of proteins with different N-terminal regions. The Runxb proteins were highly similar to AML2 (CBFA3, PEBP2alphaC or Runx3). The expression sites of the shared region of runxb mRNAs during development were the trigeminal ganglions, dorsal neurons of the neural tube and the lateral line primordia. These findings show that expression patterns of the zebrafish Runx genes are distinct from that of the mammalian genes.

Animals↗

Cloning and expression pattern of a zebrafish homolog of forkhead activin signal transducer (FAST), a transcription factor mediating Nodal-related signals.

Forkhead activin signal transducer (FAST) is a member of the winged-helix family of DNA-binding proteins that has been implicated in mesoderm induction and left-right axis specification during embryonic development in Xenopus and mouse. We have cloned and characterized a zebrafish FAST homolog. Zebrafish fast is expressed maternally and zygotically. Transcripts start regionalizing and decline in level during gastrulation. During somitogenesis, fast is expressed bilaterally in the lateral plate mesoderm, like its mouse homolog. In addition, zebrafish fast is also expressed bilaterally in the dorsal diencephalon, where the nodal-related cyclops gene is only expressed on the left side. It remains to be demonstrated whether FAST expression in the brain can mediate Nodal-induced asymmetric development.

Amino Acid Sequence↗

Expression of proneural and neurogenic genes in the zebrafish lateral line primordium correlates with selection of hair cell fate in neuromasts.

Expression of a mouse atonal homologue, math1, defines cells with the potential to become sensory hair cells in the mouse inner ear (Science 284 (1999) 1837) and Notch signaling limits the number of cells that are permitted to adopt this fate (Nat. Genet. 21 (1999) 289; J. Neurocytol. 28 (1999) 809). Failure of lateral inhibition mediated by Notch signaling is associated with an overproduction of ear hair cells in the zebrafish mind bomb (mib) and deltaA mutants (Development 125 (1998a) 4637; Development 126 (1999) 5669), suggesting a similar role for these genes in limiting the number of hair cells in the zebrafish ear. This study extends the analysis of proneural and neurogenic gene expression to the lateral line system, which detects movement via clusters of related sensory hair cells in specialized structures called neuromasts. We have compared the expression of a zebrafish atonal homologue, zath1, and neurogenic genes, deltaA, deltaB and notch3, in neuromasts and the posterior lateral line primordium (PLLP) of wild-type and mib mutant embryos. We describe progressive restriction of proneural and neurogenic gene expression in the migrating PLLP that appears to correlate with selection of hair cell fate in maturing neuromasts. In mib mutants there is a failure to restrict expression of zath1 and Delta homologues in the neuromasts revealing similarities with the phenotype previously described in the ear.

Animals↗

Expression of BMP signalling pathway members in the developing zebrafish inner ear and lateral line.

In this paper we describe the mRNA expression patterns of members of the bone morphogenetic protein (BMP) signalling pathway in the developing zebrafish ear. bmp2b, 4, and 7 are expressed in discrete areas of otic epithelium, some of which correspond to sensory patches. bmp2b and 4 mark the developing cristae before and during the appearance of differentiated hair cells. bmp4 is also expressed in a dorsal, non-sensory region of the ear. Expression of bmps in cristae is conserved between zebrafish, chick, and mouse, but there are also notable differences in ear expression patterns between these species. Of five zebrafish BMP antagonists, only one (follistatin) shows significant expression in the otic epithelium. The type I receptor bmpr-IB shows localised expression in the ear epithelium. Mediators of BMP signalling, smad1 and smad5, are expressed in statoacoustic and lateral line ganglia; smad5 is also expressed at low levels throughout the ear epithelium. An inhibitory smad, smad6, is expressed laterally in the ear epithelium. Lateral line primordia and neuromasts also express bmp2b, 4, follistatin, smad1, and smad5. The conservation of bmp expression in cristae among different species adds weight to the growing evidence that BMPs are required for the development of the vertebrate ear.

Animals↗

Cloning, expression, and alternative splicing of neogenin1 in zebrafish.

Caenorhabditis elegans UNC-40, Drosophila Frazzled, and vertebrate Neogenin and DCC constitute a subgroup of the immunoglobulin superfamily (IgSF). They possess four immunoglobulin-like domains and six fibronectin-type III repeats at the extracellular region, a single transmembrane region, and a approximately 300 amino-acid intracellular region. UNC-40, Frazzled and DCC can function in axon guidance as the receptor of Netrin (Cell Mol. Life Sci. 56 (1999) 62; Curr. Opin. Cell Biol. 10 (1998) 609). Neogenin binds to Netrin-1 with the same affinity as DCC in vitro (Cell 87 (1996) 175), and is expressed by neurons as they project axons (J. Cell Biol. 127 (1994) 2009), suggesting that it is also a DCC-like Netrin receptor. A zebrafish homologue of DCC (zDCC) is reported recently (Mech. Dev. 109 (2001) 105), but so far there is no report of zebrafish Neogenin. To elucidate a possible neural function of vertebrate Neogenin, we cloned and characterized a zebrafish homologue of neogenin, zneo1, and identified four alternative splice sites within it. In the adult, despite broad tissue distribution, our reverse transcription polymerase chain reaction and Northern analyses demonstrated the dominant expression of zneo1 mRNA in brain. We detected zneo1 mRNA in the embryos from 10 hpf onward and revealed its spatiotemporally regulated expression pattern in both neuronal and non-neuronal tissues by in situ hybridization. Our data showed that during early brain development, zneo1 mRNA was not only present in the proliferative ventricular zones but also in the domains of several first postmitotic neuron clusters when they extended axons. Alternative splicing generates several isoforms of zneo1. Most of them are developmentally regulated, showing distinct distribution in brain and other tissues.

Alternative Splicing↗

Cloning, mapping, and developmental expression of a sixth zebrafish Na,K-ATPase alpha1 subunit gene (atp1a1a.5).

We have identified and characterized cDNAs encoding a novel zebrafish Na,K-ATPase alpha subunit. The full-length cDNA encodes a 1023-amino-acid-long peptide which shows greatest homology to zebrafish alpha1 polypeptides. Radiation hybrid mapping localized the new gene (atp1a1a.5) to linkage group 1 in close proximity to the previously identified cluster of Na,K-ATPase alpha1 genes. The expression of atp1a1a.5 in zebrafish embryos was analyzed using whole-mount in situ hybridization. From mid-somitogenesis through 48 h post fertilization (hpf), atp1a1a.5 transcripts were detected in the pronephric duct, ear, and mucous cells. This expression pattern continues through 108 hpf, when high levels of expression were also detected in the intestinal bulb.

Amino Acid Sequence↗

Expression of two zebrafish homologues of the murine Six3 gene demarcates the initial eye primordia.

The murine homeobox gene Six3 and its Drosophila homologue sine oculis both have regulatory functions in eye development. We report the isolation and characterization of two zebrafish genes, six3 and six6, that are closely related to the murine Six3 gene. Zebrafish six3 may be the structural orthologue, while the six6 gene is more similar with respect to embryonic expression. Transcripts of both zebrafish six genes are first detected in involuting axial mesendoderm and, subsequently, in the overlying anterior neural plate from which the optic vesicles and the forebrain will develop. Direct correspondence between six3/six6 expression boundaries and the optic vesicles indicate essential roles in defining the eye primordia. During later stages only the six6 gene displays similar features of expression in the eyes and rostral brain as reported previously for murine Six3.

Amino Acid Sequence↗

Restricted expression of the homeobox gene prox 1 in developing zebrafish.

Prox 1 is a vertebrate homeobox gene which is homologous to the Drosophila transcription factor, prospero. We have isolated a prox 1 cDNA from zebrafish, which encodes a protein that has 82%, 84% and 83% amino acid identity with chicken, mouse and human Prox 1, respectively. Antibodies raised against human Prox 1 cross-react with zebrafish Prox 1 and are used here to determine the expression patterns of Prox 1 during zebrafish embryogenesis by whole-mount immunohistochemistry. In the 10-somite embryo, Prox 1 is expressed over the prospective lens placode and over a broad region of epithelium extending from the eye to the otic vesicle. As embryogenesis proceeds, Prox 1 expression in the eye lens becomes intense, and is detected in maturing muscle pioneer cells and superficial muscle cells. In the CNS, Prox 1 is expressed in a stripe along the forebrain-midbrain boundary, in a segmented pattern in the ventral hindbrain, and in selected cells of the ventral spinal cord. Additional sites of Prox 1 expression include the lateral line primordium, the trigeminal ganglia, the otic vesicle and occasional endodermal cells.

Amino Acid Sequence↗

Zebrafish contains two pax6 genes involved in eye development.

The Pax6 genes of both vertebrates and invertebrates are expressed in the developing eye and in the central nervous system. These genes encode transcription factors with two DNA-binding domains, an N-terminal paired domain and a homeodomain separated by a flexible linker region. Ectopic eye structures are obtained upon targeted expression of Drosophila, squid, ascidian or mouse Pax6 genes in various imaginal disc primordia of Drosophila. We have previously cloned a Pax6 cDNA from zebrafish. Here we report the cloning of a novel Pax6 homolog from zebrafish denoted Pax6.2. The coding sequences of the two genes show 82% identity whereas the deduced amino acid sequences are 95% identical with complete conservation of the paired- and homeodomains. The embryonic expression patterns of Pax6.1 and Pax6.2 reveal both overlapping and discrete expression domains suggesting a division of labor between these two very similar gene products during development of brain and eye structures. Both Pax6.1 and Pax6.2 can act as transcriptional activators with Pax6.2 being more efficient than Pax6.1. Both Pax6.1 and Pax6.2 are able to induce ectopic eyes in Drosophila, while Pax2 is not, suggesting that eye induction is not a general feature of Pax family genes but a distinct characteristic of Pax6 and its direct homologs. Attempts to detect Pax6. 2 homologs in chick, mice or humans proved unsuccessful suggesting that this gene either was lost during evolution of higher vertebrates or, more likely, arose as part of a larger scale duplication of chromosome segments occurring in the zebrafish lineage.

Amino Acid Sequence↗

The development of the paired fins in the zebrafish (Danio rerio).

In the present study, we describe the structure and normal development of the zebrafish (Danio rerio) paired fins. Particularly, we focus on the structure of the apical epidermis and on endoskeletal morphogenesis. Endoskeletal development proceeds differently in the pectoral and pelvic fins. Whereas in both fins major parts of the endoskeletal girdle develop within the fin bud mesenchyme, the pattern of chondrogenic condensations observed in the pelvic fins directly reflects the adult endoskeletal pattern. In the pectoral fin, a morphogenetic detour is taken via a functional larval endoskeleton, the endoskeletal disc. It arises in the fin bud mesenchyme from a chondrogenic anlage common with the girdle. The disc chondrifies and represents the functional endoskeleton of the larval pectoral fin. The pectoral fin endoskeleton is expanded as well as restructured during larval stages in a process which involves decomposition of cartilage matrix in the endoskeletal disc. Our comparisons of apical fold morphology with reports on other teleosts and tetrapod apical ridges show them to be homologous on the structural level. Comparisons of endoskeletal development of the zebrafish with reports on teleosts, actinopterygians and chondrichthyans show that endoskeletal morphogenesis in the zebrafish pectoral fin follows a morphogenetic process which is wide-spread among actinopterygians.

Animals↗

Use of the Gal4-UAS technique for targeted gene expression in the zebrafish.

The most common way to analyze the function of cloned genes in zebrafish is to misexpress the gene product or an altered variant of it by mRNA injection. However, mRNA injection has several disadvantages. The GAL4-UAS system for targeted gene expression allows one to overcome some of these disadvantages. To test the GAL4-UAS system in zebrafish, we generated two different kinds of stable transgenic lines, carrying activator and effector constructs, respectively. In the activator lines the gene for the yeast transcriptional activator GAL4 is under the control of a given promoter, while in the effectors the gene of interest is fused to the sequence of the DNA-binding motif of GAL4 (UAS). Crosses of animals from the activator and effector lines show that effector genes are transcribed with the spatial pattern of the activators. This work smoothes the way for a novel method of misexpression of gene products in zebrafish in order to analyze the function of genes in developmental processes.

Actins↗

Maternal and embryonic expression of zebrafish lef1.

Transcription factors of the TCF/LEF family interact with the Wnt signaling pathway to control transcription of downstream genes (Clevers, H., van de Wetering, M., 1997. TCF/LEF factor earn their wings. Trends Genet. 13, 485-489). We were interested in cloning family members which were expressed in zebrafish neural crest, because Wnt signaling modulates specification of neural crest fate (Dorsky, R.I., Moon, R.T., Raible, D.W., 1998. Control of neural crest cell fate by the Wnt signalling pathway. Nature 396, 370-373). We cloned a zebrafish homolog of lef1 and localized its chromosomal position by radiation hybrid mapping. lef1 is expressed in the neural crest as well as the tailbud and developing mesoderm, and is maternally expressed in zebrafish, unlike mouse and Xenopus homologs. In addition, we cloned two tcf3 genes and a homolog of tcf4, neither of which were strongly expressed in premigratory neural crest.

Amino Acid Sequence↗

Early appearance of pancreatic hormone-expressing cells in the zebrafish embryo.

Adult pancreatic islets comprise four cell types, alpha, beta, delta and PP, expressing glucagon, insulin, somatostatin and pancreatic-polypeptide, respectively, arising from cell lineages whose relationships during endocrine pancreas differentiation are still uncertain [Edlund, 1998. Diabetes 47, 1817-1823]. As zebrafish (Danio rerio) represents an attractive vertebrate model to study mutants affecting pancreatic organogenesis [Pack et al., 1996. Development 123, 321-328], we have investigated the expression patterns of islet hormones in zebrafish embryos, from the 16-somite (17 h) to 48-h stages, by whole-mount in situ hybridization and immunofluorescence. Results showed that in the zebrafish pancreatic primordium (a) insulin is the first hormone gene to be expressed, and (b) somatostatin colocalizes with insulin while glucagon-expressing cells, since their appearance, are distinct from insulin- or insulin/somatostatin-expressing cells. Notably, both somatostatin and glucagon, but not insulin, are first expressed in extrapancreatic regions.

Animals↗

Developmental regulation of Tbx5 in zebrafish embryogenesis.

T-box (tbx) genes constitute a large family of transcriptional regulators involved in developmental patterning processes. In tetrapods, tbx5 has been implicated in specifying forelimb type identity. Here, we report the cloning of the zebrafish tbx5.1 gene and characterise its expression during zebrafish embryogenesis and early larval development of wild type and mutant embryos that affect pectoral fin patterning. tbx5.1 is expressed during development of the heart, the pectoral fins and the eye. Notably, its expression in the lateral plate mesoderm defines a single and continuous region of heart and pectoral fin precursor cells, and constitutes the earliest specific marker for pectoral fin development in the zebrafish.

Amino Acid Sequence↗

Transposition of the nematode Caenorhabditis elegans Tc3 element in the zebrafish Danio rerio.

BACKGROUND: Transposable elements of the Tc1/mariner family are found in many species of the animal kingdom. It has been suggested that the widespread distribution of this transposon family resulted from horizontal transmission among different species. RESULTS: To test the ability of Tc1/mariner to cross species barriers, as well as to develop molecular genetic tools for studying zebrafish development, we determined the ability of the Tc3 transposon, a member of the Tc1/mariner family, to function in zebrafish. Tc3 transposons carrying sequences encoding the green fluorescent protein (GFP) were able to integrate in the fish genome by transposition. Integrated transposons expressed the GFP marker after germline transmission, and were capable of being mobilized upon introduction of transposase protein in trans. CONCLUSIONS: Our findings support models of horizontal transmission of Tc1/mariner elements between species. The work also establishes the basis for a novel method of transposon-mediated genetic transformation and for transposon-mediated genetic screens in zebrafish and other organisms.

Animals↗

Analysis of gene function in the zebrafish retina.

Mutagenesis screens in zebrafish have uncovered several hundred mutant alleles affecting the development of the retina and established the zebrafish as one of the leading models of vertebrate eye development. In addition to forward genetic mutagenesis approaches, gene function in the zebrafish embryo is being studied using several reverse genetic techniques. Some of these rely on the overexpression of a gene product, others take advantage of antisense oligonucleotides to block function of selected loci. Here we describe these methods in the context of the developing eye.

Animals↗