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PubMed2004

Molecular cloning and characterization of the catalytic domain of zebrafish homologue of the ataxia-telangiectasia mutated gene.

Inherited biallelic mutations of the ATM (ataxia-telangiectasia mutated) gene in humans cause ataxia-telangiectasia, a rare autosomal recessive disorder associated with progressive neuro-degeneration, cancer predisposition, immunodeficiency, and hypersensitivity to ionizing radiation. The ATM gene is highly conserved across a wide range of species. In an attempt to establish a zebrafish (Danio rerio) model of ataxia-telangiectasia, we cloned the coding sequence of the catalytic domain of the zebrafish homologue of ATM and found it to contain an open reading frame encoding 907 amino acids at the carboxyl terminus of the zebrafish ATM (zATM). The catalytic domain of zATM shares 67% and 66% homology with human ATM (hATM) and mouse ATM (mATM), respectively. The full-length mRNA encoding zATM is found to be approximately 11 kb by Northern hybridization, and the expression of zATM is observed in different adult and embryonic tissues. Overexpression of a kinase-inactive zATM domain in human cells has a dominant-negative effect against hATM function. Expression of the altered zATM in ZF4 cells leads to an A-T-like phenotype in response to ionizing radiation. These results taken together indicate that zATM is the homologue of hATM. Furthermore, using the kinase-inactive form of zATM should allow manipulation of zATM function in fish cells.

Amino Acid Sequence↗

PubMed1996

Secretion and mitogenic activity of zebrafish FGF3 reveal intermediate properties relative to mouse and Xenopus homologues.

Zebrafish (Brachyodanio rerio) Fgf-3 cDNAs expressed in COS-1 cells give rise to the heterogeneous set of secreted proteins with relative molecular masses in the range of 29-30.5 kDa. These proteins associate strongly with the extracellular matrix but are quantitatively released into the culture medium in the presence of heparin (5 micrograms/ml). Extracellular zebrafish FGF3 (ZFGF3) also contains a smaller sized component that appears to result from an amino-terminal proteolytic cleavage. These properties are similar to those described for Xenopus FGF3 (XFGF3). Receptor binding experiments indicate that ZFGF3 has a higher affinity for the IIIb rather than the IIIc isoform of FGFR2; properties that are more reminiscent of the mouse than the Xenopus homologue. Consistent with the FGF receptor binding properties, ZFGF3 shows a restricted mitogenic potential and a reduced transforming activity on NIH3T3 cells compared to XFGF3. Hybrid proteins made between Xenopus and zebrafish FGF3 implicate the C-terminal region in determining the differences in receptor potential affinities, mitogenic potency and transforming activity. Thus, ZFGF3 shows the structural and secretory properties of XFGF3, but has biological properties more akin to those of the mouse homologue.

Amino Acid Sequence↗

PubMed1996

Expression of axial and sonic hedgehog in wildtype and midline defective zebrafish embryos.

We present a description of the expression of the HNF-3 beta homolog axial (axl) in the developing zebrafish up to larva stages and compare it with that of sonic hedgehog (shh). Both genes are expressed in derivatives of all three germ layers in dynamic patterns that show substantial overlap, consistent with mutual regulatory interactions between the two genes. However, we also describe unique sites of expression of both axl and shh indicating that some aspects of their regulation are independent of one another. In no tail (ntl, zebrafish Brachyury) and floating head (flh, zebrafish Xnot1) mutants, both of which affect notochord development, early expression of axl in the organizer is unaffected, excluding a function for these genes in establishment of axl expression. At later stages, ntl and flh mutants show different effects on the expression of both axl and shh in the neuroectoderm of the trunk and tail reflecting their distinct contributions to the development of the midline mesoderm; in contrast to flh and ntl mutations whose effects are restricted to the trunk and tail, mutation of cyclops (cyc) affects axl and shh expression along the entire midline of the neuroectoderm. Endodermal expression of axl and shh is not affected by the mutations showing that development of the endoderm is under distinct control.

Animals↗

PubMed1997

Linkage of LMP, TAP, and RING3 with Mhc class I rather than class II genes in the zebrafish.

The LMP2 and LMP7 genes code for subunits of the proteasome, a multimeric enzymatic complex that degrades proteins into peptides. The two subunits replace corresponding constitutively expressed subunits during the immune response. Some of the peptides generated by the proteasome in the cytosol are transported by the products of the TAP1 and TAP2 genes into the lumen of the endoplasmic reticulum and are loaded onto the assembling MHC class I molecules. In mammals, the LMP2, LMP7, TAP1, and TAP2 genes reside in the class II region of the Mhc, closely linked to the RING3 gene. In the present study we identified, cloned, and sequenced the LMP, TAP2, and RING3 genes of the zebrafish, Danio rerio. We identified variants of these genes and used them in a segregation analysis of haploid embryos derived from heterozygous mothers. The analysis revealed that in zebrafish, the LMP2, LMP7, TAP12, and RING3 loci are closely linked but, in contrast to mammals, the LMP/TAP/RING3 cluster resides not in the Mhc class II but in the class I region. We also confirmed that in the zebrafish, the class I and class II regions are not linked to each other. In this species, therefore, the LMP/TAP/RING3 genes are clustered with the class I genes on a chromosome that apparently does not contain any class II genes. The linkage of LMP/TAP/RING3/class I may be the original and the LMP/TAP/RING3/class II a derived arrangement of these genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed1997

Domains of retinoid signalling and neurectodermal expression of zebrafish otx1 and goosecoid are mutually exclusive.

Retinoid signalling plays an important role in embryonic pattern formation. Excess of retinoic acid during gastrulation results in axial defects in vertebrate embryos, suggesting that retinoids are involved in early anteroposterior patterning. To study retinoid signalling in zebrafish embryos, we developed a novel method to detect endogenous retinoids in situ in embryos, using a fusion protein of the ligand inducible transactivation domain of a retinoic acid receptor and a heterologous DNA binding domain. Using this method, we show that retinoid signalling is localized in zebrafish embryos in the region of the embryonic shield, and towards the end of gastrulation in a posterior dorsal domain. To investigate the relationships between the spatial distribution of retinoid signalling and the regulation of retinoid target genes, we studied the downregulation by retinoic acid of two genes expressed in anterior regions of the embryo, goosecoid and otx1. These experiments show that expression of both genes is strongly downregulated in the anterior neurectoderm of zebrafish embryos treated with retinoic acid, whereas mesendodermal expression is only mildly affected. Interestingly, a significant downregulation of goosecoid expression by retinoic acid was observed only during midgastrulation but not in earlier stages. In agreement with these results, spatial expression of goosecoid and otx1 does not overlap with the region of retinoid signalling in the late gastrula. Our data support the hypothesis that a localized retinoid signal is involved in axial patterning during early development, at least in part through the repression of anterior genes in posterior regions of the embryo. Furthermore, our data suggest that the action of retinoids is spatially as well as temporally regulated in the developing embryo.

Animals↗

PubMed1996

Nuclear internalization of foreign DNA by zebrafish spermatozoa and its enhancement by electroporation.

Mature sperm cells of zebrafish (Danio rerio) incubated with foreign DNA have the capacity to take up foreign DNA. Such uptake can be enhanced by electroporation. Mature spermatozoa of zebrafish were incubated and electroporated in the presence of either radiolabeled or unlabeled plasmid DNA at voltages of 500 or 1,000 or 1,500 V/cm. From the percentage of radiolabeled plasmids retained on the spermatozoa, some sperm showed an ability to spontaneously take up the plasmid DNA, and the ability was enhanced one- to twofold by electroporation. Fertilization of mature eggs with the treated sperm resulted in transmission of the plasmid DNA to the resulting offspring. Frequency of transgenic individuals, as monitored by polymerase chain reaction, increased marginally, more than doubled and nearly doubled in 500 V/cm, 1,000 V/cm, and 1,500 V/cm electroporated groups, respectively, when compared to the non-electroporated group. These results indirectly implied that electroporation enhanced the capacity of spermatozoa to take up plasmid DNA. The increased field strength, however, had a deleterious effect on the motility of the sperm, causing clumping of sperms at high voltages. Light microscopic autoradiography of treated spermatozoa was able to show that the plasmid DNA was associated with the majority of sperm but was unable to differentiate whether it was present inside the nucleus or not. Ultrastructural in situ hybridization on thin sections of zebrafish spermatozoa, however, was able to show that the exogenous DNA was internalized into the nucleus and that electroporation enhanced this internalization. The results provide direct evidence for nuclear internalization of foreign DNA by non-mammalian sperm as in mammalian sperm.

Animals↗

PubMed1996

Endogenous retinoids in the zebrafish embryo and adult.

Retinoic acid and its isoforms are considered to be endogenous compounds which regulate embryonic development. In the work reported here we have determined which retinoids are present in zebrafish embryos and how their levels change throughout development and into adulthood. All-trans-RA is present and its level does not change significantly during embryogenesis. We failed to detect other retinoic acid isomers such as 9-cis-RA and 4-oxo-RA, but we did observe a rapid rise in the level of didehydroretinol after gastrulation. The most striking result is that the zebrafish embryo, like Xenopus and tunicates, contains a vast excess of t-retinal whereas the embryos of higher vertebrates have an excess of t-retinol. However, as the zebrafish grows, the levels of t-retinol rise so that by adulthood t-retinol and t-retinal concentrations are more equivalent, indicating a changing pattern of retinoid metabolism with growth. To examine the significance of the use of t-retinal as a precursor of t-RA we treated embryos with disulphiram, an inhibitor of retinaldehyde dehydrogenase. This resulted in embryos with an undulating notochord and correspondingly abnormal somites and ventral floor plate. In contrast to this effect, 4-methylpyrazole, which inhibits alcohol dehydrogenases, had no effect on development. This effect of disulphiram suggests that t-RA may be involved in the establishment of the anteroposterior axis of the embryo.

Alcohol Dehydrogenase↗

PubMed1996

Secondary axis induction by heterospecific organizers in zebrafish.

To investigate the inductive activities of the vertebrate organizer, we transplanted the chicken organizer (Hensen's node) into zebrafish gastrula and analyzed resulting secondary axes. Grafted Hensen's node did not differentiate or participate in the secondary axis. It also did not induce a secondary notochord or expression of the genes normally expressed by the fish organizer including no tail, axial, goosecoid. Nevertheless, it recruited fish cells to organize a variety of tissues: the dorsal portion of the central nervous system including Rohon-Beard sensory neurons, otic vesicles, dorsal pigment stripe, dorsal fin, somites, heart, and pronephric ducts. Enlarged neural plate induced by the organizer was shown by the expression pattern of dlx3 and msxB genes, which demarcates the early presumptive neural tissue. In addition, Hensen's node of an earlier stage chicken embryo displayed differential movement in zebrafish from that of a later stage. This might reflect unknown differences in properties between the organizer at two different developmental stages related to its normal organizer activity. To create a model system to study the molecular mechanisms of the organizer, we next transplanted genetically modified mouse cells into zebrafish embryos. We found that Wnt3A-transfected NIH3T3 cells are much more potent in inducing a secondary axis than NIH3T3 cells alone. These results suggest that formation of a variety of tissues are controlled by signalling from the organizer itself with no requirement of participation of the organizer-derived tissues. Additionally, the activities of the organizer may involve a function of Wnt-family genes.

3T3 Cells↗

PubMed1998

Anterior-posterior subdivision of the somite in embryonic zebrafish: implications for motor axon guidance.

The ventral branches of the segmental peripheral nerves in the zebrafish embryo are pioneered by the caudal primary (CaP) motor axons, which extend midsegmentally at the interface of the somite and the notochord. The signals that define the CaP pathway are not well understood. To gain insight into the nature of the guidance cues, we have examined the environment of the CaP motor axons by using electron microscopy and histochemistry. Specifically, we have mapped the distribution of the transcription factor engrailed, of a chondrotin sulfate epitope, and of the recognition molecules zebrafish semaphorin z1b and zebrafish tenascin C. Ultrastructural examination of dye-labeled CaP motor axons revealed a close association with the medial surface of the somite but not with the notochord. The CaP axons were always accompanied by cells that appeared to migrate at the interface of somite and notochord. These cells were confined to the posterior half of the somite. Some of the cells may be neural crest derived, many others are probably of sclerotomal origin. The putative migratory cells expressed a chondroitin sulfate epitope that is a marker of sclerotome in the chick. The pathway of the CaP axon and the distribution of the putative neural crest and sclerotome cells correlated with a subdivision of the myotome into an anterior and posterior components, which were evident at the histological level and by the expression of the markers engrailed, semaphorin z1b, chondroitin sulfate, and tenascin C. We suggest that both the pathway choice of the CaP axon and the route of migratory cells reflect this anterior-posterior bipartition of the myotome.

Animals↗

PubMed1998

Ectopic expression of hoxb2 after retinoic acid treatment or mRNA injection: disruption of hindbrain and craniofacial morphogenesis in zebrafish embryos.

To investigate pattern formation in the vertebrate hindbrain, we isolated a full length hoxb2 cDNA clone from zebrafish. In a gene phylogeny, zebrafish hoxb2 clusters with human HOXB2, and it maps on linkage group 3 along with several other loci whose orthologues are syntenic with human HOXB2. In the hindbrain, hoxb2 is expressed at high levels in rhombomere 3 (r3), lower levels in r4, still lower in r5, and at undetectable levels in r6. In r7, r8, and the rostral spinal cord, hoxb2 is expressed at a lower level than in r5. Lateral cells appearing to emanate from r4 express both hoxb2 and dlx2, suggesting that they are neural crest. Overexpression of hoxb2 by mRNA injections into early cleavage stage embryos resulted in abnormal morphogenesis of the midbrain and rostral hindbrain, abnormal patterning in r4, fusion of cartilage elements arising from pharyngeal arches 1 and 2, and ectopic expression of krx20 and valentino (but not pax2, rtk1, or hoxb1) in the rostral hindbrain, midbrain, and, surprisingly, the eye. Treatments with retinoic acid produced a phenotype similar to that of ectopic hoxb2 expression, including ectopic krx20 (but not valentino) expression in the eye, and fusion of cartilages from pharyngeal arches 1 and 2. The results suggest that hoxb2 plays an important role in the patterning of hindbrain and pharyngeal arches in the zebrafish.

Amino Acid Sequence↗

PubMed2000

Inherited retinal degeneration and apoptosis in mutant zebrafish.

The mechanism of retinal cell death was studied in mutant zebrafish (Danio rerio) which undergo inherited degeneration of the retina and the brain. The shrunken head (shr(m33)) mutation was isolated as part of a large scale mutagenesis screen. The yellow head (yhd) mutation arose spontaneously among inbred wild type zebrafish. Although the mutants share many morphological features, including small eyes, a small brain and an enlarged pericardial sac, crossing shr(m33) and yhd heterozygotes results in phenotypically normal fish. The retinae of both mutant lines of fish begin to develop normally and then undergo massive degenerative changes. Pyknotic cells first appear in the retina of the shr(m33) fish by 3 days post-fertilization and in the yhd fish by 1.5 days post-fertilization. By 5 days post-fertilization the outer nuclear layer containing the photoreceptor cells has largely disappeared in both mutants. The inner nuclear layer and ganglion cell layer are also severely affected. By 6-7 days post-fertilization, the retina has been largely cleared of pyknotic cells by retinal pigment epithelial cells and by rare macrophage-like cells. Both mutations are lethal by 7-8 days post-fertilization. Two independent measures, TdT-mediated dUTP-biotin nick end label (TUNEL) and transmission electron microscopy, indicate that the pyknotic cells in the mutant retinae are apoptotic. Apoptosis is very rarely observed during normal development of the teleost retina and was not observed in age-matched wild type zebrafish retinae examined for comparison. Our results indicate that a genetic defect can induce massive apoptosis in cell populations that do not normally undergo apoptosis during development.

Animals↗

PubMed1998

Cloning of two loci for synapse protein Snap25 in zebrafish: comparison of paralogous linkage groups suggests loss of one locus in the mammalian lineage.

Synaptosome-associated protein of 25 kDa (Snap25) is an intracellular protein that is defined as a target receptor for synapse vesicles prior to neurotransmitter release. Snap25 is highly conserved, with 61% identity between human and Drosophila melanogaster. Whereas mammals and chicken have a single locus for Snap25, the tetraploid goldfish has at least three loci. We report that the zebrafish has two loci with 91% amino acid identity to each other. The alternative splicing of exon 5 arose before the gene duplication. The expression patterns of the two loci are virtually identical in adult zebrafish. The two zebrafish snap25 loci are located in paralogous linkage groups that seem to correspond to human chromosome 20, which harbors the SNAP locus, and human chromosome 14. Because no additional Snap25 homologue has been reported for any mammal or chicken, snap25.2 may have been lost in the amniote or even tetrapod lineage.

Amino Acid Sequence↗

PubMed2000

Glowing zebrafish: integration, transmission, and expression of a single luciferase transgene promoted by noncovalent DNA-nuclear transport peptide complexes.

The development of vehicles driving foreign DNA into the cell nucleus is essential for effective cellular gene transfer applications. We report that noncovalent binding of nuclear localization signal (NLS) peptides to plasmid DNA enhances nuclear uptake of the DNA and promotes germline integration, inheritance, and expression of a single copy of a luciferase reporter gene in zebrafish. As few as 10 DNA-NLS complexes (0.06 fg plasmid DNA) cytoplasmically injected are sufficient to produce germline-transgenic zebrafish bearing a single copy of the transgene. This corresponds to a 10(5)-fold reduction in DNA concentration compared to commonly used procedures. Use of 10(3) or 10(4) DNA-NLS complexes augments the number of transgene integrations, which occur mostly within 1-4 distinct insertion sites in the genome. In situ hybridization analyses and transmission studies show that transgene integration into the germline and somatic tissues is mosaic, and that the extent of mosaicism is negatively correlated with the amount of DNA-NLS injected. In addition, a larger proportion of zebrafish harboring a single copy of the transgene expresses luciferase, albeit at a 10-fold lower level than those containing numerous transgene insertions. The data demonstrate the potential use of nuclear targeting peptides noncovalently bound to vector DNA to enhance the efficiency of biotechnological nonviral gene transfer applications.

Animals↗

PubMed1998

Screening mosaic F1 females for mutations affecting zebrafish heart induction and patterning.

The genetic pathways underlying the induction and anterior-posterior patterning of the heart are poorly understood. The recent emergence of the zebrafish model system now allows a classical genetic approach to such challenging problems in vertebrate development. Two large-scale screens for mutations affecting zebrafish embryonic development have recently been completed; among the hundreds of mutations identified were several that affect specific aspects of cardiac morphogenesis, differentiation, and function. However, very few mutations affecting induction and/or anterior-posterior patterning of the heart were identified. We hypothesize that a directed approach utilizing molecular markers to examine these particular steps of heart development will uncover additional such mutations. To test this hypothesis, we are conducting two parallel screens for mutations that affect either the induction or the anterior-posterior patterning of the zebrafish heart. As an indicator of cardiac induction, we examine expression of nkx2.5, the earliest known marker of precardiac mesoderm; to assess anterior-posterior patterning, we distinguish ventricle from atrium with antibodies that recognize different myosin heavy chain isoforms. In order to expedite the examination of a large number of mutations, we are screening the haploid progeny of mosaic F1 females. In these ongoing screens, we have identified four mutations that affect nkx2.5 expression as well as 21 that disrupt either ventricular or atrial development and thus far have recovered several of these mutations, demonstrating the value of our approach. Future analysis of these and other cardiac mutations will provide further insight into the processes of induction and anterior-posterior patterning of the heart.

Animals↗

PubMed1998

Cloning and genetic mapping of zebrafish BMP-2.

The BMP family of polypeptide growth factors has been shown to play diverse roles in establishing embryonic patterning and tissue fates. We report the cloning of the zebrafish homologue of BMP-2, examine its expression during embryogenesis, and find that it is localized to the distal end of the long arm of zebrafish chromosome 20. A missense mutation of the bmp2 gene has recently been shown to be responsible for the early dorsalized phenotype of the zebrafish swirl mutant [Kishimoto et al., 1997]. Given the dynamic expression of bmp2 in the developing embryo and the complex interactions of BMP signaling response in vertebrates, it is possible that other mutant phenotypes, due to altered bmp2 gene expression, will eventually map to or interact with this genetic locus.

Amino Acid Sequence↗

PubMed2001

Müller cell differentiation in the zebrafish neural retina: evidence of distinct early and late stages in cell maturation.

The vertebrate neural retina is mainly composed of cells of neuroectodermal origin. The primary cell types found in all vertebrate retinas are several categories of neurons and the archetypical retina glial cell the Müller cell. Although the neurons and the single glial cell type of the retina are specialized for very distinct functions, they all have a common developmental origin within the tissue. How the distinctions between cell types, in particular between neurons and glia, arise during embryonic development remains a central issue in neurobiology. In this report, we examine the genesis of Müller glial cells during zebrafish (Danio rerio) eye development. Particular emphasis is placed on the expression of the Müller cell maturation markers carbonic anhydrase and glutamine synthetase. In addition, we report that the HNK-1 monoclonal antibody, which identifies a particular glycoconjugate frequently found on cell surface recognition molecules, also identifies zebrafish retina Müller cells early in development. The expression patterns of these three markers clearly show that the Müller cells mature in stages: HNK-1 labeling and glutamine synthetase arise earlier than carbonic anhydrase expression. In addition, the embryonic zebrafish neural retina is characterized by the presence of amoeboid, carbonic anhydrase-positive microglial cells even before the genesis of retinal neuroectodermal glia. The stepwise maturation of the glia is likely to be indicative of an overall retinal maturational program in which cell differentiation and the expression of certain phenotype-defining gene products may be separately regulated.

Animals↗

PubMed2000

Distinct requirements for zebrafish angiogenesis revealed by a VEGF-A morphant.

Angiogenesis is a fundamental vertebrate developmental process that requires signalling by the secreted protein vascular endothelial growth factor-A (VEGF-A). VEGF-A functions in the development of embryonic structures, during tissue remodelling and for the growth of tumour-induced vasculature. The study of the role of VEGF-A during normal development has been significantly complicated by the dominant, haplo-insufficient nature of VEGF-A-targeted mutations in mice. We have used morpholino-based targeted gene knock-down technology to generate a zebrafish VEGF-A morphant loss of function model. Zebrafish VEGF-A morphant embryos develop with an enlarged pericardium and with major blood vessel deficiencies. Morphological assessment at 2 days of development indicates a nearly complete absence of both axial and intersegmental vasculature, with no or reduced numbers of circulating red blood cells. Molecular analysis using the endothelial markers fli-1 and flk-1 at 1 day of development demonstrates a fundamental distinction between VEGF-A requirements for axial and intersegmental vascular structure specification. VEGF-A is not required for the initial establishment of axial vasculature patterning, whereas all development of intersegmental vasculature is dependent on VEGF-A signalling. The zebrafish thus serves as a quality model for the study of conserved vertebrate angiogenesis processes during embryonic development.

Animals↗

PubMed2000

Light-induced rod and cone cell death and regeneration in the adult albino zebrafish (Danio rerio) retina.

Light-induced photoreceptor cell degeneration has been studied in several species, but not extensively in the teleost fish. Furthermore, the continual production of rods and cones throughout the teleost's life may result in regeneration of lost rods and cones. We exposed adult albino zebrafish to 7 days of constant darkness, followed by 7 days of constant 8000 lux light, followed by 28 days of recovery in a 14-h light:10-h dark cycle. We characterized the resulting photoreceptor layer cell death and subsequent regeneration using immunohistochemistry and light microscopy. Within the first 24 h of constant light, the zebrafish retina exhibited widespread rod and cone cell apoptosis. High levels of cell proliferation within the inner nuclear layer (INL) were observed within the first 3 days of constant light, as assessed by immunodetection of proliferating cell nuclear antigen and BrdU labeling. The proliferating cells within the INL were closely associated with the radial processes of Müller glia, similar to the pluripotent retinal stem cells observed during embryonic development. Using antibodies generated against the individual zebrafish opsins, we determined that rods and the green, blue, and ultraviolet cone cells were replaced within the 28 day recovery period. While both rods and cones were replaced, the well-ordered cone cell mosaic was not reestablished.

Animals↗