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At least 19 recordsLinked to original sources
PubMed1997

High-frequency generation of transgenic zebrafish which reliably express GFP in whole muscles or the whole body by using promoters of zebrafish origin.

Despite a number of reports on transgenic zebrafish, there have been no reports on transgenic zebrafish in which the gene is under the control of a promoter of zebrafish origin. Neither have there been reports on transgenic zebrafish in which the gene is under the control of a tissue-specific promoter/enhancer. To investigate whether it is possible to generate transgenic zebrafish which reliably express a reporter gene in specific tissues, we have isolated a zebrafish muscle-specific actin (alpha-actin) promoter and generated transgenic zebrafish in which the green fluorescent protein (GFP) reporter gene was driven by this promoter. In total, 41 GFP-expressing transgenic lines were generated with a frequency of as high as 21% (41 of 194), and GFP was specifically expressed throughout muscle cells in virtually all of the lines (40 of 41). Nonexpressing transgenic lines were rare. This demonstrates that a tissue-specific promoter can reliably drive reporter gene expression in transgenic zebrafish in a manner identical to the control of the endogeneous expression of the gene. Levels of GFP expression varied greatly from line to line; i.e., fluorescence was very weak in some lines, while it was extremely high in others. We also isolated a zebrafish cytoskeletal beta-actin promoter and generated transgenic zebrafish using a beta-actin-GFP construct. In all of the four lines generated, GFP was expressed throughout the body like the beta-actin gene, demonstrating that consistent expression could also be achieved in this case. In the present study, we also examined the effects of factors which potentially affect the transgenic frequency or expression levels. The following results were obtained: (i) expression levels of GFP in the injected embryo were not strongly correlated to transgenic frequency; (ii) the effect of the NLS peptide (SV40 T antigen nuclear localization sequence), which has been suggested to facilitate the transfer of a transgene into embryonic nuclei, remained to be elusive; (iii) a plasmid vector sequence placed upstream of the construct might reduce the expression levels of the reporter gene.

Actins↗
PubMed2006

Human melanoma cells transplanted into zebrafish proliferate, migrate, produce melanin, form masses and stimulate angiogenesis in zebrafish.

In this research, we optimized parameters for xenotransplanting WM-266-4, a metastatic melanoma cell line, including zebrafish site and stage for transplantation, number of cells, injection method, and zebrafish incubation temperature. Melanoma cells proliferated, migrated and formed masses in vivo. We transplanted two additional cancer cell lines, SW620, a colorectal cancer cell line, and FG CAS/Crk, a pancreatic cancer cell line and these human cancers also formed masses in zebrafish. We also transplanted CCD-1092Sk, a human fibroblast cell line established from normal foreskin and this cell line migrated, but did not proliferate or form masses. We quantified the number of proliferating melanoma and normal skin fibroblasts by dissociating xenotransplant zebrafish, dispensing an aliquot of CM-DiI labeled human cells from each zebrafish onto a hemocytometer slide and then visually counting the number of fluorescently labeled cancer cells. Since zebrafish are transparent until approximately 30 dpf, the interaction of labeled melanoma cells and zebrafish endothelial cells (EC) can be visualized by whole-mount immunochemical staining. After staining with Phy-V, a mouse anti-zebrafish monoclonal antibody (mAb) that specifically labels activated EC and angioblasts, using immunohistology and 2-photon microscopy, we observed activated zebrafish EC embedded in human melanoma cell masses. The zebrafish model offers a rapid efficient approach for assessing human cancer cells at various stages of tumorigenesis.

Animals↗
PubMed2004

The zebrafish metaxin 3 gene (mtx3): cDNA and protein structure, and comparison to zebrafish metaxins 1 and 2.

The metaxin genes of zebrafish have been investigated by determining the sequences of metaxin cDNAs and analyzing the translated amino acid sequences. A zebrafish cDNA corresponding to a third metaxin gene was identified. Zebrafish cDNAs representing metaxins 1 and 2, previously described in human and mouse, were also identified. The zebrafish metaxin genes are designated mtx1, mtx2 and mtx3, following zebrafish nomenclature guidelines. The zebrafish metaxin 3 (ZMTX3) cDNA codes for a protein of 313 amino acids (MW 35,208), while the ZMTX1 and ZMTX2 cDNAs specify proteins of 317 residues (MW 35,906) and 274 residues (MW 30,852), respectively. Alignment of the ZMTX3 and ZMTX1 amino acid sequences revealed 40% identities, while 26% identities were found for the ZMTX3/ZMTX2 alignment. A phylogenetic tree showed that the metaxins share a common ancestry, with the grouping of the zebrafish sequences with the homologous human and mouse sequences. Analysis of the domain structure of the zebrafish metaxins uncovered a glutathione S-transferase (GST) domain for each protein and, in addition, a thioredoxin-like domain for ZMTX2. A region of transmembrane helices was found near the C-terminus for the ZMTX1 protein. In addition, regions of alpha helix were seen to be the predominant feature of zebrafish metaxin secondary structure, particularly for ZMTX2 and ZMTX3. The ZMTX3 cDNA sequence has the greatest homology to a human sequence at cytogenetic location 5q14.1, close to the thrombospondin 4 gene (THBS4). Also, the mouse metaxin 3 homologue is adjacent to Thbs4 at 13C3.

Amino Acid Sequence↗
PubMed2001

Characterization of zebrafish caspase-3 and induction of apoptosis through ceramide generation in fish fathead minnow tailbud cells and zebrafish embryo.

Caspase-3 was cloned from zebrafish embryos and its properties were characterized to identify the biological implications of caspase in embryogenesis and apoptosis in zebrafish, which is a model organism in vertebrate developmental biology and genetics. The predicted amino acid sequence, totalling 282 amino acid residues, consisted of the prodomain and large and small subunits. Phylogenetic analysis showed that the cloned zebrafish caspase was a member of the caspase-3 subfamily with approx. 60% identity with caspase-3 from Xenopus, chicken and mammals. In addition, recombinant zebrafish caspase hydrolysed acetyl-Asp-Glu-Val-Asp-4-methyl-coumaryl-7-amide, and exhibited similar substrate specificity to the mammalian caspase-3 subfamily. Therefore this caspase was designated zebrafish caspase-3. Overexpression of zebrafish caspase-3 induced apoptosis and increased ceramide levels in fish fathead minnow tailbud cells and zebrafish embryos. Both ceramide generation and apoptosis induction were inhibited by treatment with a caspase inhibitor, benzyloxycarbonyl-Asp-Glu-Val-Asp-fluoromethylketone. Moreover, zebrafish caspase-3 mRNA was present in early embryos up to the 1000-cell stage as a maternal factor, and was then expressed throughout the body after the gastrula stage by zygotic expression. These findings indicate that the isolated caspase-3 plays an important role in the induction of ceramide generation as well as apoptosis in fish cells and the zebrafish embryo, and suggest that caspase-3 functions as a modulator of the pro-apoptotic signal in development.

Amino Acid Sequence↗
PubMed2005

Slit-like 2, a novel zebrafish slit homologue that might involve in zebrafish central neural and vascular morphogenesis.

Nervous and vascular systems grow as parallel networks, indicating common cues in distal targets. We have identified a novel zebrafish gene slit-like 2 (slitl2) that might involve in zebrafish central neural and vascular morphogenesis. Whole-mount in situ hybridization of zebrafish embryo detected distinct signals of slitl2 transcripts in zebrafish midline structure of central nervous system similar to that of slits. Strong expression is also observed in zebrafish vasculature. Zebrafish slitl2 shares amino acid sequence identity of 41% with Homo sapiens slitl2 (vasorin) and Mus musculus slitl2, and 35%, 33% with Danio rerio slit3, slit2. Analysis of zebrafish slitl2 cripto growth factor domain, extracellular matrix protein slit domain, and putative signal peptide confirms that as a secreted and cell-surface protein slitl2 may be essential in axon guidance, vessel development, and axis patterning. These results provide evidence that slitl2 may play important roles in zebrafish central nervous system and vascular morphogenesis.

Amino Acid Sequence↗
PubMed2005

Zebrafish gonadotropins and their receptors: II. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone subunits--their spatial-temporal expression patterns and receptor specificity.

Gonadotropins, follicle-stimulating hormone (FSH), and luteinizing hormone (LH) play critical roles in vertebrate reproduction. In the present study, we cloned and characterized zebrafish FSHbeta (fshb), LHbeta (lhb), and GTHalpha (cga) subunits. Compared with the molecules of other teleosts, the cysteine residues and potential glycosylation sites are fully conserved in zebrafish Lhb and Cga but not in Fshb, whose cysteines exhibit unique distribution. Interestingly, in addition to the pituitary, fshbeta, lhbeta, and cga were also expressed in some extrapituitary tissues, particularly the gonads and brain. In situ hybridization showed that zebrafish fshbeta and lhbeta were expressed in two distinct populations of gonadotrophs in the pituitary. Real-time reverse transcription-polymerase chain reaction (RT-PCR) analysis revealed that all the three subunits increased expression before ovulation (0100-0400) when the germinal vesicles in the full-grown follicles were migrating toward the periphery, but the levels dropped at 0700, when ovulation occurred. Recombinant zebrafish FSH (zfFSH) and LH (zfLH) were produced in the Chinese hamster ovary (CHO) cells and their effects on the cognate receptors (zebrafish Fshr and Lhr) tested. Interestingly, zfFSH specifically activated zebrafish Fshr expressed together with a cAMP-responsive reporter gene in the CHO cells, whereas zfLH could stimulate both Fshr and Lhr. In conclusion, the present study systematically investigated gonadotropins in the zebrafish in terms of their structure, spatial-temporal expression patterns, and receptor specificity. These results, together with the availability of recombinant zfFSH and zfLH, provide a solid foundation for further studies on the physiological relevance of FSH and LH in the zebrafish, one of the top biological models in vertebrates.

Amino Acid Sequence↗
PubMed1997

Contribution of zebrafish-mouse cell hybrids to the mapping of the zebrafish genome.

The zebrafish, Danio rerio, is becoming an increasingly popular model for the study of vertebrate development. Indeed, the biology of the fish offers great advantages for such studies. The life cycle of the zebrafish is relatively short (2-3 months) and the embryos develop outside the mother, facilitating the visualization of any mutated phenotype. At present, more than 1000 embryonic mutations have been reported. However, until recently, there was no physical or genetic map for this organism. In an effort to generate such a map, we have produced and characterized a panel of zebrafish-mouse cell hybrids. We have used whole-cell fusion to transfer zebrafish chromosomes from two different zebrafish cell lines into mouse recipient cells, thus generating more than 100 hybrids. Using fluorescence in situ hybridization and polymerase chain reaction analysis, we have determined the zebrafish chromosome composition of these hybrids. Here we report that elements from the 25 linkage groups of the zebrafish genome are present in our hybrids. These hybrids could identify the chromosomal location of genes affected in zebrafish mutants.

Animals↗
PubMed2003

Cloning of zebrafish BAD, a BH3-only proapoptotic protein, whose overexpression leads to apoptosis in COS-1 cells and zebrafish embryos.

The BH3-only proapoptotic protein, BAD, was cloned from zebrafish embryos and its properties were characterized. Zebrafish BAD (zBAD) is a protein with 147 amino acids that contains a BH3 domain and a putative 14-3-3 binding site with the sequence of RPRSRS(84)AP, corresponding to S(136) in mouse BAD (mBAD). zBAD shares 34%, 28%, and 29% amino acid sequence identity to the human, mouse, and rat BAD, respectively. RT-PCR analysis revealed that the expression of zBAD gene is found in various parts of zebrafish tissues. The treatment with the z-VAD fmk, a broad-range caspase inhibitor, in COS-1 cells significantly increased the expression of zebrafish BAD fusion proteins (GFP-zBAD and HA-zBAD), indicating that zebrafish BAD fusion proteins may be cleaved by caspase(s). zBAD was shown to induce apoptosis when it was overexpressed in COS-1 cells. In addition, zBAD was also expressed in muscle cells under the muscle-specific promoter from zebrafish alpha-actin gene. Abnormality in the skeletal muscles and the loss of green fluorescence signal in the same region were observed. Taken together, our results indicate that zBAD could induce apoptosis in vitro and in vivo and may have biological implications in apoptosis during zebrafish development.

Actins↗
PubMed1999

Faithful expression of green fluorescent protein (GFP) in transgenic zebrafish embryos under control of zebrafish gene promoters.

Although the zebrafish has become a popular model organism for vertebrate developmental and genetic analyses, its use in transgenic studies still suffers from the scarcity of homologous gene promoters. In the present study, three different zebrafish cDNA clones were isolated and sequenced completely, and their expression patterns were characterized by whole-mount in situ hybridization as well as by Northern blot hybridization. The first clone encodes a type II cytokeratin (CK), which is specifically expressed in skin epithelia in early embryos and prominently expressed in the adult skin tissue. The second clone is muscle specific and encodes a muscle creatine kinase (MCK). The third clone, expressed ubiquitously in all tissues, is derived from an acidic ribosomal phosphoprotein P0 (arp) gene. In order to test the fidelity of zebrafish embryos in transgenic expression, the promoters of the three genes were isolated using a rapid linker-mediated PCR approach and subsequently ligated to a modified green fluorescent protein (gfp) reporter gene. When the three hybrid GFP constructs were introduced into zebrafish embryos by microinjection, the three promoters were activated faithfully in developing zebrafish embryos. The 2.2-kb ck promoter was sufficient to direct GFP expression in skin epithelia, although a weak expression in muscle was also observed in a few embryos. This pattern of transgenic expression is consistent with the expression pattern of the endogenous cytokeratin gene. The 1.5-kb mck promoter/gfp was expressed exclusively in skeletal muscles and not elsewhere. By contrast, the 0.8-kb ubiquitous promoter plus the first intron of the arp gene were capable of expressing GFP in a variety of tissues, including the skin, muscle, lens, neurons, notochord, and circulating blood cells. Our experiments, therefore, further demonstrated that zebrafish embryos can faithfully express exogenously introduced genes under the control of zebrafish promoters.

Animals↗
PubMed2003

Faithful expression of a tagged Fugu WT1 protein from a genomic transgene in zebrafish: efficient splicing of pufferfish genes in zebrafish but not mice.

The teleost fish are widely used as model organisms in vertebrate biology. The compact genome of the pufferfish, Fugu rubripes, has proven a valuable tool in comparative genome analyses, aiding the annotation of mammalian genomes and the identification of conserved regulatory elements, whilst the zebrafish is particularly suited to genetic and developmental studies. We demonstrate that a pufferfish WT1 transgene can be expressed and spliced appropriately in transgenic zebrafish, contrasting with the situation in transgenic mice. By creating both transgenic mice and transgenic zebrafish with the same construct, we show that Fugu RNA is processed correctly in zebrafish but not in mice. Furthermore, we show for the first time that a Fugu genomic construct can produce protein in transgenic zebrafish: a full-length Fugu WT1 transgene with a C-terminal beta-galactosidase fusion is spliced and translated correctly in zebrafish, mimicking the expression of the endogenous WT1 gene. These data demonstrate that the zebrafish:Fugu system is a powerful and convenient tool for dissecting both vertebrate gene regulation and gene function in vivo.

Alternative Splicing↗
PubMed2003

Molecular cloning of zebrafish (Danio rerio) MutS homolog 6(MSH6) and noncoordinate expression of MSH6 gene activity and G-T mismatch binding proteins in zebrafish larvae.

Eukaryotic MutS homolog 6(MSH6) is a DNA mismatch recognition protein associated with mismatch repair of simple base-base mispairs and small insertion-deletion loops. As replication or recombination errors generated during embryonic development of living organisms should be efficiently corrected to maintain the integrity of genetic materials, we attempted to study MSH6 gene expression in developing zebrafish (Danio rerio) and the influence of MSH6 expression on the production of mismatch binding factors. A full-length cDNA encoding zebrafish MSH6 (zMSH6) was first obtained by rapid amplification of cDNA ends (RACE). The deduced amino acid sequence of zMSH6 shares 57% and 56% identity with human and mouse MSH6, respectively. The 190-kDa recombinant zMSH6 containing 1,369 amino acids bound preferentially to a heteroduplex than to a homoduplex DNA. Northern blot and semiquantitative RT-PCR analysis detected apparent levels of zMSH6 mRNA expression in 12 and 36-hr-old zebrafish embryos, while this expression in 84-hr-old larvae was dramatically reduced to 23% of that in 12-hr-old embryos when beta-actin mRNA was constitutively synthesized. Incubation of G-T and G-G heteroduplex probes with 12 to 60-hr-old zebrafish extracts produced predominantly high-shifting binding complexes with very similar band intensity. Although low in zMSH6 mRNA production, the extracts of 84-hr-old larvae interacted significantly stronger than the embryonic extracts with both G-T and G-G mispairs, producing high and low-shifting complexes. Heteroduplex-recognition proteins in 108-hr-old larvae gave a similar pattern of mismatch binding. The intensities of G-T complexes produced by 84 and 108-hr-old zebrafish extracts were 2.5 to 3-fold higher than those of G-G complexes. Our data indicate that the production of efficient MSH6-independent binding factors, particularly G-T-specific recognition proteins, is upregulated in zebrafish at the larval stage when MSH6 gene activity is downregulated.

Amino Acid Sequence↗
PubMed2002

Construction of a zebrafish cDNA microarray: gene expression profiling of the zebrafish during development.

Vertebrate embryogenesis is a complex process controlled by a transcriptional hierarchy that coordinates the action of thousands of genes. To identify and analyze the expression patterns of these genes, we constructed a zebrafish cDNA microarray containing 4512 unique genes identified from zebrafish embryonic heart, adult hearts, and skeletal muscle cDNA libraries. We examined the patterns of gene expression during development in the zebrafish between five time points relative to 12h post-fertilization (hpf). Differentially expressed genes can be grouped into two categories, early genes that are expressed at 5hpf and genes expressed at 48/72/120hpf. Furthermore, we report the utilization of cDNA microarray technology to investigate the adaptive molecular responses of zebrafish to hypoxia during development. Our study provides the first utilization of cDNA microarray in the zebrafish and reveals dynamic changes in levels of gene expression in relation to development and survival of the zebrafish embryos under hypoxic stress.

Animals↗
PubMed2003

Genetic and genomic tools for zebrafish research: the NIH zebrafish initiative.

The National Institutes of Health (NIH) has been a leading advocate of the zebrafish as a model organism for the study of vertebrate development, physiology, and disease. Genomic tools, developed with the support of NIH funding, have made zebrafish even more attractive as a genetic system and have stimulated research using this model. The NIH continues to provide support for new and existing community resources, such as the Zebrafish International Resource Center and the Zebrafish Information Network, research tool development and mutant screens, and a wide array of investigator-initiated studies that are using zebrafish to elucidate many aspects of vertebrate biology.

Animals↗
PubMed2006

Differential expression of photoreceptor-specific genes in the retina of a zebrafish cadherin2 mutant glass onion and zebrafish cadherin4 morphants.

Cadherins are Ca2+ -dependent transmembrane molecules that mediate cell-cell adhesion through homophilic interactions. Cadherin2 (also called N-cadherin) and cadherin4 (also called R-cadherin), members of the classic cadherin subfamily, have been shown to be involved in development of a variety of tissues and organs including the visual system. To gain insight into cadherin2 and cadherin4 function in differentiation of zebrafish photoreceptors, we have analyzed expression patterns of several photoreceptor-specific genes (crx, gnat1, gnat2, irbp, otx5, rod opsin, rx1, and uv opsin) and/or a cone photoreceptor marker (zpr-1) in the retina of a zebrafish cadherin2 mutant, glass onion (glo) and in zebrafish embryos injected with a cadherin4 specific antisense morpholino oligonucleotide (cdh4MO). We find that expression of all these genes, and of zpr-1, is greatly reduced in the retina of both the glo and cadherin4 morphants. Moreover, in these embryos, expression of some genes (e.g. gnat1, gnat2 and irbp) is more affected than others (e.g. rod opsin and uv opsin). In embryos with both cadherins functions blocked (glo embryos injected with the cdh4MO), the eye initially formed, but became severely and progressively disintegrated and expressed little or no crx and otx5 as development proceeded. Our results suggest that cadherin2 and cadherin4 play important roles in the differentiation of zebrafish retinal photoreceptors.

Animals↗
PubMed2005

Zebrafish gonadotropins and their receptors: I. Cloning and characterization of zebrafish follicle-stimulating hormone and luteinizing hormone receptors--evidence for their distinct functions in follicle development.

In the present study, we cloned and characterized zebrafish FSH receptor (Fshr) and LH receptor (Lhr). Both fshr and lhr were abundantly expressed in the zebrafish gonads; however, they could also be detected in the kidney and liver, respectively. When overexpressed in mammalian cell lines together with a cAMP-responsive reporter gene, zebrafish Fshr responded to goldfish pituitary extract but not hCG, whereas Lhr could be activated by both. It was further demonstrated that Fshr was specific to bFSH, while Lhr could be stimulated by both bovine FSH and LH. Low level of fshr expression could be detected in the immature ovary, but the level steadily increased during vitellogenesis of the first cohort of developing follicles. In contrast, the expression of lhr could barely be detected in the immature ovary, but it became detectable at the beginning of vitellogenesis and steadily increased afterward with the peak level reached at the full-grown stage. At the follicle level, the expression of fshr was very weak in the follicles of primary growth stage but significantly increased with the follicles entering vitellogenesis. However, after reaching the maximal level in the midvitellogenic follicles, the level of fshr expression dropped slightly but significantly at the full-grown stage. In comparison, the expression of lhr obviously lagged behind that of fshr. Its expression became detectable only when the follicles started to accumulate yolk granules, but the level rose steadily afterward and reached the peak at the full-grown stage before oocyte maturation. These results suggest differential roles for Fshr and Lhr in zebrafish ovarian follicle development.

Amino Acid Sequence↗
PubMed2005

Identification of zebrafish ARNT1 homologs: 2,3,7,8-tetrachlorodibenzo-p-dioxin toxicity in the developing zebrafish requires ARNT1.

To use the zebrafish (Danio rerio) as a model to study 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) developmental toxicity, it is essential to know which proteins are involved in mediating toxicity. Previous work has identified zfAHR2 as the receptor that binds TCDD mediating downstream responses. Although zfARNT2b can form a functional heterodimer with zfAHR2 in vitro, zfarnt2 null mutants show no protection against endpoints of TCDD developmental toxicity, demonstrating that zfARNT2b cannot be the physiological dimerization partner for zfAHR2 mediating responses to TCDD in zebrafish embryos. The purpose of the current study was to identify an alternate dimerization partner(s) for zfAHR2 that may function to mediate TCDD developmental toxicity. By searching zebrafish genomic sequence and using the polymerase chain reaction-based rapid amplification of cDNA ends technique, three forms of cDNA that seem to be alternate mRNA splice variants of a zebrafish homolog of ARNT1 were detected. Analysis of the zfARNT1 proteins in vitro demonstrates that the two longest forms of zfARNT1, zfARNT1b and zfARNT1c, can form functional heterodimers with zfAHR2. However, the shortest form, zfARNT1a, seems to be nonfunctional with zfAHR2 in vitro. To determine whether a zfARNT1 protein functions with zfAHR2 in vivo, a morpholino targeted against the 5' end of zfARNT1 (zfarnt1-MO) was used. Injection of the zfarnt1-MO before TCDD treatment significantly decreases the induction of zfCYP1A mRNA and protein. In addition, zfarnt1 morphants show complete protection against TCDD-induced pericardial edema and show partial protection against reduced blood flow and craniofacial malformations caused by TCDD, demonstrating the role of zfARNT1 proteins in mediating these responses.

Amino Acid Sequence↗
PubMed2001

Zebrafish tiggy-winkle hedgehog promoter directs notochord and floor plate green fluorescence protein expression in transgenic zebrafish embryos.

Zebrafish tiggy-winkle hedgehog (twhh) is a member of the hedgehog gene family that plays an important role in patterning brain, neural tube, somites, and eyes. To better understand the regulation of its tissue-specific expression, the activity of the twhh promoter was determined in zebrafish embryos by transient and transgenic expression analysis. Transient expression studies revealed that the 5.2-kb twhh promoter drove green fluorescence protein (GFP) expression in the notochord, floor plate, and branchial arches. Deletion analysis showed that distinct regions of the twhh promoter regulated the respective notochord or floor plate specific expression. To confirm the tissue specificity of the twhh promoter, transgenic zebrafish containing the twhh-GFP transgene were generated. GFP expression was analyzed in the F1, F2, and F3 generations of the transgenic embryos. The results confirmed the tissue-specific expression of the transgene in the notochord, floor plate, and branchial arches. In addition, GFP expression was also found in the pectoral fin buds, retina, and epithelial lining cells of the Kupffer's vesicle in the transgenic fish embryos. The expression pattern of the twhh-GFP transgene mimicked the expression of the endogenous twhh mRNAs in the floor plate, fin buds, branchial arches, retina, and epithelial lining cells of the Kupffer's vesicle. The expression in the notochord, however, did not mimic the pattern of the endogenous twhh expression. To determine whether no tail (ntl) or floating head (flh) mutants that have developmental defect in the notochord or the Kupffer's vesicle may affect the GFP expression in these regions, GFP expression was analyzed in ntl or flh transgenic embryos. No GFP expression could be detected in the midline region of the ntl transgenic embryos. However, in flh transgenic embryos, although GFP expression was affected in the midline region, its expression in the Kupffer's vesicle appeared normal. Together, these data indicated that the 5.2-kb twhh promoter contains regulatory elements for tissue-specific expression of twhh in the floor plate, pectoral fin bud, branchial arches, retina, and Kupffer's vesicle.

Animals↗
PubMed2005

Determination of the expression pattern of the dual promoter of zebrafish fushi tarazu factor-1a following microinjections into zebrafish one cell stage embryos.

The zebrafish fushi tarazu factor-1a (ff1a) is a transcription factor belonging to the NR5A subgroup of nuclear receptors. The NR5A receptors bind DNA as monomers and are considered to be orphans due to their ability to promote transcription of downstream genes without ligands. In zebrafish, four ff1 homologues (Ff1a, Ff1b, Ff1c, and Ff1d) have been identified so far. The gene coding for Ff1a is driven by two separate promoters, and give rise to four splice variants. Ff1a is expressed in the somites and pronephric ducts during somitogenesis and in the brain, liver, and mandibular arch during later embryonic stages. In adults the gene is highly expressed in gonads, liver, and intestine, but can be detected in most tissues. The broad variety of embryonic expression domains indicates several important developmental features. One of the mammalian fushi tarazu factor-1 genes, steroidogenic factor-1 (SF-1), is essential for the development of gonads and adrenals. SF-1 is together with Sox9, WT1, and GATA4 a positive transcriptional regulator of human anti-mullerian hormone (AMH) and thereby linked to the male sex-determining pathway. The zebrafish ff1a dual promoter contains several GATA binding sites and E-boxes, a site for DR4, XFD2, MyoD, Snail, HNF3, S8, and an HMG-box recognition site for Sox9. In a first attempt to dissect the ff1a promoter in vivo we have produced first generation transgenes in order to determine the correlation between the expression of the endogenous ff1a gene and the microinjected ff1a dual promoter coupled to the pEGFP reporter vector. Our results show that the microinjected constructs are expressed in the correct tissues.

Animals↗