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Zebrafish: a new model on the pharmaceutical catwalk.

Zebrafish is recognized as one of the most important vertebrate model organisms; however, its value in pharmacological studies has not been extensively explored and exploited. In this review, I summarize significant findings about the effects of drugs and medicines on important physiological processes in zebrafish. Our experiments have shown that cardiovascular, anti-angiogenic and anti-cancer drugs elicit comparable responses in zebrafish embryos to those in mammalian systems. Similar observations have been reported by other laboratories, exposing zebrafish to a variety of pharmaceutical active compounds affecting a range of different processes. All the data summarized indicate that zebrafish represents a very valuable organism for different kinds of pharmacological studies, such as screenings of chemical libraries, lead validation and optimization, mode-of-action studies, analysis of gene function, predictive toxicology and teratogenicity, pharmacogenomics and toxicogenomics. Zebrafish pharmacological assays have specific advantages compared to in vitro cell culture studies and in vivo experiments using mice, complementing these assays to give valuable guides for future tests of new drugs for human therapy.

Animals↗

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

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

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine↗

A critical period for functional vestibular development in zebrafish.

We have determined a critical period for vestibular development in zebrafish by using a bioreactor designed by NASA to simulate microgravity for cells in culture. A critical period is defined as the briefest period of time during development when stimulus deprivation results in long lasting or permanent sensory deficits. Zebrafish eggs were collected within 3 hours of being laid and fertilized. In experiment 1, eggs were placed in the bioreactor at 3, 24, 30, 36, 48, or 72 hours postfertilization (hPF) and maintained in the bioreactor until 96 hPF. In experiment 2, eggs were placed in the bioreactor immediately after they were collected and maintained in the bioreactor until 24, 36, 48, 60, 66, 72, or 96 hPF. Beginning at 96 hPF, all larvae had their vestibulo-ocular reflexes (VOR) evaluated once each day for 5 days. Only larvae that hatched from eggs that were placed in the bioreactor before 30 hPF in experiment 1 or removed from the bioreactor later than 66 hPF in experiment 2 had VOR deficits that persisted for at least 5 days. These data suggest a critical period for vestibular development in the zebrafish that begins before 30 hPF and ends after 66 hPF. To confirm this, zebrafish eggs were placed in the bioreactor at 24 hPF and removed at 72 hPF. VORs were evaluated in these larvae once each day for 5 days beginning at 96 hPF. These larvae had VOR deficits that persisted for at least 5 days. In addition, larvae that had been maintained in the bioreactor from 24 to 66 hPF or from 30 to 72 hPF, had only temporary VOR deficits. In a final experiment, zebrafish eggs were placed in the bioreactor at 3 hPF and removed at 96 hPF but the bioreactor was turned off from 24 hPF to 72 hPF. These larvae had normal VORs when they were removed from the bioreactor at 96 hPF. Taken as a whole, these data support the idea that there is a critical period for functional maturation of the zebrafish vestibular system. The developmental period identified includes the timeframe during which the vestibular primary afferent neurons are born, innervate their central and peripheral targets, and remodel their central projections.

Animals↗

The fate of human malignant melanoma cells transplanted into zebrafish embryos: assessment of migration and cell division in the absence of tumor formation.

Certain aggressive melanoma cell lines exhibit a dedifferentiated phenotype, expressing genes that are characteristic of various cell types including endothelial, neural, and stem cells. Moreover, we have shown that aggressive melanoma cells can participate in neovascularization in vivo and vasculogenic mimicry in vitro, demonstrating that these cells respond to microenvironmental cues and manifest developmental plasticity. To explore this plasticity further, we transplanted human metastatic melanoma cells into zebrafish blastula-stage embryos and monitored their behavior post-transplantation. The data show that human metastatic melanoma cells placed in the zebrafish embryo survive, exhibit motility, and divide. The melanoma cells do not form tumors nor integrate into host organs, but instead become scattered throughout the embryo in interstitial spaces, reflecting the dedifferentiated state of the cancer cells. In contrast to the fate of melanoma cells, human melanocytes transplanted into zebrafish embryos most frequently become distributed to their normal microenvironment of the skin, revealing that the zebrafish embryo contains possible homing cues that can be interpreted by normal human cells. Finally, we show that within the zebrafish embryo, metastatic melanoma cells retain their dedifferentiated phenotype. These results demonstrate the utility of the zebrafish embryonic model for the study of tumor cell plasticity and suggest that this experimental paradigm can be a powerful one in which to investigate tumor-microenvironment interactions.

Animals↗

Piloting the zebrafish genome browser.

This correspondence is a primer for the zebrafish research community on zebrafish tracks available in the UCSC Genome Browser at http://genome.ucsc.edu based on Sanger's Zv4 assembly. A primary capability of this facility is comparative informatics between humans (as well as many other model organisms) and zebrafish. The zebrafish genome sequencing project has played important roles in mutant mapping and cloning, and comparative genomic research projects. This easy-to-use genome browser aims to display and download useful genome sequence information for zebrafish mutant mapping and cloning projects. Its user-friendly interface expedites annotation of the zebrafish genome sequence.

Animals↗

Comparison of the expression patterns of newly identified zebrafish retinoic acid and retinoid X receptors.

Retinoic acid (RA) signaling is important for multiple aspects of embryonic development and tissue homeostasis. Heterodimers of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) transduce RA signaling. It is not yet clear how the diversity of receptor combinations relates to the diversity of functions for RA. The expression patterns of three zebrafish RARs and four RXRs were reported recently. Here, we identify an additional RAR, a zebrafish RARgamma paralog, and two additional RXRs, duplicates of the previously identified RXRalpha and RXRgamma. Thus, the zebrafish genome contains duplicates of each RAR and RXR gene. All zebrafish RAR and RXR paralogs have overlapping and distinct areas of expression, as might be expected for duplicate genes in the process of diverging in function. By representing what is potentially the complete set of zebrafish RARs and RXRs, this study provides a valuable reference for future functional studies of the individual zebrafish RARs and RXRs.

Amino Acid Sequence↗

Differential regulation of spontaneous and heat-induced HSP 70 expression in developing zebrafish (Danio rerio).

A spontaneous high expression of heat shock protein 70 (HSP 70) was found to arise in zebrafish (Danio rerio) at the larval stage (84 hr after fertilization). The level of HSP 70 in 84-hr-old larvae was estimated to be six- to eightfold that of 12-hr-old embryos. As heat-induced HSP 70 synthesis in many eukaryotic organisms is known to be mediated by a transcriptional-dependent pathway activated by heat shock factor 1 (HSF-1), we then examined if the spontaneous and heat-induced HSP 70 synthesis in zebrafish were controlled by the same mechanism. Although the transient increase of a 62-kDa HSF-1-like polypeptide in 72- to 96-hr-old larvae seemed to correlate with the onset of the spontaneous HSP 70 production, an anti-HSF-1 antibody cocktail supershifted the heat shock element (HSE) binding complex induced by stressed but not by unstressed zebrafish extracts. Northern blot and quantitative RT-PCR analysis demonstrated the predominant presence of the cognate form of hsp 70 mRNA (hsc 70 mRNA) in developing zebrafish. The extent of heat-induced HSP 70 production in 84-hr-old larvae matched well with a dramatic increase in hsp 70 mRNA accumulation, while no apparent increase in total hsp 70 mRNA could be detected in 72- to 84-hr-old unstressed larvae by northern blot analysis. The stable expression of hsc 70 mRNA specific to beta-actin mRNA in normal zebrafish was confirmed by RT-PCR analysis. Hence, the spontaneous high expression of HSP 70 in zebrafish is believed to be controlled by a mechanism different from the HSF-1-dependent transcriptional activation of hsp 70 under heat stress. J. Exp. Zool. 293:349-359, 2002.

Animals↗

The molecular cloning and characterization of potential chick DM-GRASP homologs in zebrafish and mouse.

A full-length zebrafish cDNA clone and a partial mouse cDNA clone similar to chick DM-GRASP were isolated and analyzed. The nucleotide sequence of the full-length zebrafish clone shares 54% identity, and predicts 39% amino acid identity, with chick DM-GRASP. The partial mouse clone shares 76% nucleotide identity, and predicts 76% amino acid identity, with chick DM-GRASP. The predicted proteins encoded by both of these clones exhibit conserved structural domains that are characteristic of the chick protein. These features may identify them as a distinct subfamily within the immunoglobulin superfamily of cell adhesion molecules. Expression of the zebrafish DM-GRASP protein is similar to chick DM-GRASP and is principally restricted to a small subset of developing sensory and motor neurons during axonogenesis. Zebrafish DM-GRASP expression was temporally regulated and limited to specific axon domains. This regional expression correlated with fasciculated axon domains. These results suggest that the zebrafish and mouse cDNA clones represent the respective fish and mammalian homologs of chick DM-GRASP. The highly selective expression of zebrafish DM-GRASP suggests that it is involved in the selective fasciculation and guidance of axons along their normal pathways.

Activated-Leukocyte Cell Adhesion Molecule↗

Selective labeling of zebrafish thrombocytes: quantitation of thrombocyte function and detection during development.

Zebrafish thrombocytes, the nucleated equivalents of mammalian platelets, have been characterized morphologically, but knowledge about their developmental synthesis and biochemistry is limited. Given the increasing use of zebrafish as a genetic model to study hemostasis, it is important to isolate and study the function of zebrafish thrombocytes. Therefore, the objective of this study was to isolate thrombocytes, study their function in vitro, and identify the developmental stage at which they enter circulation. To achieve these goals, we developed a method for the selective labeling of thrombocytes and assayed these cells for activation by known mammalian platelet agonists. In both in vitro incubations of whole blood and blood labeled in vivo with the lipophilic dye DiI-C(18), we found labeling in only a single population of cells. These cells were identified as zebrafish thrombocytes by Wright-Giemsa staining. Using selective DiI-C(18) labeling, we showed the formation of thrombocyte aggregates, filopodia, and lipid rafts in response to platelet agonists. Additionally, we showed that aggregates are labeled by binding FITC-conjugated annexin V to exposed phosphatidylserine on the thrombocyte membrane. Using these fluorescent-labeling methods, we developed the first microquantitative assay for thrombocyte aggregation. With this assay, we provided evidence for the presence of an ADP receptor, P2Y(1), in the zebrafish thrombocytes. To study the developmental stage at which thrombocytes appear, we microinjected DiI-C(18) into the circulation of zebrafish embryos and identified the presence of DiI-C(18)-labeled thrombocytes at the 36 h postfertilization stage. These findings will prove helpful in dissecting the functions of thrombocytes in hemostasis and provide further insight into the role of platelets in thrombosis.

Animals↗

Dissecting hematopoiesis and disease using the zebrafish.

The study of blood has often defined paradigms that are relevant to the biology of other vertebrate organ systems. As examples, stem cell physiology and the structure of the membrane cytoskeleton were first described in hematopoietic cells. Much of the reason for these successes resides in the ease with which blood cells can be isolated and manipulated in vitro. The cell biology of hematopoiesis can also be illuminated by the study of human disease states such as anemia, immunodeficiency, and leukemia. The sequential development of the blood system in vertebrates is characterized by ventral mesoderm induction, hematopoietic stem cell specification, and subsequent cell lineage differentiation. Some of the key regulatory steps in this process have been uncovered by studies in mouse, chicken, and Xenopus. More recently, the genetics of the zebrafish (Danio rerio) have been employed to define novel points of regulation of the hematopoietic program. In this review, we describe the advantages of the zebrafish system for the study of blood cell development and the initial success of the system in this pursuit. The striking similarity of zebrafish mutant phenotypes and human diseases emphasizes the utility of this model system for elucidating pathophysiologic mechanisms. New screens for lineage-specific mutations are beginning, and the availability of transgenics promises a better understanding of lineage-specific gene expression. The infrastructure of the zebrafish system is growing with an NIH-directed genome initiative, providing a detailed map of the zebrafish genome and an increasing number of candidate genes for the mutations. The zebrafish is poised to contribute greatly to our understanding of normal and disease-related hematopoiesis.

Animals↗

Early life stage toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin in zebrafish (Danio rerio).

Toxicity and histopathology of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in zebrafish (Danio rerio) early life stages was characterized from 12 to 240 hr postfertilization (hpf) following water-borne exposure of newly fertilized eggs. TCDD did not increase egg mortality (0-48 hpf), nor did it affect time to hatching (48-96 hpf). Egg doses of 1.5 ng [3H]TCDD/g or greater elicited toxic responses in zebrafish larvae. Pericardial edema and craniofacial malformations were first observed at 72 hpf, followed by the onset of yolk sac edema (96 hpf) and mortality (132 hpf). At 240 hpf the ED50s for pericardial edema, yolk sac edema, and craniofacial malformations were 2.2, 2.1, and 1.9 ng [3H]TCDD/g egg, respectively. The LD50, determined at 240 hpf, was 2.5 ng [3H]TCDD/g egg. Severe hemodynamic changes, observed as slowed blood flow in vascular beds of the trunk, head, and gills and slowed heart rate, occurred in TCDD-treated zebrafish prior to or coincident with the onset of gross signs of toxicity. Histological examination of TCDD-treated zebrafish revealed a variety of epithelial tissue lesions including arrested gill development and ballooning degeneration and/or necrosis of the renal tubules, hepatocytes, pancreas, and all major brain regions. Mesenchymal tissue lesions included subcutaneous edema in the head, trunk, and yolk sac, edema of the pericardium and skeletal muscle, and underdevelopment of the swim bladder. This demonstration of zebrafish responsiveness to TCDD early life stage toxicity coupled with the considerable information on developmental biology and genetics of zebrafish provides a foundation for future investigations into the mechanism of TCDD developmental toxicity.

Abnormalities, Drug-Induced↗

Zebrafish Mhc class II alpha chain-encoding genes: polymorphism, expression, and function.

Its small size and short generation time renders the zebrafish (Brachydanio rerio) an ideal vertebrate for immunological research involving large populations. A prerequisite for this is the identification of the molecules critical for an immune response in this species. In earlier studies, we cloned the zebrafish genes coding for the beta chains of the class I and class II major histocompatibility complex (Mhc) molecules. Here, we describe the cloning of the zebrafish alpha chain-encoding class II gene, which represents the first identification of a class II A gene in teleost fishes. The gene, which is less than 3 kilobases (kb) distant from one of the beta chain-encoding genes, is approximately 1.2 kb long and consists of four exons interrupted by very short (< 200 base pairs) introns. Its organization is similar to that of the mammalian class II A genes, but its sequence differs greatly from the sequence of the latter (36% sequence similarity). Among the most conserved parts is the promoter region, which contains X, Y, and TATA boxes with high sequence similarity to the corresponding mammalian boxes. The observed striking conservation of the promoter region suggests that the regulatory system of the class II genes was established more than 400 million years ago and has, principally, remained the same ever since. Like the DMA, but unlike all other mammalian class II A genes, the zebrafish gene codes for two cysteine residues which might potentially be involved in the formation of a disulfide bond in the alpha 1 domain. The primary transcript of the gene is 1196 nucleotides long and contains 708 nucleotides of coding sequence. The gene is expressed in tissues with a high content of lymphoid/myeloid cells (spleen, pronephros, hepatopancreas, and intestine). The analyzed genomic and cDNA sequences are probably derived from different loci (their overall sequence similarity in the coding region is 73% and their 3' untranslated regions are highly divergent from each other). The genes are apparently functional. Comparison of genes from different zebrafish populations reveals high exon 2 variability concentrated in positions coding for the putative peptide-binding region. Phylogenetic analysis suggests that the zebrafish class II A genes stem from a different ancestor than the mammalian class II A genes and the recently cloned shark class II A gene.

Amino Acid Sequence↗

Zebrafish--an emerging genetic model for the study of cytokines and hematopoiesis in the era of functional genomics.

Now that whole genomes are sequenced, the identification of gene function rather than gene discovery is a major challenge. Saturation mutagenesis and screening for mutant phenotypes are methods that allow sampling of the genome for lesions in genes critical for particular physiological processes. This approach promises to provide new insights into gene function, even for molecularly well-characterized processes such as hematopoiesis and cytokine signaling. Animal models for such genetic approaches have traditionally included Drosophila and the mouse. Recently, the zebrafish (Danio rerio) has emerged as a flexible and informative vertebrate for genetic studies. Zebrafish hematopoiesis has a morphological and molecular complexity closer to that of mammals than does Drosophila, providing scope for recognizing mutant zebrafish phenotypes representing finely tuned lesions in these processes. Compared to mice, zebrafish represent an economical, flexible, and genetically tractable animal model for mutagenesis studies. The structure of the teleost genome creates several phylogenetic issues in assessing zebrafish and piscine orthologues and paralogues of known mammalian genes, here exemplified by a cytokine ligand (interleukin-1beta), kinase receptors (c-kit and c-fins), and a family of intracellular signaling molecules (JAK kinases). Several anemic zebrafish mutants are now genetically characterized, and others present hematopoietic phenotypes that promise novel insights into the regulation of hematopoiesis.

Animals↗

The antigen receptor (NCCRP-1) on catfish and zebrafish nonspecific cytotoxic cells belongs to a new gene family characterized by an F-box-associated domain.

The catfish nonspecific cytotoxic cell receptor protein (NCCRP-1) provides an important function in target cell recognition and activation of cytotoxicity. This report identifies and characterizes a zebrafish orthologue of the catfish NCCRP-1. The zebrafish NCCRP-1 cDNA contains an open reading frame that encodes a predicted protein of 237 amino acids with a MW of 27 kDa and a pI of 5.5. Sequence similarities comparisons show that the NCCRP-1 receptors from these two phylogenetically distant species share a high degree of identity. These results suggested that NCCRP-1 performs a crucial function in innate immunity in teleosts. Further, a zebrafish 17-mer peptide corresponding to the catfish NCCRP-1 antigen-binding domain inhibited (catfish) cytotoxicity toward conventional tumor target cells (HL-60). These data appeared to indicate that the zebrafish NCCRP-1 protein may function as an antigen recognition molecule and, as such, may participate in innate immunity in teleosts. A homology search of the zebrafish NCCRP-1 protein revealed that it shares a significant level of identity with another group of proteins belonging to an F-box subfamily. These proteins share an F-box domain in the N terminus (not present in NCCRP-1) and an extremely conserved C-terminal region that has been termed the F-box-associated domain (FBA). The FBA is currently of unknown function. A new gene family is proposed in this work, based on similarities in the FBA sequences with the catfish and zebrafish NCCRP-1 peptides. This new gene family includes several F-box domain-containing proteins and a predicted C. elegans protein.

Amino Acid Motifs↗

Isolation, characterization, expression and functional analysis of the zebrafish ortholog of MEN1.

Mutations in the MEN1 gene lead to an autosomal dominant disorder, multiple endocrine neoplasia type 1 (MEN1), which is characterized by tumors of the parathyroid, entero-pancreatic neuroendocrine, and pituitary tissues. The protein encoded by MEN1, 610-amino acid menin, resides primarily in the nucleus and binds to the transcription factor JunD, resulting in the repression of JunD-induced transcription. We report here a detailed characterization of the zebrafish men1 gene and its full-length (2551 nt) transcript, encoding a 617-amino acid protein with 67% identity and 80% similarity to human menin. Of the 81 missense mutations and in-frame deletions reported in MEN1 patients, 72 occur in residues that are identical in zebrafish, suggesting the importance of the conserved regions. The zebrafish men1 gene maps 61 cM from the top of linkage group 7 (LG7), a region that appears to show conserved synteny to the MEN1 loci at human 11q13. A 2.7-kb men1 message is detected at all stages of zebrafish development analyzed, from one-cell embryos to adult fish. Whole-mount in situ hybridization showed ubiquitous distribution of men1 message in zebrafish embryos at cleavage, blastula, gastrula, and early segmentation stages, with relatively abundant expression in blood cell progenitors (24 h post fertilization) and mesenchymal tissues (48 h post fertilization) at later stages. Zebrafish menin binds both human and mouse JunD, and represses JunD-induced transcription, indicating that the JunD-binding ability of menin is evolutionarily conserved.

Amino Acid Sequence↗

Expression of three zebrafish orthologs of human FMR1-related genes and their phylogenetic relationships.

The human fragile X mental retardation syndrome is caused by expansions of a CGG repeat in the FMR1 gene. FXR1 and FXR2 are autosomal paralogs of FMR1. The products of the three genes, FMRP, FXR1P, and FXR2P, respectively, belong to a family of RNA-binding proteins. While the FMR1-related gene family is well described in human, mouse and Drosophila, little is known about zebrafish (Danio rerio) orthologs of these genes. Here we collate the known FMR1-related gene sequences from zebrafish, examine their regions of structural conservation, and define their orthologies with the human genes. We demonstrate that zebrafish possess only three FMR1-related genes, fmr1, fxr1 and fxr2, and these are orthologous to the human FMR1, FXR1 and FXR2 genes respectively. We examine the spatiotemporal pattern of transcription of the zebrafish genes from 0 hours post fertilisation (hpf) until 24 hpf. Expression of fmr1, fxr1 and fxr2 is widespread throughout this time. However, relative to surrounding tissues, expression of fxr2 is raised in adaxial and somitic cells by 12 hpf while fxr1 expression is high in the anterior of the embryo, and is raised in adaxial cells by 12 hpf. Distinct patterns (and levels) of expression are seen for the different genes later in development. At 24 hpf, fxr1 and fxr2 transcripts show complex distribution patterns in somites. The expression of the FMR1-related gene family in zebrafish tissues is broadly consistent with expression in mouse and human, supporting the idea that zebrafish should be an excellent model organism in which to study the functions of the vertebrate FMR1-related gene family.

Amino Acid Sequence↗

Characterisation of Fmrp in zebrafish: evolutionary dynamics of the fmr1 gene.

Fragile X syndrome is the most common inherited form of mental retardation. It is caused by the lack of the Fragile X Mental Retardation Protein (FMRP), which is encoded by the FMR1 gene. Although Fmr1 knockout mice display some characteristics also found in fragile X patients, it is a complex animal model to study brain abnormalities, especially during early embryonic development. Interestingly, the ortholog of the FMR1 gene has been identified not only in mouse, but also in zebrafish (Danio rerio). In this study, an amino acid sequence comparison of FMRP orthologs was performed to determine the similar regions of FMRP between several species, including human, mouse, frog, fruitfly and zebrafish. Further characterisation of Fmrp has been performed in both adults and embryos of zebrafish using immunohistochemistry and western blotting with specific antibodies raised against zebrafish Fmrp. We have demonstrated a strong Fmrp expression in neurons of the brain and only a very weak expression in the testis. In brain tissue, a different distribution of the isoforms of Fmrp, compared to human and mouse brain tissue, was shown using western blot analysis. Due to the high similarity between zebrafish Fmrp and human FMRP and their similar expression pattern, the zebrafish has great potential as a complementary animal model to study the pathogenesis of the fragile X syndrome, especially during embryonic development.

Amino Acid Sequence↗

Different spatio-temporal expressions of three otx homeoprotein transcripts during zebrafish embryogenesis.

Three zebrafish otx homeoproteins containing a homeodomain homologous to that of the Drosophila orthodenticle head gap gene product have been identified by cloning and sequencing of cDNAs. The zebrafish otx2 homeoprotein shares high amino-acid sequence identity with the mouse Otx2 homeoprotein, whereas the zebrafish otx1 and otx3 homeoproteins exhibit moderate homology with the mouse Otx1 and Otx2 homeoproteins. Three otx homeoprotein mRNAs show different spatio-temporal expression patterns during zebrafish embryogenesis as revealed by Northern blot and whole mount in situ hybridization analyses. Large amounts of the otx1 homeoprotein mRNA are found in fertilized uncleaving eggs. The otx3 homeoprotein mRNA appears in the embryonic shield, the site of the organizer. In the developing brain, three zebrafish otx mRNAs are distributed in the diencephalon and the midbrain, but their fine expression patterns are different. These results suggest that three zebrafish otx homeoproteins, alone or in combination, may play roles in very early embryogenesis, gastrulation, and the development and subdivision of the diencephalon and the midbrain.

Amino Acid Sequence↗