BackgroundUnderstanding the functional role(s) of the more than 20,000 proteins of the vertebrate genome is a major next step in the post-genome era. The approximately 4,000 co-translationally translocated (CTT) proteins – representing the vertebrate secretome – are important for such vertebrate-critical processes as organogenesis. However, the role(s) for most of these genes is currently unknown.ResultsWe identified 585 putative full-length zebrafish CTT proteins using cross-species genomic and EST-based comparative sequence analyses. We further investigated 150 of these genes (Figure 1) for unique function using morpholino-based analysis in zebrafish embryos. 12% of the CTT protein-deficient embryos resulted in specific developmental defects, a notably higher rate of gene function annotation than the 2%–3% estimate from random gene mutagenesis studies.Conclusion(s)This initial collection includes novel genes required for the development of vascular, hematopoietic, pigmentation, and craniofacial tissues, as well as lipid metabolism, and organogenesis. This study provides a framework utilizing zebrafish for the systematic assignment of biological function in a vertebrate genome.
SHBG (sex hormone binding globulin) transports androgens and estrogens in the blood of vertebrates including fish. Orthologs of SHBG in fish are poorly defined, and we have now obtained a zebrafish SHBG cDNA and characterized the zebrafish SHBG gene and protein through molecular biological, biochemical, and informatics approaches. Amino-terminal analysis of zebrafish SHBG indicated that its deduced precursor sequence includes a 25-residue secretion polypeptide and exhibits 22-27% homology with mammalian SHBG sequences and 41% with a deduced fugufish SHBG sequence. The 356-residue mature zebrafish SHBG (39,243 Da) sequence comprises a tandem repeat of laminin G-like domains typical of SHBG sequences; contains three N-glycosylation sites; and exists as a 105,000 +/- 8700 Da homodimer. Zebrafish SHBG exhibits a high affinity and specificity for sex steroids. An RT-PCR indicated that SHBG mRNA first appears in zebrafish larva, and SHBG mRNA was localized within the liver and gut at this stage of development by whole-mount in situ hybridization. In adult fish, SHBG mRNA was found in liver, testis, and gut. In the liver, immunoreactive SHBG was present in hepatocytes and concentrated in intrahepatic bile duct cells, whereas in the testis it was confined to cells surrounding the seminiferous tubule cysts. In the intestine, immunoreactive SHBG was present in the stroma and epithelial cells of the villous projections and the surrounding muscle. The production and presence of SHBG in the gut of developing and adult zebrafish suggests a novel role for this protein in regulating sex steroid action at this site.
Background: The zebrafish is a powerful model vertebrate amenable to high throughput in vivo genetic analyses. Examples include reverse genetic screens using morpholino knockdown, expression-based screening using enhancer trapping and forward genetic screening using transposon insertional mutagenesis. We have created a database to facilitate web-based distribution of data from such genetic studies.
Cellular processes rely on dynamic events occurring between the cortical cytoskeleton and plasma membrane. Members of the Band 4.1 superfamily, which are best known for their ability to tether the cytoskeleton to the plasma membrane, play prominent structural and regulatory roles that influence cell-cell and cell-substrate interactions, endo- and exocytosis, cell polarity, migration, proliferation, and differentiation. We have identified a new member of the zebrafish Band 4.1 superfamily, which is the homolog of human myosin regulatory light chain interacting protein (MIR), and have examined its role in embryonic development. Zebrafish Mir contains the conserved amino-terminal plasma membrane-binding FERM (Band 4.1/ezrin/radixin/moesin) domain as well as other putative protein-protein interacting domains, including a RING finger. Overall, zebrafish Mir is 71% identical to human MIR located at chromosome 6p23-p22.3, and maps on linkage group 19 to a region of synteny with human chromosome 6. In situ hybridization and RT-PCR revealed that mir is expressed maternally and ubiquitously throughout development. Blocking Mir translation using a mir-specific, morpholino-based, knock-down strategy or expressing Mir constructs lacking the RING finger domain disrupts gastrulation and leads to subsequent trunk and tail defects. In severe cases, morphants exogastrulate. The synergistic effect seen when two mir-specific morpholinos are used in conjunction reflects the specific knock-down of mir. In addition, morphant phenotypes induced by mir-specific morpholinos are rescued by overexpression of the full-length Mir. In situ hybridization analysis with mesodermal- and neural-specific markers shows that morphants exhibit a delay in cell movements associated with gastrulation, epiboly, convergence, and extension. A yeast two-hybrid analysis was performed to identify binding partners that may participate with Mir during gastrulation, and Annexin V, a calcium channel protein, was isolated. At early developmental stages, annexin V transcripts colocalize with mir, but after gastrulation, annexin V mRNA becomes localized to the distal tail region and an area in the olfactory placode. At the protein level, Mir colocalizes with Annexin V when expressed in COS cells. Together, these results indicate that Mir is essential for embryonic development and that its role in early embryonic development likely involves calcium-dependent mechanisms essential during the extensive cell movements associated with gastrulation.
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