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Functional study of human genes in zebrafish
The zebrafish (Danio rerio) is a vertebrate model system suitable for fishing novel genes in post-genome era. Two neural mutants isolated from the ENU-based chemical mutagenesis, were characterized and the point mutations were identified by the positional cloning method. The first mutant is headless: Wnt antagonists secreted by the organizer have been identified as head inducers. The severe head defects in headless are due to a mutation in Tcf-3, a component of the Wnt pathway. The second is mind bomb: Reduced lateral inhibition in mutant permits too many neural progenitors to differentiate as neurons. Mind bomb encodes a novel gene in the Notch signaling pathway. In addition to chemical mutagenesis, we developed a novel transgene reporter system and tried to apply its use for the insertional mutagenesis. The first mutant "ondal" from this new screening approach, was characterized and mapped to a gene involved in axonal myelination and human schizophrenia. The other mutants, samdori and hayan-4 are being mapped and characterized. As a separate approach, we are challenging a genome-wide screen against the human UniGene collections. Currently, more than 15,000 human full-length cDNAs are available from the Korean UniGene Information (KUGI). After in silico screening of functionally unknown genes through database search, we performed overexpression experiments by microinjecting the single human genes into the zebrafish embryos. So far, we performed microinjections more than 2,700 human full-length cDNAs and identified 50 functionally active genes. Zebrafish embryos display variable phenotypes from developmental defects to neuronal cell death in injected embryos. Many of these genes were involved in not only developmental process but also diseases, such as cancer. After these phenotype-based screening, homologous zebrafish genes were cloned and further functional studies were performed by expression analysis, overexpression and antisense knockdown approaches. From these functionally active genes, we found that the human clone #462, now named it as "Ottogi", plays a role in vertebrate head formation during early development. Overexpression of Ottogi caused a big head phenotype, indicating its possible role in the Wnt signaling pathway. Ottogi physically interacted with the Wnt receptor Frizzled 8 within the endoplasmic reticulum. These findings suggest that Ottogi is a novel component negatively regulating the Wnt signal pathway. In addition, the human clone #498, now named as ZNF312b, plays a role in tumor progression and metastasis in gastric cancer via transcriptional activation of the K-ras oncogene. ZNF312b seems to be specifically overexpressed in gastric cancer tissues and cell lines. The overexpression of ZNF312b induces cancer-like phenotypes, including accelerated proliferation and increased tumor masses in nude mice, which are reversed by its knockdown in gastric cancer cell lines, implying involvement in gastric tumor progression.
