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.

HSP70 has widely been induced in in vivo hyperthermia conditions in various organisms to study gene regulation and recently neuroprotectve roles of the induced gene expression under varying conditions. We investigated different responses among various tissues in zebrafish under heat shock to evaluate whether spatial and temporal expression pattern of zebrafish (z) hsp70 in transcriptional and translational level under heat shock stress in different brain regions. Heat shock groups were given for 1 h at after recovery by transferring the treated animals back to for 1, 2 and 24 h for recovery, respectively. Control (CTRL) group was kept at . At the end of treatments, five animals were collected and used for isolation of total RNAs and peptides from the corresponding tissues. Expression of zhsp70 mRNA showed different patterns in recovery periods in the tissues including the brain, eye, intestines, muscles, heart and testis by RT-PCR. Unlike the RT-PCR analysis, Northern blot analysis demonstrated nearly 30-fold increase in zhsp70 at 1 h heat shock, suggesting that RT-PCR may not be appropriate in unmasking regulation of the time-dependent zhsp70 expression. In the experiment involving different brain regions, the cerebellum showed gradual activation at 1 h to R1h and decreases in R2h and R24h, while the medulla oblongata and optic tectum showed gradual increase at R1h and decrease at R24h, indicating that different brain tissues respond specifically to heat shock in inducing zhsp70 and recovering from the heat shock status. Western blot analysis also demonstrated that the intracellular levels of zHSP70 in three different brain regions including the cerebellum, medulla oblongata and optic tectum are differently induced and recovered to normal state. These results clearly demonstrate that different regions of the body and the brain tissues are responding differently to heat shock in the aspects of its level of expression and speed of recovery.

제브라피쉬 배아 유래세포의 최적 성장조건을 확립하기 위해 3종류의 배지 DMEM, K-NAC, D-NAC 그룹에서의 세포 성장률을 비교하였다. 실험 결과, 접종밀도에 따른 성장률의 경우, DMEM에 비해 K-NAC 그룹에서 초기 접종 효율이 높게 나타났으며, 후기 성장률은 DMEM 그룹에서 높게 나타났다. K-NAC, DMEM 그룹 모두 FBS 농도에 의한 성장차는 보이지 않았으며, 0.1% embryo extract를 첨가한 배지에서 효과는 낮게 나타

We investigated the relationship between the TiO2 photocatalytic decomposition of bisphenol A in water and biological toxicity to zebrafish (Danio rerio) during 1～28 weeks post development stage. The bisphenol A in water was completely degraded by the TiO2 photocatalysis in 50 hours. After the photocatalysis, no toxic effects on the morphogenesis of the zebrafish were observed during the development, growth, and maturate stages. Catalase activity of control group was not different from 1～5 week post fertilized group. However, toxic effect on the catalase activity of adult stage(28 weeks) decreased 50% than control group.

대량의 발생 유전학적 연구가 가능한 척추동물로서 최근 제브라피쉬가 새로운 동물모델로 급부상하고 있다 다양한 형태의 돌연변이들로부터 새로운 유전자들이 발굴되어지고 있으며, 인간 유전체의 기능 분석 수단으로 활용되어지고 있다. 신경계의 형성과 분화에 이상이 있는 hendless와 mind bomb이라는 두 가지 돌연변이주에서 positional cloning에 의한 원인 유전자의 발굴과 기능 분석의 예로써 현재 제브라피쉬의 연구 현황을 살펴보고자 한다. h

The zebrafish (Danio rerio) is now the pre-eminent vertebrate model system for clarification of the roles of specific genes and signaling pathways in development. I will talk about positional cloning of two developmental mutants in zebrafish. The first mutant is headless: The vertebrate organizer can induce a complete body axis when transplanted to the ventral side of a host embryo by virtue of its distinct head and trunk inducing properties. Wingless/Wntantagonists secreted by the organizer have been identified as head inducers. Their ectopic expression can promote head formation, whereas ectopic activation of Wnt signalling during early gastrulation blocks head formation. These observations suggest that the ability of head inducers to inhibit Wntsignalling during formation of anterior structures is what distinguishes them from trunk inducers that permit the operation of posteriorizing Wnt signals. I describe the zebrafish headless (hdl) mutant and show that its severe head defects are due to a mutation in T-cell factor-3 (Tcf3), a member of the Tcf/Lef family. Loss of Tcf3 function in the hdl mutant reveals that hdl represses Wnt target genes. I provide genetic evidence that a component of the Wntsignalling pathway is essential in vertebrate head formation and patterning. Second mutant is mind bomb: Lateral inhibition, mediated by Notch signaling, leads to the selection of cells that are permitted to become neurons within domains defined by proneuralgene expression. Reduced lateral inhibition in zebrafish mib mutant embryos permits too many neural progenitors to differentiate as neurons. Positional cloning of mib revealed that it is a gene in the Notch pathway that encodes a RING ubiquitin ligase. Mib interacts with the intracellular domain of Delta to promote its ubiquitylation and internalization. Cell transplantation studies suggest that mib function is essential in the signaling cell for efficient activation of Notch in neighboring cells. (중략)

The effects of bisphenol A on the catalase activities in the development stage of zebrafish were investigated. In this study, the catalase activities for zebrafish fries exposed to bisphenol A of 1×10-10 g/ℓ during 1 week, 2 week, and 4 week post-hatching were examined. Also, the changes of organs weight and the catalase activities for adult zebrafishes exposed to bisphenol A during 3 weeks were investigated. Catalase activities for zebrafish fries exposed to bisphenol A of 1×10-10 g/ℓ during 1 week post-hatching were significantly lower, compared to the control. Somewhat, for zebrafish fries exposed to bisphenol A during 4 week post-hatching, catalase activities were significantly increased. For adult zebrafishes, the effects of bisphenol A were higher for female than male. Specially, catalase activities were significantly increased in the ovary of zebrafishes exposed to bisphenol A during 3 weeks. The ovary weight were increased for zebrafishes exposed to bisphenol A during 3 weeks. Catalase activities were increased in the intestine of female exposed to bisphenol A during 3 weeks. Catalase activities were increased in testis exposed to bisphenol A during 3 weeks but there was no significance. In conclusion, the damages of an endocrine disrupter were higher in the earlier development stage compared with adult. The damages were higher for female exposed to an endocrine disrupter compared with male.