| Budget Amount *help |
¥3,500,000 (Direct Cost: ¥3,500,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 2002: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Research Abstract |
Many organs are formed in the process of the development, while unnecessary ones are eliminated. During the amphibian metamorphosis, tadpole tail and gills are regressing, as levels of plasma thyroid hormone are rising. By our research the following results are obtained.
1.We injected dominant-negative thyroid hormone receptor gene together with a maker gene into tail muscle cells in living tadpoles to interrupt thyroid hormone signaling, thereby demonstrating that muscle cell death in tadpole tail commit suicide till NF-stage 62. Furthermore, we have shown that muscle cell death in rapidly shortening tails after NF-stage 62 is executed not only by suicide, but also by murder mechanism where cells lose the anchorage and die by the degradation of the extracellular matrix due to the increase of the matrix metalloproteinases.
2.The matrix metalloproteinases(MMP) are believed to play a main role in the murder mechanism during the tail regression. A MMP inhibitor represses completely the resolution of the notochord that is constituted by the basement membrane, but partially the regression of tail. This result supports that dual mechanisms, suicide and murder, contribute to tail regression.
3.We have isolated two clones by the subtraction library and differential hybridization as genes correlated with tail muscle cell death, obtained their full-length cDNAs, and analyzed. Both of them are actin-binding proteins, and not considered as suicide genes, because cell survival was not reduced by their overexpression in tadpole tail-derived myoblastic cell line.
4.A cDNA library has been constructed by placing cDNAs downstream of the tetracycline-inducible promoter. Tadpoles are coinjected with clones of the cDNA library and a reporter gene, and reared in the presence of tetracycline derivative, doxycycline. We are searching for cDNA clones which diminishes the expression of reporter gene after doxycycline treatment.
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