The Molecular Mechanism of Organ Regression during the Development

许多器官在发育过程中形成，而不必要的器官则被淘汰。在两栖动物的变态过程中，蝌蚪的尾巴和鳃在退化，因为血浆甲状腺激素的水平在上升。本研究获得了以下结果：1.将显性负性甲状腺激素受体基因和标记基因共同注入活体蝌蚪尾肌细胞，阻断甲状腺激素信号传导，从而证实蝌蚪尾肌细胞死亡至NF-62期。此外，我们已经表明，在NF-62期后迅速缩短的尾部中，肌细胞死亡不仅是通过自杀，而且还通过谋杀机制来执行，即细胞失去锚定，并且由于基质金属蛋白酶的增加而通过细胞外基质的降解而死亡。2.基质金属蛋白酶（MMP）被认为在尾部消退期间的谋杀机制中起主要作用。MMP抑制剂完全抑制由基底膜构成的脊索的消退，但部分抑制尾部的消退。这一结果支持了自杀和谋杀双重机制对尾退化的影响。3.利用消减文库和差异杂交技术分离了两个尾肌细胞死亡相关基因的克隆，获得了它们的全长cDNA，并进行了分析。这两个基因都是肌动蛋白结合蛋白，不被认为是自杀基因，因为它们在蝌蚪尾源性成肌细胞系中的过表达不会降低细胞存活率。4.通过将cDNA置于四环素诱导型启动子的下游构建cDNA文库。将cDNA文库的克隆和报告基因共注射到蝌蚪中，并在四环素衍生物多西环素存在下饲养。我们正在寻找cDNA克隆，减少报告基因的表达后，强力霉素治疗。

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.