Coen L, du Pasquier D, Le Mevel S, Brown S, Tata J, Mazabraud A, Demeneix B A
Laboratoire de Physiologie Générale et Comparée, Muséum National d'Histoire Naturelle, Unité Mixte de Recherche, Centre National de la Recherche Scientifique 8572, 7, Rue Cuvier, 75231 Paris, Cedex 5, France.
Proc Natl Acad Sci U S A. 2001 Jul 3;98(14):7869-74. doi: 10.1073/pnas.141226798. Epub 2001 Jun 26.
Amphibian metamorphosis involves extensive, but selective, neuronal death and turnover, thus sharing many features with mammalian postnatal development. The antiapoptotic protein Bcl-X(L) plays an important role in postnatal mammalian neuronal survival. It is therefore of interest that accumulation of the mRNA encoding the Xenopus Bcl-X(L) homologue, termed xR11, increases abruptly in the nervous system, but not in other tissues, during metamorphosis in Xenopus tadpoles. This observation raises the intriguing possibility that xR11 selectively regulates neuronal survival during postembryonic development. To investigate this hypothesis, we overexpressed xR11 in vivo as a green fluorescent protein (GFP)-xR11 fusion protein by using somatic and germinal transgenesis. Somatic gene transfer showed that the fusion protein was effective in counteracting, in a dose-dependent manner, the proapoptotic effects of coexpressed Bax. When GFP-xR11 was expressed from the neuronal beta-tubulin promoter by germinal transgenesis we observed neuronal specific expression that was maintained throughout metamorphosis and beyond, into juvenile and adult stages. Confocal microscopy showed GFP-xR11 to be exclusively localized in the mitochondria. Our findings show that GFP-xR11 significantly prolonged Rohon-Beard neuron survival up to the climax of metamorphosis, even in the regressing tadpole tail, whereas in controls these neurons disappeared in early metamorphosis. However, GFP-xR11 expression did not modify the fate of spinal cord motoneurons. The selective protection of Rohon-Beard neurons reveals cell-specific apoptotic pathways and offers approaches to further analyze programmed neuronal turnover during postembryonic development.
两栖动物变态发育涉及广泛但具有选择性的神经元死亡和更替，因此与哺乳动物出生后的发育有许多共同特征。抗凋亡蛋白Bcl-X(L)在出生后的哺乳动物神经元存活中起重要作用。因此，有趣的是，在非洲爪蟾蝌蚪变态发育期间，编码非洲爪蟾Bcl-X(L)同源物（称为xR11）的mRNA在神经系统中突然增加，但在其他组织中没有增加。这一观察结果提出了一个有趣的可能性，即xR11在胚胎后发育过程中选择性地调节神经元存活。为了研究这一假设，我们通过体细胞和生殖系转基因在体内过表达xR11，将其作为绿色荧光蛋白(GFP)-xR11融合蛋白。体细胞基因转移表明，该融合蛋白能以剂量依赖的方式有效对抗共表达的Bax的促凋亡作用。当通过生殖系转基因从神经元β-微管蛋白启动子表达GFP-xR11时，我们观察到神经元特异性表达，这种表达在整个变态发育过程中一直保持，直至幼体和成体阶段。共聚焦显微镜显示GFP-xR11仅定位于线粒体。我们的研究结果表明，即使在退化的蝌蚪尾巴中，GFP-xR11也能显著延长罗霍恩-比尔神经细胞的存活时间，直至变态发育的高潮，而在对照组中，这些神经元在变态发育早期就消失了。然而，GFP-xR11的表达并没有改变脊髓运动神经元的命运。罗霍恩-比尔神经细胞的选择性保护揭示了细胞特异性凋亡途径，并为进一步分析胚胎后发育过程中程序性神经元更替提供了方法。
