STUDIES ON THE MECHANISMS BY WHICH HELIX-LOOP-HELIX TRANSCRIPTION FACTORS REGULATE NEURAL DEVELOPMENT

为了了解神经发育的分子机制，我们重点研究了碱性螺旋-环-螺旋（bHLH）激活蛋白和抑制蛋白的作用。Xenopus MyT1的表达受bHLH蛋白的正调控和Notch/Delta信号转导通路的负调控，最近被证明在神经元分化中起关键作用。在果蝇中，编码bHLH转录因子的前基因和Delta/Notch信号通路的作用被研究得最为广泛。为了了解c2hc型锌指基因在果蝇神经发生中的功能作用，我们克隆了果蝇c2hc型锌指基因dNZF-1的cDNA。dNZF-1蛋白由1220个氨基酸组成，只含有两个C2HC锌指，而所有脊椎动物的MyT/NTF蛋白都含有6个锌指。dNZF-1的第一个C2HC锌指与NZF-3的第四个锌指的氨基酸序列同源性为96.3%，但dNZF-1与两个锌指以外的其他成员的结构同源性很小。凝胶阻滞实验表明，dNZF-1蛋白能够结合AAAGTTT序列的双拷贝；此外，它能够通过该序列在Schneider细胞中激活热休克70最小启动子荧光素酶报告基因的转录。胚胎全安装原位杂交表明，dNZF-1转录本在胚胎后期中枢神经系统的神经元亚群中表达。此外，使用Gal4/UAS系统表达dNZF-1的显性阴性形式可诱导胚胎致死。综上所述，这些结果表明dNZF-1在神经元分化中的功能作用。

To understand the molecular mechanisms of neural development, we are focusing on the studies of actions of basic helix-loop-helix (bHLH) activator and repressor proteins. Xenopus MyT1, whose expression is positively regulated by the bHLH protein and negatively regulated by the Notch/Delta signal transduction pathway, has recently been shown to play a critical role in neuronal differentiation. In Drosophila, the roles of the proneural genes encoding bHLH transcriptional factors and the Delta/Notch signaling pathway have been the most extensively studied. To understand the functional roles of C2HC-type zinc finger gene(s) in Drosophila neurogenesis, we cloned the cDNA of a Drosophila C2HC-type zinc finger gene (termed dNZF-1). The dNZF-1 protein is composed of 1220 amino acids and contains only two C2HC zinc fingers, whereas all vertebrate MyT/NTF proteins contain six zinc fingers. The first C2HC zinc finger of dNZF-1 shows 96.3% amino acid sequence identity with the fourth one of NZF-3, but there is little structural homology of dNZF-1 to other members outside the two zinc fingers. Gel retardation assays showed that the dNZF-1 protein is able to bind to the double copy of an AAAGTTT sequence; furthermore, it is able to activate transcription of the heat-shock 70 minimal promoter-luciferase reporter gene through this sequence in Schneider cells. Whole-mount in situ hybridization to embryos showed that dNZF-1 transcripts are expressed in neuronal subsets of the central nervous system at the late embryonic stage. Moreover, expression of a dominant negative form of dNZF-1 using the Gal4/UAS system induced embryonic lethality. Taken together, these results suggest a functional role of dNZF-1 in neuronal differentiation.