THE DROSOPHILA CNS: A MODEL FOR PCB NEUROXICITY

无脊椎动物和脊椎动物胚胎中产生神经元的细胞必须在整个发育过程中正确地接收和解释位置信息，以便准确地确定终末分化的神经元。对位置提示的错误解释可能会导致严重的发育异常，在最极端的情况下会导致死亡。腹侧神经系统缺陷(VND)是一种NK-2型同源框基因，它识别果蝇中线近端腹侧神经外胚层细胞和神经母细胞。由于该基因的脊椎动物同源基因在脊椎动物的中枢神经系统中以平行的结构域表达，这意味着调节和功能上的保守。这一建议的总体目标是了解果蝇vnd基因如何在转录和翻译后水平整合位置信息，以便它适合于涉及中枢神经系统背腹规范的层级网络。具体地说，我们想要定位负责VND在转基因胚胎中表达的增强子，从两个相关的果蝇物种中鉴定VND调节域中的保守序列岛，进行与VND增强子结合的调节器的DNA结合研究，并通过突变相关DNA结合位点并测试它们在转基因胚胎中的活性来确认DNA结合位点的功能意义。表皮生长因子(EGF)途径还通过诱导大部分未知目标蛋白的磷酸化，沿着背腹轴形成发育中的中枢神经系统。由于VND在EGF受体突变体中的表达受到影响，我们认为VND是受磷酸化调控的。我们想要确定VND是否被磷酸化，并识别被修饰的氨基酸，长期目标是确定磷酸化在VND作为转录因子的功能中的作用。最后，我们想要讨论NK-2盒在VND作为转录因子的作用中的作用。NK-2盒是同源结构域下游18个氨基酸的保守延伸。缺乏NK-2盒的突变的VND蛋白驱动报告表达的能力将在组织培养模型中进行测试。我们最近发现，野生型VND的过度表达会导致神经元前体身份的转变。过表达突变型VND、缺乏NK-2盒的转基因胚胎将被检测神经元命运的变化。这些研究直接涉及VND的结构和功能，VND是早期神经元发育的关键调节因子，并可能作为脊椎动物VND样基因的原型。

Cells of both invertebrate and vertebrate embryos that give rise to neurons must correctly receive and interpret positional information throughout development so that terminally differentiated neurons are precisely determined. The mis-interpretation of positional cues can lead to severe developmental abnormalities and in the most extreme case death. Ventral nervous system defective (vnd), an NK-2 type homeobox gene, specifies the identity of midline proximal ventral neuroectodermal cells and neuroblasts in Drosophila. Since the vertebrate homologues of this gene are expressed in parallel domains in the vertebrate CNS, this suggests both regulatory and functional conservation. The overall goal of this proposal is to understand how the Drosophila vnd gene integrates positional information at the transcriptional and post-translational level so that it fits into the hierarchical network involved in CNS dorsal-ventral specification. Specifically, we want to localize the enhancers responsible for vnd expression in transgenic embryos, identify conserved sequence islands in the vnd regulatory domain from two related Drosophila species, perform DNA binding studies with regulators that bind vnd enhancers and confirm the functional significance of the DNA binding sites by mutating the relevant DNA binding sites and testing their activity in transgenic embryos. The epidermal growth factor (EGF) pathway also patterns the developing CNS along the dorsal-ventral axis by inducing the phosphorylation of largely unidentified target proteins. Since expression of Vnd is affected in EGF receptor mutants, we believe that Vnd is regulated by phosphorylation. We want to detemine if Vnd is phosphorylated, and to identify the amino acids that are modified, with the long term goal of determining the role of phosphorylation in Vnd's function as a transcription factor. Finally, we want to address the role of the NK-2 box, a conserved stretch of 18 amino acids downstream of the homeodomain, in Vnd's role as a transcription factor. The capacity of mutated Vnd protein, lacking the NK-2 box, to drive reporter expression will be assayed in a tissue culture model. We recently showed that over-expression of wild-type Vnd leads to a transformation in neuronal precursor identity. Transgenic embryos that over-express mutant Vnd, lacking the NK-2 box, will be assayed for alteration in neuronal fate. These studies directly address the structure and function of vnd, a critical regulator of early neuronal development, and may serve as a prototype for vertebrate vnd-like genes.