THE DROSOPHILA CNS--A MODEL FOR PCB NEUROTOXICITY

果蝇中枢神经系统——多氯联苯神经毒性模型

• 批准号: 6031263
• 负责人: DERVLA M MELLERICK-DRESSLER
• 金额: $ 7.58万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-09-30 至 2001-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6031263
• 关键词: DNA binding protein; Drosophilidae; binding sites; biological models; central nervous system; developmental neurobiology; embryo /fetus; epidermal growth factor; gene expression; genetic enhancer element; genetic mapping; genetic transcription; genetically modified animals; growth factor receptors; halobiphenyl /halotriphenyl compound; homeobox genes; invertebrate embryology; mutant; neurotoxicology; nucleic acid sequence; phosphorylation; posttranslational modifications; protein sequence; reporter genes; tissue /cell culture; transcription factor

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.

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，以便精确地确定终末分化的神经元。 对位置线索的错误解释会导致严重的发育异常，在最极端的情况下会导致死亡。果蝇神经系统缺陷基因（vnd）是一个NK-2型同源异型盒基因，它决定了果蝇中线近腹侧神经外胚层细胞和成神经细胞的身份。 由于该基因的脊椎动物同源物在脊椎动物CNS中的平行结构域中表达，这表明调节和功能保守。 本提案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，使其适合参与中枢神经系统背腹侧规范的层次网络。 具体来说，我们要本地化的增强子负责vnd在转基因胚胎中的表达，确定保守的序列岛的vnd调节结构域从两个相关的果蝇物种，进行DNA结合的研究与监管机构结合vnd增强子和确认的功能意义的DNA结合位点突变相关的DNA结合位点，并测试其在转基因胚胎中的活性。 表皮生长因子（EGF）途径还通过诱导大量未鉴定的靶蛋白的磷酸化，使发育中的CNS沿背腹轴沿着形成模式。 由于Vnd的表达在EGF受体突变体中受到影响，我们认为Vnd受磷酸化调节。 我们想要确定Vnd是否被磷酸化，并鉴定被修饰的氨基酸，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，我们想解决的作用NK-2盒，保守的延伸18个氨基酸的同源结构域下游，在Vnd的作用作为一个转录因子。 将在组织培养模型中测定缺乏NK-2盒的突变的Vnd蛋白驱动报告基因表达的能力。 我们最近发现，野生型Vnd的过度表达导致神经元前体身份的转变。 将测定过表达突变体Vnd（缺乏NK-2盒）的转基因胚胎的神经元命运改变。 这些研究直接阐明了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。