HOW DOES VND INTEGRATE POSITIONAL INFORMATION?

VND 如何整合位置信息？

• 批准号: 6198468
• 负责人: DERVLA M MELLERICK-DRESSLER
• 金额: $ 19.99万
• 依托单位: UNIVERSITY OF MICHIGAN AT ANN ARBOR
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-15 至 2004-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6198468
• 关键词: Drosophilidae; developmental genetics; developmental neurobiology; epidermal growth factor; gene mutation; genetic enhancer element; genetically modified animals; growth factor receptors; homeobox genes; interneurons; intracellular transport; invertebrate embryology; molecular cloning; motor neurons; nerve stem cell; neurogenesis; neurogenetics; nucleic acid sequence; phosphoproteins; phosphorylation; protein transport; reporter genes; site directed mutagenesis; 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 that give rise to motor neuron and interneurons. Since the vertebrate homologues of this gene, Nkx2.2 and Nkx 2.1, play parallel roles in vertebrate neuronal patterning, this suggests both regulatory and functional conservation. The overall goal of this proposal is to understand how the Drosophila vnd gene integrates positional information so that it fits into the hierarchical network involved in CNS dorsal-ventral specification. Specifically, we will localize the enhancers responsible for vnd- specific expression by dissecting the 5' region of vnd in transgenic embryos. In addition we will identify conserved sequence islands in the vnd regulatory domain from two related Drosophila species. We will perform DNA binding studies with those regulators that bind vnd enhancers. We will confirm the functional significance of the DNA binding sites by mutating these regions and testing the activity of mutated enhancers in a transgenic embryo reporter assay system. We will also address the role of the NK-2 box, a conserved stretch of 18 amino acids, downstream of the homeobox 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 both tissue culture and transgenic embryos. 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 neural precursor identity. Finally, we will illucidate the relationship between the epidermal growth factor (EGF) pathway and Vnd. EGF patterns the developing CNS along the dorsal- ventral axis by inducing the phosphorylation of largely unidentified target proteins. Since expression of Vnd is altered when the EGF receptor is missing or the EGF ligand, Spitz is over-expressed, Vnd is likely regulated by phosphorylation. The amino acids that are phosphorylated in Vnd will be identified by acid hydrolysis and peptide mapping. Following their mutation, the role of phosphorylation in Vnd's function will be analyzed by examining neuronal marker expression in gain-of-function embryos that over-express wild-type and mutant Vnd. These studies 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.

产生神经元的无脊椎动物和脊椎动物胚胎细胞必须在整个发育过程中正确地接收和解释位置信息，以便精确地确定最终分化的神经元。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。腹侧神经系统缺陷（vnd）是一种NK-2型同源盒基因，它指定了果蝇中线近侧腹侧神经外胚层细胞和神经母细胞的同一性，这些细胞产生运动神经元和中间神经元。由于该基因的脊椎动物同源物Nkx2.2和nkx2.1在脊椎动物神经元模式中发挥平行作用，这表明既有调控作用，也有功能保护作用。本提案的总体目标是了解果蝇vnd基因如何整合位置信息，使其适合中枢神经系统背-腹侧规范的分层网络。具体来说，我们将通过解剖转基因胚胎中vnd的5'区来定位负责vnd特异性表达的增强子。此外，我们将从两个相关的果蝇物种中确定vnd调控域的保守序列岛。我们将对那些结合vnd增强子的调节因子进行DNA结合研究。我们将通过对这些区域进行突变，并在转基因胚胎报告分析系统中测试突变增强子的活性，来确认DNA结合位点的功能意义。我们还将讨论NK-2盒的作用，这是同源盒下游的一个保守的18个氨基酸延伸，在Vnd作为转录因子的作用中。缺乏NK-2盒子的突变Vnd蛋白驱动报告基因表达的能力将在组织培养和转基因胚胎中进行检测。我们最近发现野生型Vnd的过表达导致神经元前体身份的转变。过表达缺乏NK-2盒子的Vnd突变体的转基因胚胎，将检测其神经前体特性的改变。最后，我们将阐明表皮生长因子（EGF）通路与Vnd之间的关系。EGF通过诱导大部分未知靶蛋白的磷酸化，沿背腹轴影响发育中的中枢神经系统。由于当EGF受体缺失或EGF配体Spitz过表达时，Vnd的表达发生改变，因此Vnd可能受磷酸化调节。在Vnd中被磷酸化的氨基酸将通过酸水解和肽图谱来鉴定。在它们发生突变之后，我们将通过检测过表达野生型和突变型Vnd的功能获得胚胎中的神经元标记物表达来分析磷酸化在Vnd功能中的作用。这些研究解决了vnd的结构和功能，它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。