HOW DOES VND INTEGRATE POSITIONAL INFORMATION?

VND 如何整合位置信息？

• 批准号: 6402698
• 负责人: DERVLA M MELLERICK-DRESSLER
• 金额: $ 20.45万
• 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/6402698
• 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.

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，它指定了果蝇中线近端腹侧神经外胚层细胞和神经母细胞的身份，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基因Nkx2.2和Nkx2.1在脊椎动物的神经元模式中扮演着平行的角色，这表明调节和功能上的保守。这个建议的总体目标是了解果蝇vnd基因是如何整合位置信息的，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，我们将通过解剖转基因胚胎中VND的5‘端区域来定位负责VND特异性表达的增强子。此外，我们还将从两个相关的果蝇物种中鉴定VND调节域中的保守序列岛。我们将与那些结合VND增强子的调节器进行DNA结合研究。我们将通过突变这些区域并在转基因胚胎报告实验系统中测试突变的增强子的活性来确认这些DNA结合位点的功能意义。我们还将讨论NK-2盒在VND作为转录因子的作用中的作用。NK-2盒是一个由18个氨基酸组成的保守片段，位于同源盒下游。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和转基因胚胎中进行检测。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。过度表达突变型VND、缺乏NK-2盒的转基因胚胎将被检测神经前体身份的变化。最后，我们将阐明表皮生长因子(EGF)途径与VND之间的关系。EGF通过诱导大部分未知目标蛋白的磷酸化，使发育中的中枢神经系统沿着背腹轴形成模式。由于当EGF受体缺失或EGF配体Spitz过度表达时，VND的表达会发生变化，因此VND可能受到磷酸化的调节。在VND中被磷酸化的氨基酸将通过酸解和肽图来鉴定。在它们突变后，将通过检测过度表达野生型和突变型VND的功能获得胚胎中神经元标记物的表达来分析磷酸化在VND功能中的作用。这些研究涉及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。