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HOW DOES VND INTEGRATE POSITIONAL INFORMATION?

VND 如何整合位置信息？

基本信息

批准号：
6611373
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
项目类别：
财政年份：
2000
资助国家：
美国
起止时间：
2000-08-15 至 2005-07-31
项目状态：
已结题

项目摘要

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接收和解释位置信息，以便精确确定终末分化的神经元。对位置线索的误解可能导致严重的发育异常，最极端的情况下甚至导致死亡。腹神经系统缺陷 (vnd) 是一种 NK-2 型同源盒基因，指定果蝇中产生运动神经元和中间神经元的中线近端腹侧神经外胚层细胞和神经母细胞的身份。由于该基因的脊椎动物同源物 Nkx2.2 和 Nkx 2.1 在脊椎动物神经元模式中发挥平行作用，这表明调节和功能保守。该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规范所涉及的分层网络。具体来说，我们将通过解剖转基因胚胎中vnd的5'区域来定位负责vnd特异性表达的增强子。此外，我们将鉴定来自两个相关果蝇物种的 vnd 调节域中的保守序列岛。我们将使用那些结合 vnd 增强子的调节剂进行 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，NK-2 盒是 18 个氨基酸的保守序列，位于 Vnd 作为转录因子的同源盒下游。将在组织培养物和转基因胚胎中检测缺乏 NK-2 盒的突变 Vnd 蛋白驱动报告基因表达的能力。我们最近表明，野生型 Vnd 的过度表达会导致神经元前体身份的转变。将检测过度表达突变体 Vnd（缺乏 NK-2 盒）的转基因胚胎的神经前体身份的改变。最后，我们将阐明表皮生长因子（EGF）途径与Vnd之间的关系。 EGF 通过诱导大部分未识别的靶蛋白的磷酸化，使中枢神经系统沿着背腹轴发育。由于当 EGF 受体缺失或 EGF 配体 Spitz 过度表达时，Vnd 的表达会发生改变，因此 Vnd 可能受到磷酸化的调节。 Vnd 中磷酸化的氨基酸将通过酸水解和肽图谱进行鉴定。突变后，将通过检查过度表达野生型和突变型 Vnd 的功能获得胚胎中的神经元标记表达来分析磷酸化在 Vnd 功能中的作用。这些研究解决了早期神经元发育的关键调节因子 vnd 的结构和功能，并可能作为脊椎动物 vnd 样基因的原型。