HOW DOES VND INTEGRATE POSITIONAL INFORMATION?

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物Nkx 2.2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。 这个建议的总体目标是了解果蝇vnd基因如何整合位置信息，使其适合参与中枢神经系统背腹侧规范的层次网络。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，我们将确定两个相关的果蝇物种的vnd调控域中的保守序列岛。 我们将用那些结合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基因测定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，这是一个保守的18个氨基酸的延伸，位于Vnd作为转录因子的同源框下游。 将在组织培养物和转基因胚胎中测定缺乏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通过诱导大部分未鉴定的靶蛋白的磷酸化，使发育中的CNS沿背腹轴沿着。 由于当EGF受体缺失或EGF配体Spitz过度表达时，Vnd的表达会改变，因此Vnd可能受磷酸化调节。 将通过酸水解和肽图谱鉴定Vnd中磷酸化的氨基酸。 突变后，磷酸化在Vnd功能中的作用将通过检查过度表达野生型和突变型Vnd的功能获得性胚胎中的神经元标志物表达来分析。 这些研究阐明了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。

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.