GROWTH CONE GUIDANCE--INTERACTIONS AND SURFACE MOLECULES

我们已经鉴定并开始了分子和功能 两种新的细胞表面分子的表征 神经发生。通过制备单抗发现了Lazarillo和Conlin 特定于发育中的神经系统，然后利用这些 单抗作为生化试剂用于单抗的纯化和鉴定 抗原。我们已经克隆了编码Lazarillo的基因 建议克隆该应用中的Conlin编码基因。 Lazarillo代表了一种新的分子，一种脂钙蛋白，涉及 在神经元通路中的发现并提出了一种新的分子信号 路径。我们正在提议进行实验，以确定 Lazarillo调节生长锥和丝状体行为以指导 沿着特定路径的神经元。这将需要开发一种 “分子敲除”反义技术特异地抑制 Lazarillo在已鉴定神经元中的表达。我们将测试 Lazarillo介导其对生长锥调节的假说 通过信号转导途径的行为，包括一个小的 亲脂配体和蛋白质配体。结构和功能 建议进行实验来鉴定这两种小的亲脂配体 它结合在Lipocalin疏水口袋和蛋白质配体上 它与假定的蛋白质相互作用结构域结合 由我们的建模研究建议。我们预测这些同源基因 Lazarillo存在于包括脊椎动物在内的其他动物中，并具有 在他们的神经系统发育过程中也是如此。 我们已经提出了一种直接的基于PCR的方法来 识别这些同源基因，并将使用序列信息和 原位杂交验证同源性。康诺林独特的空间 以及对神经元生长锥体的子集的时间定位 这使它成为一种新的分子功能的很好的候选者。 鉴定刀豆素的实验包括免疫亲和力和 生化提纯、表达克隆。我们假设 柯林素作为生长锥体在神经元寻路中的作用 在中央神经节内从一个轴突切换到另一个轴突 神经痛。我们将通过观察以下行为来检验这一假设 已确定已从其生长锥体中移除了球藻糖浆 表面。很明显，许多基本的分子机制 在发育过程中是高度保守的 对各种神经系统的评估，如线虫、苍蝇和 老鼠。我们建议拉扎里洛和科诺林将带领我们进入新的 所有发育中的神经系统所使用的分子机制。

We have identified and begun the molecular and functional characterization of two new cell surface molecules involved in neurogenesis. Lazarillo and Conulin were found by making mAbs specific to the developing nervous system and then using those mAbs as biochemical reagents to purify and characterize each antigen. We have cloned the gene encoding Lazarillo and we are proposing to clone the gene encoding Conulin in this application. Lazarillo represents a new class of molecule, a lipocalin, involved in neuronal path findings and suggests a new molecular signaling pathway. We are proposing experiments to characterize how Lazarillo regulates growth cone and filopodial behavior to guide neurons along a specific pathway. This will entail developing a "molecular knockout" anti-sense technology to specifically inhibit the expression of Lazarillo in identified neurons. We will test the hypothesis that Lazarillo mediates its regulation of growth cone behavior via a signal transduction pathway that includes a small lipophilic ligand and a protein ligand. Structural and functional experiments are proposed to identify both the small lipophilic ligand that binds in the lipocalin hydrophobic pocket and the protein ligand that binds to the putative protein protein interaction domain suggested by our modeling studies. We predict that homologues of Lazarillo exist in other animals, including vertebrates, and function in a similar way during the development of their nervous systems. We have proposed a straightforward PCR based approach to identify these homologues and will use sequence information and in situ hybridization to verify the homology. Conulin's unique spacial and temporal localization to a subset of neuronal growth cones makes it a good candidate for a novel molecular function. Experiments to identify Conulin include immunoaffinity and biochemical purification, and expression cloning. We hypothesize that Conulin functions in neuronal path finding as growth cones switch from one axonal fascicle to another in the central ganglionic neuropil. We will test this hypothesis by observing the behavior of identified growth cones that have had conulin removed from their surfaces. It is clear that many of the basic molecular mechanisms in developmental processes have been highly conserved during the evaluation of nervous systems as diverse as nematode, fly and mouse. We propose that Lazarillo and Conulin will lead us to new molecular mechanisms used by all developing nervous systems.