CHEMOREPULSION MEDIATED NETRIN RECEPTORS UNC5H AND DCC

神经生物学的一个核心问题是确定神经系统功能的基础——复杂的神经元连接网络是如何建立的。这种复杂的模式至少在一定程度上是由神经元细胞体及其轴突在扩散趋化信号的引导下迁移到目标细胞时产生的。我的实验室的长期目标是了解细胞如何在引导信号的梯度中检测和定位自己，并将这些信息在细胞内转化为形状和运动的变化，从而导致定向迁移。Netrin-1就是这种引导线索的一个例子，已经确定了两个受体家族，它们指定对Netrin-1的引诱和排斥反应。细胞或轴突对netrin-1的吸引是由DCC（在结直肠癌中缺失）介导的。对秀丽隐杆线虫的研究表明，对netrin-1的排斥是由UNC5受体指定的，并且UNC5与DCC一起调节这种排斥。初步数据显示，UNC5H和DCC物理相互作用形成一个排斥蛋白复合物。基于这些研究，该资助提案的主要假设是，在脊椎动物体内，UNC5的同源物（UNC5Hs）与DCC一起介导了netrin-1依赖性的化学排斥。为了解决这个假设，这个提议的具体目的有两个方面。I)确定UNC5H/DCC复合物在指导netrin-1介导的排斥中的体内作用。提出了netrin-1在指导小脑颗粒细胞迁移中的作用的研究。这些研究检查了netrin- 1在体内功能丧失和功能获得的后果，以及小脑颗粒细胞在体外的迁移。II)为了理解指定排斥与吸引的分子机制，将使用酵母双杂交和亲和色谱方法鉴定与UNC5Hs结合并转导排斥信号的分子。这些相互作用蛋白在响应netrin-1介导排斥中的功能将在轴突引导或细胞迁移试验中进行测试。这些实验为理解细胞如何将netrin-1信号转化为定向迁移提供了基础。

A central problem in neurobiology is to determine how the complex networks of neuronal connections, that underlie the functioning of the nervous system, are established. This intricate pattern is generated, at least in part, when neuronal cell bodies and their axons migrate to their targets, guided by diffusible chemotropic cues. The long term goal for my laboratory is to understand how cells detect and orient themselves in gradients of guidance cues, and transduce this information intracellularly into changes in shape and motility that lead to directed migrations. Netrin-1 is an example of such a guidance cue, and two families of receptors that specify attractant and repellent responses to netrin-1 have been identified. The attraction of a cell or axon toward netrin-1 is mediated by DCC (Deleted in Colorectal Carcinoma). Studies in C. elegans demonstrate that repulsion away from netrin-1 is specified by UNC5 receptors, and that UNC5 functions together with DCC to mediate this repulsion. In the preliminary data, it is shown that UNC5H and DCC physically interact to form a repellent protein complex. Based on these studies, the major hypothesis of the grant proposal is that vertebrate homologs of UNC5 (UNC5Hs), together with DCC, mediate netrin-1 dependent chemorepulsion in vertebrates in vivo. To address the hypothesis, the specific aims of this proposal are two-fold. I) To define the in vivo role of the UNC5H/DCC complex in directing netrin-1 mediated repulsion. Studies are proposed on the role of netrin-1 in guiding the migration of cerebellar granule cells. These studies examine the consequences of netrin- 1 loss-of-function and gain-of-function in vivo, and the migration of cerebellar granule cells in vitro. II) To understand the molecular mechanisms that specify repulsion versus attraction, the molecules that bind to UNC5Hs and transduce a repellent signal will be identified using yeast two-hybrid and affinity chromatography approaches. The function(s) of these interacting proteins in mediating repulsion in response to netrin-1 will be tested in axon guidance or cell migration assays. These experiments provide the foundation for understanding how cells translate a netrin-1 signal into directed migrations.