Role of mPar6 Polarity in CNS Neuronal Migration

描述(申请人提供)：年轻的神经元从它们产生的部位迁移到它们建立成人大脑回路的位置是发育中的关键一步。迁徙缺陷会导致许多人类先天缺陷，从严重的智力低下到轻微的学习障碍，以及大量的癫痫。我们的实验室一直致力于了解控制迁移的基因，希望能深入了解这一正常发育的关键步骤。我们以小脑颗粒神经元的迁移为模型系统，研究了神经胶质引导的神经元迁移的分子调控。神经元极性的建立是启动神经元沿神经胶质引导迁移的关键步骤。对颗粒神经元迁移功能基因的筛选显示，极性信号复合体mPar6a神经元高水平表达，退出周期并建立极性。在线虫中，一组6个PAR蛋白建立前/后不对称，并控制随后的不对称细胞分裂。PAR蛋白在整个进化过程中是保守的。在《初步研究》(Solecki et al，2004)中，我们发现mPar6a信号复合体定位于迁移中的小脑颗粒神经元的中心体，当神经元沿胶质纤维迁移时，它协调中心体和细胞核的运动。在这项拟议的研究中，我们将研究mPar6a复合体的其他成分，aPKcheeta和Par3，在迁移颗粒神经元的极性中。MPar6a在细胞分裂中的作用表明，靶向功能缺失突变体和shRNA实验将是不可行的。因此，我们将使用Tsien开发的一种新方法，将结合染料ReAshS的基因标签(TC)整合到mParGq中，并使用发色团辅助的光灭活和单色激光灭活中心体中的mParGa。对于这些实验，我们将产生TC-mPar6a BAC转基因小鼠，使对颗粒细胞和皮质神经元的研究成为可能。在最后一组实验中，我们将研究在颗粒细胞中表达的受体/配体系统，它与mParGa复合体、EphB配体ePhin-B1和ePhin-B2相互作用。总之，这些实验将为大脑发育中皮质区域神经元迁移的调控提供新的信息。

DESCRIPTION (provided by applicant): The migration of young neurons from sites where they are generated into the positions where they establish the circuitry of the adult brain is a critical step in development. Defects in migration cause a host of human birth defects, ranging from severe mental retardation to subtle learning disabilities, as well as a large number of the epilepsies. Our lab has focused on understanding the genes that control migration, in the hope that insights into this key step in normal development. We use the migration of the cerebellar granule neuron as a model system to examine the molecular control of glial-guided neuronal migration. The establishment of neuronal polarity is a key step in initiating neuronal migration along the glial guide. Screens for genes that function in granule neuron migration revealed high levels of expression of the polarity signaling complex mPar6a neurons exiting the cycle and establishing polarity. In C. elegans, a set of 6 PAR proteins establish anterior/posterior asymmetries and control subsequent asymmetric cell divisions. PAR proteins are conserved throughout evolution. In Preliminary Studies (Solecki et al, 2004), we discovered that the mPar6a signaling complex is localized in the centrosome of migrating cerebellar granule neurons, where it coordinates the movement of the centrosome and the nucleus as the neuron migrates along the glial fiber. In the proposed research, we will study the other components of the mPar6a complex, aPKCzeta and Par3, in the polarity of migrating granule neurons. The role of mPar6a in cell division suggests that targeted loss of function mutants and shRNA experiments will not be feasible. We will therefore use a novel method developed by Roger Tsien to incorporate a genetic tag (TC) which binds the dye ReAshS, into mParGq and use chromophore-assisted light inactivation with a monochromatic laser to inactivate mParGa in the centrosome. For those experiments, we will generate TC-mPar6a BAC transgenic mice, enabling studies on granule cells and cortical neurons. In a final group of experiments, we will study a receptor/ligand system expressed in granule cells which interacts with the mParGa complex, the EphB ligands ephrin-B1 and ephrin-B2. Together, these experiments will provide novel information on the regulation of neuronal migration in cortical regions of developing brain.