Rac GTPases in the Mammalian Brain Development

描述(由申请人提供)：Rac GTP酶在神经发育的许多方面都是重要的信号转导。过去对Rac GTP酶的研究主要是利用结构活性突变体或显性负性突变体的过度表达。这种方法不能区分rac1和其密切相关的亚型rac3，并且可能干扰其他Rho GTP酶的功能，如CDC42。为了克服这些障碍，我们采用了一种有条件的基因靶向策略，使用FOXG1-CRE小鼠来删除端脑祖细胞中的成簇的rac1或cdc42。这些研究揭示了rac1独特的和新的功能(见初步结果)。根据这些结果，我们推测，rac1在腹侧端脑前体细胞的增殖和分化中具有重要作用，它为中线连合束的形成传递Netrin/DCC介导的信号。此外，在轴突生长和脊柱形态发生方面，rac1可能与rac3具有关键但重叠的功能。我们将在三个具体目标上测试我们的假设。 在目标1中，我们将使用条件性rac1缺失、rac3缺失和rac1/rac3复合突变来研究前脑的发育。此外，我们还将利用神经球培养来研究RAC在神经前体细胞中的特定功能及其相关机制。在目标2中，我们将使用一系列的外植体和细胞培养试验来检验是否在netrin诱导的信号转导和生长锥功能中需要rac1。在目标3中，我们还将利用rac1/rac3复合突变胚胎和在rac3缺失的遗传背景中的rac1的出生后缺失来确定rac1和rac3是否共同调节轴突生长和树突棘的形态发生。 总而言之，这个项目将解决大脑发育的三个重要方面，在这些方面，RAC GTP酶要么具有初步结果所表明的新功能(目标1和2)，要么预期发挥关键作用并值得特别关注(目标3)。我们的目标是将功能特异性分析与对这些关键信号转导的机械理解联系起来。 公共卫生进展：RAC GTP酶在神经发育的许多方面都是重要的信号转导，但在以前的研究中，它们的生理功能可能被显性突变体的过度表达所掩盖。利用条件性基因打靶，首席研究人员发现了神经前体细胞中Rac GTP酶的新功能和Netrin介导的轴突引导。本项目将确定这些重要功能的机制，并进一步探索Rac GTP酶在轴突生长和树突棘形态发生中的作用。

DESCRIPTION (provided by applicant): Rac GTPases are important signal transducers in many aspects of neural development. Past studies of Rac GTPases mainly used over-expression of constitutive-active or dominant- negative mutants of Rac1. This approach cannot distinguish between Rac1 and its closely related isoform Rac3, and may interfere with the functions of other Rho GTPases such as Cdc42. To overcome these obstacles, we have employed a conditional gene-targeting strategy using Foxg1-Cre mice to delete the floxed Rac1 or Cdc42 in telencephalic progenitors. These studies have revealed unique as well as novel functions of Rac1 (see Preliminary Results). Based on these results, we hypothesize that Rac1 has important functions in the proliferation and differentiation of ventral telencephalic progenitors, and it transduces Netrin/DCC-mediated signal for the midline commissural tract formation. Moreover, Rac1 may have critical, but overlapping functions with Rac3 in axonal growth and spine morphogenesis. We will test our hypotheses in three specific aims. In Aim 1 we will use conditional Rac1-null, Rac3-null, and Rac1/Rac3 compound mutants to examine the development of the forebrain. In addition, we will use neurosphere cultures to study the specific functions of Rac in neural progenitors and the involved mechanisms. In Aim 2 we will use a series of explant and cell culture assays to examine whether Rac1 is needed for netrin-induced signal transduction and growth cone functions. In Aim 3 we will also use the Rac1/Rac3 compound mutant embryos and postnatal deletion of Rac1 in the Rac3-null genetic background to determine whether Rac1 and Rac3 collectively regulate axonal outgrowth and the dendritic spine morphogenesis. In summary, this project will address three important aspects of brain development in which Rac GTPases either have novel functions as indicated by preliminary results (Aim 1 & 2) or are expected to play a critical role and warrant special attention (Aim 3). Our goal is to bridge functional specificity analysis to mechanistic understanding of these critical signal transducers. PUBLIC HEALTH REVELANCE: Rac GTPases are important signal transducers in many aspects of neural development, but their physiologic functions could be masked in previous studies by over-expression of dominant mutants. Using conditional gene-targeting, the principal investigator has uncovered novel functions Rac GTPases in neural progenitors and the Netrin-mediated axon guidance. The present project will determine the mechanism of these important functions, and further explore the roles of Rac GTPases in axonal outgrowth and the dendritic spine morphogenesis.