Pathfinding and target recognition by cortical axons

我们建议研究皮层第5层和第6层传入脑干和脊髓的不同发育阶段的控制机制，包括它们的寻路和分子控制它们对目标的识别和神经支配。我们将检验这一假说，即皮层的主要进出通道由皮层底板轴突通过这一通道。这将通过制造转基因小鼠来实现，这些转基因小鼠具有发育不良的亚板轴突，或者完全缺乏亚板神经元，并通过分析无法形成丘脑皮质投影的Mash-1突变小鼠来实现。为了验证轴突化学引诱剂Netrin-1在分子上定义了第5层皮质脊髓轴突穿过前脑、中脑和后脑的皮层下通路这一观点，我们将把这一轴突通路与Netrin-1的表达模式联系起来，并分析缺乏Netrin-1或其受体DCC的突变小鼠皮质脊髓轴突异常寻路的原因。其余的目标集中在皮质脊髓轴突的目标识别，其特征是沿轴突轴的分支重新形成并定向生长到目标。我们将研究BMP-3和一种具有6个Ig结构域和一个MAM结构域的新型分泌cam样蛋白（暂时命名为“Ig6M”）的作用，这些蛋白是用差异显示PCR筛选分离出来的，以确定皮质脊髓轴突的候选靶识别分子。我们将使用体外轴突引导和分支分析，以及小鼠功能获得和功能丧失基因实验，来确定BMP-3和Ig6M在控制皮质脊髓轴突分支的形成和定向生长中的充足性和必要性。

We propose to study the mechanisms that control distinct phases of the development of cortical layer 5 and layer 6 efferent projections to the brainstem and spinal cord, including their pathfinding and the molecular control of their recognition and innervation of targets. We will test the hypothesis that the pioneering of the major path in and out of the cortex by cortical subplate axons through this path. This will be done by creating transgenic mice engineered to have stunted subplate axons, or lack subplate neurons altogether, and by analyzing Mash-1 mutant mice that fail to form a thalamocortical projection. To test the idea that the axonal chemoattractant, Netrin-1, molecularly defines the subcortical path of layer 5 corticospinal axons through the forebrain, midbrain and hindbrain, we will correlate this axonal path with the pattern of Netrin-1 expression, and will analyze mutant mice deficient for Netrin-1 or its receptor, DCC, for aberrant pathfinding by corticospinal axons. The remaining aims focus on target recognition by corticospinal axons, which is characterized by a de novo formation of branches along the axon shaft and their directed growth into targets. We will study the roles of BMP-3 and a novel secreted CAM-like protein with 6 Ig domains and a MAM domain (temporarily named "Ig6M"), which were isolated using a differential display PCR screen to identify candidate target recognition molecules for corticospinal axons. We will use in vitro axon guidance and branching assays, as well as gain-of-function and loss-of-function genetic experiments in mice, to determine the sufficiency and requirement of BMP-3 and Ig6M in controlling the formation and directed growth of corticospinal axon branches.