Planar cell polarity control of axon guidance

项目摘要 背连合轴突穿过中线是脊椎动物和无脊椎动物神经系统的一个显著特征。 感觉和运动系统、运动和姿势的左右协调所必需的系统。在 脊椎动物脊髓后连合轴突向中线底板延伸并穿过中线底板，然后转向 纵向上升到大脑。虽然生长锥的航行和整个地板已经 经过深入研究，它的最终决定--从中线出来后是上升还是下降-- 很好理解。遗传研究清楚地表明，这一决定涉及平面细胞极性（PCP）途径，但 我们对PCP信号如何引导生长锥的理解是不完整的。PCP途径是一种细胞-细胞 接触介导的信号通路，在细胞之间传递极性信息，以定向它们 迁移然而，我们对PCP信号在连合轴突引导中的作用的机械理解是 这主要是由体外分离的生长锥的研究所提供的信息。因此，在我们的理解的一个主要差距， 连合轴突引导是细胞接触介导的线索在纵向引导中发挥的作用。使用 透明的斑马鱼胚胎，以可视化单个识别的先驱连合的轴突和生长锥 在PCP突变体的中间神经元中，我们发现PCP信号通路的核心成分是必需的， 同样地在连合神经元内和其环境中用于正确的轴突靶向。PCP蛋白定位 到生长锥和其轨迹上的细胞。我们假设生长锥利用PCP信号， 在其直接神经上皮环境中，其生长响应于平面极化线索。目标1 我们将通过在空间和时间上定位PCP核心部件的需求来检验这一假设， 生长锥环境，并通过生长锥膜和肌动蛋白动力学的定量实时成像， 正在做出前方瞄准的决定在目标2中，我们将扩大我们的范围，以发现连合轴突 通过靶向G 0 CRISPR在别处参与PCP信号传导的蛋白质的指导作用 屏幕最后，在目标3中，我们将再次扩大我们的范围，以测试PCP信号功能的假设 在脊髓中的纵向轴突引导中广泛地。这项工作的成功结果将是一个深刻的 背连合神经元生长锥是如何被极化为向前生长的机制的理解 在体内通过平面细胞极性途径，使其能够建立控制运动的感觉回路， 姿势

PROJECT SUMMARY Midline crossing by dorsal commissural axons is a prominent feature of vertebrate and invertebrate nervous systems, necessary for the left-right coordination of sensory and motor systems, locomotion, and posture. In the vertebrate spinal cord, dorsal commissural axons extend towards and cross the midline floorplate, and then turn longitudinally to ascend towards the brain. While the growth cone’s voyage to and across the floorplate has been intensively studied, its final decision—whether to ascend or descend after emerging from the midline—is less well understood. Genetic studies clearly implicate the Planar Cell Polarity (PCP) pathway in this decision, but our understanding of how PCP signaling guides the growth cone is incomplete. The PCP pathway is a cell-cell contact-mediated signaling pathway that transmits polarity information between cells to orient them for directed migration. Yet our mechanistic understanding of the role of PCP signaling in commissural axon guidance is largely informed by studies of isolated growth cones in vitro. Thus, A major gap in our understanding of commissural axon guidance is the role that cell contact-mediated cues play in longitudinal guidance. Using the transparent zebrafish embryo to visualize the axons and growth cones of single identified pioneer commissural interneurons in PCP mutants, we have found that core components of the PCP signaling pathway are required equally within the commissural neuron and in its environment for correct axon targeting. PCP proteins localize to the growth cone and to the cells on its trajectory. We hypothesize that the growth cone uses PCP signaling to polarize its growth in response to planar-polarized cues in its immediate neuroepithelial environment. In Aim 1 we will test this hypothesis by locating, in space and time, the requirement for PCP core components in the growth cone environment, and by quantitative live imaging of growth cone membrane and actin dynamics as it is making its anterior targeting decision. In Aim 2 we will expand our scope to discover the commissural axon guidance role of proteins that have been implicated in PCP signaling elsewhere through a targeted G0 CRISPR screen. Finally, in Aim 3 we will expand our scope once again to test the hypothesis that PCP signaling functions broadly in longitudinal axon guidance in the spinal cord. The successful outcome of this work will be a deep mechanistic understanding of how the dorsal commissural neuron growth cone is polarized for anterior growth in vivo by the Planar Cell Polarity pathway, to enable it to build sensory circuits controlling locomotion and posture.