Molecular regulation of dendrite morphogenesis

描述（由申请人提供）：感觉神经元利用复杂的树突状突起局部网络来检测外部刺激。试图解释这些复杂结构的模型必须包括控制分支起始、延伸和终止等关键事件的机制。这些特征在脊椎动物和无脊椎动物系统中普遍观察到，因此可能受进化上保守的成分支配。简单模式生物C. elegans，显示了一对PVD伤害感受神经元，其用网状阵列的树突状分支包裹动物。我们使用延时成像来显示每个分支所占据的离散局部区域由接触依赖性机制定义，其中姐妹树突（即，来自同一神经元的树突）彼此排斥以阻止生长。在哺乳动物和昆虫中也观察到“自我回避”，但对这种基本模式事件的分子基础知之甚少。我们的工作揭示了一种新的机制，其中可扩散的提示信号β 4 -6/Netrin在PVD树突的尖端被捕获，以介导涉及受体β 4 -40/DCC和β 4 -5的途径中的自我回避。这一发现意义重大，因为它描述了这些高度保守的蛋白质在树突自我回避中的作用的第一个例子。现在，我们已经扩展了这些发现，以确定β-6/Netrin自我回避通路的多个下游组件。在这些新结果的基础上，我们提出，β-6/Netrin触发肌动蛋白丝生长在接触姐妹树突的尖端，从事非肌肉肌球蛋白马达，驱动回缩。具体目标1中描述的实验利用TIRF显微镜在活生物体中的新应用来测试该模型。这些研究是有意义的，因为我们对细胞膜上的信号如何在自我回避过程中触发树突退缩知之甚少。我们的工作揭示了保守的膜蛋白tomoregulin在自我回避中的关键作用。具体目标2将定义这种作用的机制，并确定tomoregulin是否是其他已知的短距离β- 6/Netrin信号传导事件所必需的。目标2是重要的，因为它解决了一个基本问题，即β-6/Netrin通路如何独特地适应接触依赖性自我回避。为了解决树突状生长的机制，我们利用强大的细胞特异性分析方法来识别树突状细胞的靶点。 保守的LIM同源结构域转录因子MEC-3，其是PVD分支所需的。具体目标3将测试基于这些结果的模型，即树突状分支通过以下方式稳定： 与相邻表皮中的密蛋白样蛋白和其他特定细胞表面组分的相互作用。这些实验很重要，因为感觉神经元的生长通常是在与表皮组织密切接触的情况下进行的，但在这个位置形成树突状结构的细胞间机制却定义不清。这项工作在C。elegans被期望鉴定出在人类神经系统中也指定树突结构的关键决定因素。

DESCRIPTION (provided by applicant): Sensory neurons utilize complex, topical networks of dendritic processes to detect external stimuli. Models that seek to explain the creation of these elaborate structures must include mechanisms that control the key events of branch initiation, elongation and termination. These features are universally observed in both vertebrate and invertebrate systems and are therefore likely governed by evolutionarily conserved components. The simple model organism, C. elegans, displays a single pair of PVD nociceptive neurons that envelop the animal with a net-like array of dendritic branches. We used time-lapse imaging to show that the discrete topical region occupied by each branch is defined by a contact-dependent mechanism in which sister dendrites (i.e., dendrites from the same neuron) repel each other to stop outgrowth. "Self-avoidance" is also observed in mammals and insects but the molecular underpinning of this fundamental patterning event is poorly understood. Our work has revealed a novel mechanism in which the diffusible cue UNC-6/Netrin is captured at the tips of PVD dendrites to mediate self-avoidance in a pathway involving the receptors UNC-40/DCC and UNC-5. This discovery is significant because it describes the first example of a role for these highly conserved proteins in dendrite self-avoidance. Now, we have extended these findings to identify multiple downstream components of the UNC-6/Netrin self-avoidance pathway. On the basis of these new results, we propose that UNC-6/Netrin triggers actin filament growth at the tips of contacting sister dendrites to engage a non-muscle myosin motor that drives retraction. Experiments described in Specific Aim 1 exploit the novel application of TIRF microscopy to a living organism to test this model. These studies are significant because little is known of how signals at the cell membrane trigger dendrite withdrawal during self-avoidance. Our work has uncovered a key role for a conserved membrane protein, tomoregulin, in self-avoidance. Specific Aim 2 will define the mechanism of this effect and determine if tomoregulin is necessary for other known short-range UNC- 6/Netrin signaling events. Aim 2 is significant because it addresses the fundamental question of how the UNC-6/Netrin pathway has been uniquely adapted for contact-dependent self-avoidance. To address the mechanism of dendritic outgrowth, we exploited powerful cell-specific profiling methods to identify targets of a conserved LIM-homeodomain transcription factor, MEC-3, that is required for PVD branching. Specific Aim 3 will test a model, based on these results, that dendritic branches are stabilized by interaction with claudin-like proteins and other specific cell-surface components in the adjacent epidermis. These experiments are important because sensory neuron outgrowth is typically executed in close contact with epidermal tissue but the intercellular mechanisms that pattern dendritic architecture in this location are poorly defined. This work in C. elegans is expected to identify key determinants that also specify dendritic architecture in the human nervous system.