Molecular regulation of dendrite morphogenesis

描述(申请人提供)：感觉神经元利用复杂的局部树突网络来检测外部刺激。试图解释这些复杂结构的创建的模型必须包括控制分支起始、伸长和终止的关键事件的机制。这些特征在脊椎动物和无脊椎动物系统中都能普遍观察到，因此很可能是由进化上保守的成分控制的。这种简单的模式生物--秀丽线虫--展示了一对PVD伤害性感受性神经元，这些神经元用网状的树突分支阵列包裹着动物。我们使用时间推移成像来表明，每个分支占据的离散局部区域是由接触依赖机制定义的，在这种机制中，姐妹树突(即来自同一神经元的树突)相互排斥以阻止生长。“自我回避”在哺乳动物和昆虫中也被观察到，但这一基本模式事件的分子基础却鲜为人知。我们的工作揭示了一种新的机制，其中可扩散的提示UNC-6/Netrin被捕获在PVD树突的顶端，以在涉及受体UNC-40/DCC和UNC-5的途径中介导自我回避。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发挥作用的第一个例子。现在，我们已经将这些发现扩展到确定UNC-6/Netrin自我避免途径的多个下游组件。在这些新结果的基础上，我们认为UNC-6/Netrin在接触姐妹树突的顶端触发肌动蛋白细丝生长，从而参与驱动收缩的非肌肉肌球蛋白马达。具体目标1中描述的实验探索了TIRF显微镜在活体上的新应用，以测试这一模型。这些研究意义重大，因为人们对细胞膜上的信号如何在自我回避过程中触发树突撤回知之甚少。我们的工作揭示了一种保守的膜蛋白TomoRegin在自我回避中的关键作用。具体目标2将定义这种效应的机制，并确定TomoRegin是否对其他已知的短程UNC-6/Netrin信号事件是必需的。目标2具有重要意义，因为它解决了UNC-6/Netrin途径如何独特地适应接触依赖型自我回避这一根本问题。为了解决树突状突起的生长机制，我们利用了强大的细胞特异性图谱方法来识别 保守的LIM同源结构域转录因子MEC-3，是PVD分支所必需的。基于这些结果，《特指目标3》将测试一个模型，即树突分支通过以下方式稳定 与邻近表皮中的棒状蛋白和其他特定的细胞表面成分相互作用。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结构的细胞间机制尚不清楚。这项在线虫身上的工作有望确定关键决定因素，这些因素也指定了人类神经系统中的树突结构。

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.