Peripheral territory selection by zebrafish trigeminal sensory neurons

描述（由申请人提供）：本提案侧重于躯体感觉神经元选择其周围乔木区域的机制。面部的触觉是由三叉神经感觉神经元完成的。每个三叉神经都投射出一个外周轴突，在头部的离散部分形成一个错综复杂的分支。所有三叉神经的树突一起用感觉纤维网络覆盖了整个表皮。在之前的工作中，我们以斑马鱼三叉神经系统为模型，研究了三叉神经如何相互协调以形成均匀而全面的乔木排列。利用成像和胚胎学方法，我们证明了生长乔木之间的排斥相互作用限制了它们的领土，并确保了头部表皮的全面神经支配。我们通过行为实验证明，去除这些排斥约束会破坏乔木区域地图，并损害动物在环境中定位刺激的能力。在人类中，由于发育或损伤导致的三叉神经支配区域排列缺陷也可导致触觉缺陷，包括疼痛性神经病变。在此，我们建议进一步利用斑马鱼三叉神经系统的清晰度、可及性和分子可溯性来解决三叉神经神经元在发育期间和损伤后如何选择其外周区域的三个相关问题，强调排斥性相互作用在塑造树梢区域中的作用。首先，我们将探讨感觉神经元亚型是否使用独立的排斥系统来选择它们的领土，以及神经元形状和功能之间是否存在相关性。我们将通过创造转基因来独立监测活胚胎中每个三叉神经感觉亚型的发育和形态来实现这一目标。其次，我们将描述三叉神经在外周损伤后区域重建过程中所采用的细胞和分子策略。在这些实验中，我们将采用我们开发的一种新方法，用激光对三叉神经细胞进行精确的损伤。斑马鱼三叉神经的清晰性和可及性使我们能够以高分辨率实时跟踪单个受损三叉神经的再生。第三，我们将利用斑马鱼中可用的分子和基因组工具来识别在三叉神经中介导相互排斥的分子。我们将进行全面的表达筛选，以确定哪些基因是参与这一过程的最佳候选基因，并采用功能丧失方法来测试它们的功能。通过这些研究，我们不仅希望提供神经元协调选择其神经支配区域的基本机制的描述，而且希望揭示可能导致人类面部神经病变的三叉神经支配缺陷的原因。公共卫生相关性：外周触觉神经元的发育缺陷，或在以后的生活中对它们的损伤，可引发疼痛性神经病变。周围神经病变是一种常见且异质性的疾病，影响了约2000万美国人，可导致疼痛感增强且通常持续存在，但在细胞和分子水平上对其知之甚少。本研究旨在了解控制触觉神经元发育和修复的基本机制，我们相信这将最终为治疗周围神经病变患者提供更好的方法。

DESCRIPTION (provided by applicant): This proposal focuses on the mechanisms by which somatosensory neurons choose their peripheral arbor territories. Touch sensation in the face is accomplished by trigeminal sensory neurons. Each trigeminal neuron projects one peripheral axon that elaborates an intricately branched arbor in a discrete portion of the head. Together, the arbors of all trigeminal neurons blanket the entire epidermis with a network of sensory fibers. In previous work, we used the zebrafish trigeminal system as a model to investigate how trigeminal neurons coordinate with one another to create their even and comprehensive arbor arrangement. Using imaging and embryological approaches, we demonstrated that repulsive interactions between growing arbors limit their territories and ensure comprehensive innervation of the head epidermis. We showed with a behavioral experiment that removing these repulsive constraints disrupts the arbor territory map and impairs the ability of animals to locate stimuli in the environment. In humans, defects in the arrangement of trigeminal innervation territories that occur developmentally or as a result of injury can also lead to defects in touch sensation, including painful neuropathies. We propose here to take further advantage of the clarity, accessibility and molecular tractability of the zebrafish trigeminal system to address three related questions about how trigeminal neurons choose their peripheral territories during development and after injury, emphasizing the role of the repulsive interactions that shape arbor territories. First, we will explore whether subtypes of sensory neurons use independent repulsive systems to choose their territories and whether there is a correlation between neuronal shape and function. We will accomplish this by creating transgenes for independently monitoring the development and morphologies of each trigeminal sensory subtype in live embryos. Second, we will characterize the cellular and molecular strategies employed by trigeminal neurons during the re-establishment of territories after peripheral injury. For these experiments, we will employ a new method we have developed to inflict precise damage on trigeminal neurons with a laser. The clarity and accessibility of zebrafish trigeminal neurons allows us to follow the regeneration of single damaged trigeminal arbors at high-resolution in real time. Third, we will exploit the molecular and genomic tools available in zebrafish to identify the molecules that mediate mutual repulsion among trigeminal neurons. We will perform a comprehensive expression screen to determine which genes are the best candidates for participating in this process, and a loss-of-function approach to test their functions. With these studies, we hope not only to provide a description of the basic mechanisms used by neurons to coordinately choose their innervation territories, but also to shed light on the causes of trigeminal innervation defects that can contribute to facial neuropathies in humans. PUBLIC HEALTH RELEVANCE: Developmental defects in peripheral touch-sensing neurons, or damage to them later in life, can trigger painful neuropathies. Peripheral neuropathies are a common and heterogeneous collection of diseases, affecting some 20 million Americans, that can result in heightened and often persistent pain sensation, but very little is understood about them at the cellular and molecular level. The studies in this proposal aim at understanding the basic mechanisms that control the development and repair of touch-sensing neurons, which we believe will ultimately lead to better ways for treating patients who suffer from peripheral neuropathies.