Peripheral territory selection by zebrafish trigeminal sensory neurons

描述（由申请人提供）：该提案侧重于躯体感觉神经元选择其外周乔木领土的机制。面部的触觉是由三叉神经感觉神经元完成的。每个三叉神经元投射出一个外周轴突，这些轴突在头部的一个离散部分中形成一个错综复杂的分支。所有三叉神经元的主干一起用感觉纤维网络覆盖整个表皮。在以前的工作中，我们使用斑马鱼三叉神经系统作为模型来研究三叉神经元如何相互协调，以创建其均匀和全面的乔木排列。使用成像和胚胎学的方法，我们证明了排斥生长乔木之间的相互作用限制了他们的领土，并确保全面的神经支配的头部表皮。我们的行为实验表明，消除这些排斥性的限制扰乱了乔木领土地图，并损害了动物在环境中定位刺激的能力。在人类中，在发育过程中或由于损伤而发生的三叉神经支配区域的排列缺陷也可导致触觉缺陷，包括疼痛性神经病。我们建议进一步利用斑马鱼三叉神经系统的清晰度，可访问性和分子可处理性来解决三个相关的问题，即三叉神经元如何在发育过程中和损伤后选择其外周领土，强调形成乔木领土的排斥相互作用的作用。首先，我们将探讨感觉神经元的亚型是否使用独立的排斥系统来选择它们的领土，以及神经元的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，我们将描述外周损伤后三叉神经元在重建领土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，用激光对三叉神经元造成精确的损伤。斑马鱼三叉神经元的清晰度和可访问性使我们能够在真实的时间内以高分辨率跟踪单个受损三叉神经乔木的再生。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介导三叉神经元之间相互排斥的分子。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，并采用功能丧失的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，而且还希望阐明三叉神经支配缺陷的原因，这些缺陷可能导致人类面神经病变。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，可能引发疼痛性神经病。周围神经病变是一种常见的异质性疾病，影响约2000万美国人，可导致疼痛感增强且通常持续，但在细胞和分子水平上对其了解甚少。该提案中的研究旨在了解控制触觉神经元发育和修复的基本机制，我们相信这最终将为治疗周围神经病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.