Robo-Slit Signaling in Avian Cornea Innervation

描述（由申请人提供）：拟议研究的近期目标是更好地了解胚胎发育期间角膜及其神经支配的生物学，并具有应用从这些研究中获得的知识来制定导致修复成人受损角膜神经的策略的长期意图。角膜神经具有感觉功能，并提供重要的神经营养物质，促进组织的维护和损伤后的修复。了解影响角膜神经支配的分子因素具有迫切的医学意义，原因有两个：1)人类发育过程中角膜神经支配不当与先天性眼部疾病有关。2)在与糖尿病、隐形眼镜使用、角膜移植和LASIK相关的干眼症中，角膜神经支配的改变或显著减少已被描述。由于角膜移植或LASIK手术造成的角膜神经损伤修复缓慢或无法修复。因此，目前有大量角膜神经受损的高危人群。了解引导三叉神经节（TG）衍生轴突进入角膜并在角膜内分支的引导分子，将有助于确定先天性眼病中相同的引导机制是否被破坏，以及这些引导分子是否可以被临床医生用于治疗眼部问题和角膜神经损伤手术后的治疗。robot - slit信号通路在脊椎动物系统中广泛用于引导感觉轴突到达目标组织。在此，我们将开始研究Robo-Slit信号通路在鸟类角膜神经支配中的活性。将利用原位杂交技术收集相关的Slit和Robo蛋白的基因表达数据。功能研究将通过抑制Robo-Slit信号和利用器官培养和卵内电穿孔研究发育后果来进行，以便在活体器官和发育中的鸡中研究该途径。这些研究将有助于确定该通路对角膜神经支配的整体功能，此外还有助于阐明分泌Slits所需的组织以及在发育过程中何时需要Robo和Slit相互作用。本研究将对Robo-Slit信号参与角膜神经支配多个阶段的假说进行验证，具体目的如下：(1)表征来自角膜和晶状体的Robos -Slit信号在角膜神经支配过程中的轴突引导电位；(2)阐明Robos对角膜神经支配的体内功能需求；(3)确定角膜中神经排斥剂Slit2是否与神经引诱剂Slit2N相结合，以及这两种Slit变体对tg源性神经元的轴突引导电位是否不同。

DESCRIPTION (provided by applicant): The immediate goal of the proposed research is to better understand the biology of the cornea and its innervations during embryonic development, with a long-range intention of applying the acquired knowledge gained from these studies to devise strategies that lead to repairing damaged corneal nerves in adults. Corneal nerves serve a sensory role, and also deliver critical neurotrophins that promote tissue maintenance and repair following injury. Understanding the molecular factors influencing cornea innervation is of immediate medical importance for two reasons: 1) Improper innervation of the cornea during human development is associated with congenital eye disorders. 2) Alterations, or significant reductions, in corneal innervation have been described in perceived Dry Eye associated with diabetes mellitus, contact lens use, cornea transplantation and LASIK. Damaged nerves in the cornea resulting from transplantation or LASIK surgery are slow to repair or fail to do. Thus, there is currently a large population of at-risk humans for disorders where corneal nerves are impaired. Understanding the guidance molecules that instruct trigeminal ganglion (TG)-derived axons to enter and branch within the cornea will help to determine if the same guidance mechanisms are disrupted in congenital eye disorders, and if these guidance molecules can be utilized by clinicians to remedy ocular problems and following surgeries that damage corneal nerves. The Robo-Slit signaling pathway is widely used in vertebrate systems to guide sensory axons to their target tissues. Here, we will begin to study the activity of the Robo-Slit signaling pathway on avian cornea innervation. Gene expression data will be gathered for relevant Slit and Robo proteins using in situ hybridization. Functional studies will be performed by inhibiting Robo-Slit signaling and studying the developmental consequences using organ cultures and in ovo electroporation so that the pathway may be studied in living organs and developing chickens. These studies will help to determine the overall function of the pathway on cornea innervation, in addition to elucidating the tissues that are required to secrete Slits and when during development Robo and Slit interactions are required. In this proposal the hypothesis that Robo-Slit signaling is involved in multiple stages of cornea innervation will be tested with the following Specific Aims: (1) Characterize the axon guidance potential of Robo-Slit signaling from the cornea and lens during cornea innervation, (2) Elucidate the in vivo functional requirement of Robos on cornea innervation, (3) Determine if neuro-repellant Slit2 is cleaved to neuro-attractant Slit2N in the cornea and whether the two Slit variants have different axon-guidance potentials on TG-derived neurons.