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角膜的生物学及其在胚胎发育期间的神经支配，长期意图是应用从这些研究中获得的知识来设计导致修复成人受损角膜神经的策略。角膜神经起感觉作用，并且还递送促进损伤后的组织维持和修复的关键神经营养因子。了解影响角膜神经支配的分子因素具有直接的医学重要性，原因有两个：1）人类发育期间角膜的神经支配不当与先天性眼病有关。2)在与糖尿病、接触透镜使用、角膜移植和LASIK相关的干眼症中描述了角膜神经支配的改变或显著减少。角膜中因移植或LASIK手术而受损的神经修复缓慢或无法修复。因此，目前有大量的人处于角膜神经受损的疾病的风险中。了解指导三叉神经节（TG）衍生的轴突进入角膜并在角膜内形成分支的指导分子将有助于确定在先天性眼病中是否破坏相同的指导机制，以及这些指导分子是否可被临床医生用于治疗眼部问题和损伤角膜神经的手术。Robo-Slit信号通路广泛用于脊椎动物系统，以引导感觉轴突到达其靶组织。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。将使用原位杂交收集相关Slit和Robo蛋白的基因表达数据。将通过抑制Robo-Slit信号传导和使用器官培养物和卵内电穿孔研究发育后果来进行功能研究，以便可以在活器官和发育中的鸡中研究该途径。这些研究将有助于确定角膜神经支配途径的整体功能，此外还将阐明分泌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。在本提案中，将使用以下特定目标测试Robo-Slit信号传导参与角膜神经支配的多个阶段的假设：（1）表征在角膜神经支配期间来自角膜和透镜的Robo-Slit信号传导的轴突引导潜力，（2）阐明Robos对角膜神经支配的体内功能要求，（3）确定神经排斥性Slit 2是否在角膜中裂解为神经吸引性Slit 2N，以及两种Slit变体是否对TG衍生的神经元具有不同的轴突导向潜力。

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.