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过程中角膜及其神经的生物学，并长期打算应用从这些研究中获得的知识来设计修复成人受损角膜神经的策略。角膜神经起感觉作用，并提供关键的神经营养因子，促进损伤后的组织维护和修复。了解影响角膜神经支配的分子因素具有直接的医学意义，原因有两个：1)在人类发育过程中，角膜神经支配不当与先天性眼病有关。2)在糖尿病、隐形眼镜使用、角膜移植和LASIK相关的知觉干眼中，角膜神经的变化或显著减少已被描述。由于移植或LASIK手术造成的角膜神经损伤，修复速度很慢或无法修复。因此，目前有大量高危人群患有角膜神经受损的疾病。了解指导三叉神经节(TG)来源的轴突进入角膜并在角膜内分支的指导分子将有助于确定相同的指导机制是否在先天性眼病中被破坏，以及这些指导分子是否可以被临床医生用于治疗眼睛问题和随后损害角膜神经的手术。在脊椎动物系统中，Robo-Sit信号通路被广泛用于引导感觉神经轴突到达其靶组织。在这里，我们将开始研究Robo-Sit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Sit和Robo蛋白的基因表达数据。功能研究将通过抑制Robo-Sit信号并利用器官培养和卵子电穿孔来研究发育后果，以便在活的器官和发育中的鸡身上研究这一途径。这些研究将有助于确定该通路在角膜神经支配中的整体功能，以及在发育过程中需要ROBO和SILT相互作用的组织。在这个提案中，Robo-Slip信号参与了角膜神经支配的多个阶段的假设将受到以下特定目标的检验：(1)表征来自角膜和晶状体的Robo-Sit信号在角膜神经支配过程中的轴突引导潜力，(2)阐明在体内机器人对角膜神经支配的功能要求，(3)确定神经排斥剂Slit2是否在角膜中被切割为神经诱导剂Slit2N，以及这两个狭缝变体是否对TG衍生的神经元具有不同的轴突引导潜力。

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.