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.
The cornea is the most densely innervated tissue of the body, meaning that any physical trauma or medical treatment of the cornea, elective or required, that damages these nerves has the potential for producing great pain, reducing the eye's ability to heal itself and disrupting the natural biology and health of the eye surface. While many medical advances have been made in healing corneal trauma and in corrective surgeries, little is known concerning the mechanisms of corneal innervation or how to repair corneal nerves once they become damaged. The aims proposed in this study will fill in substantial knowledge gaps regarding the nature of cornea innervation and also show the potential for the Slit family of nerve guidance molecules to be utilized by scientists and clinicians in remedying ocular problems involving impaired or damaged corneal nerves.
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