Mechanisms of Corneal Development and Wound Healing
Lwigale, Peter Y.
Doctor of Philosophy
Deep corneal injuries are one of the major causes of vision impairment and blindness. The shortage in available donor corneas has prompted researchers to look into developing alternative means of treating corneal injuries. Previously, our lab demonstrated that embryonic cornea heals scar-free. The purpose of this thesis work is to elucidate molecular pathways and signals during corneal development and to understand how the embryonic cornea heals. My current research uses the chick as a model organism to study embryonic cornea. During ocular development, periocular neural crest cells (pNC) migrate into the region between the lens and presumptive corneal epithelium to form the corneal endothelium and stromal keratocytes. Although multiple ocular dysgeneses are associated with defects in neural crest cell development, very little is known about the molecular mechanisms behind the process. To investigate the role of the environment in formation of the corneal endothelium and the stroma, I introduce a model for studying pNC development in the ocular environment. I demonstrate that upon orthotopic injection of pNC into the periocular region the cells timely migrate and differentiate into multiple ocular derivatives confirming that pNC are multipotent. Using this model, I show that the presumptive cornea is conducive to endothelial differentiation between embryonic day (E) 3-E7. At the same time, the periocular region does not induce corneal migration of the heterotopically injected pNC at E5 suggesting environmental changes in favor of an alternative ocular fate. I further demonstrate that corneal stroma at E7 may have the potential to induce pNC-to-keratocyte differentiation on the spot. To investigate specific signals required for pNC differentiation I focus on the corneal endothelium, which is the first corneal layer to be specified from the pNC in both avians,and humans. I find that the nascent cornea is competent to induce differentiation of ectopically injected pNC into corneal endothelium. Injected pNC downregulate the expression of multipotency transcription factors and upregulate endothelium-related genes. I show that TGFβ2 signaling in the nascent corneal environment plays a critical role in changing the molecular signature of pNC during the formation of the corneal endothelium. Additionally, proper arrangement of the collagen and the proteoglycans in the stromal extracellular matrix (ECM) is essential for functional transparency and light refraction of the cornea. Here, I present that the cornea is able to recapitulate its three-dimensional collagen structure during wound healing. The results of the presented work shed light on the complex molecular network involved in pNC differentiation in the eye and on the process of embryonic wound healing. Together, these findings could aid the development of alternative treatments for corneal injuries and disorders.
Cornea; Development; Wound Healing; Periocular Neural Crest; Endothelium