Dysgenesis of the anterior eye together with corneal diseases and injuries are major causes of ocular defects and loss of vision. The periocular neural crest cells (pNC) are multipotent embryonic cell population that provide crucial signals and contribute to the cellular and extracellular components of the cornea, but the molecular mechanisms underlying these processes are still not well understood. This project seeks to understand the mechanisms by which two recently identified pNC signals Cxcl12 and Npnt respectively regulate ocular vasculogenesis, and corneal development, homeostasis and wound healing. Specifically, we will: (1) Determine the role of Cxcl12/Cxcr4 signaling during development of the periocular vascular network and corneal innervation; (2) Define the function of Nephronectin (Npnt) during corneal development and in the adult cornea. (3) Determine the function of Npnt during embryonic and adult corneal wound healing. Previously, we showed that the presumptive cornea is never vascularized during development and identified several pro-and anti-angiogenic factors in the anterior eye. We showed that Semaphorin3A, PlexinD1, and Cxcl14 functioned at different spatiotemporal levels to inhibit vascular endothelial growth factor (VEGF) signaling, which is crucial for the formation of the limbal vasculature, from functioning in the cornea at different stages of development. Our ongoing studies show that despite the robust expression of VEGF in the anterior eye, vasculogenesis only occurs in the region of Cxcl12 expression, and that endothelial cells express the Cxcr4 receptor. In addition, we identified novel expression of an extracellular matrix protein, Npnt, during corneal development, and it is maintained in the Descemet's membrane in the adult cornea. We also developed a technique to access late-stage chick embryos in ovo, and showed for the first time that wounded embryonic corneas heal scar-free. Proposed experiments will take advantage of the high manipulability of chick embryos combined with the powerful mouse genetics to identify the function of Cxcl12 and Npnt during ocular development. Ocular studies in chick include loss- and gain-of- function experiments utilizing viral constructs to overexpress shRNA and genes of interest. Analysis of Cxcr4 and Npnt knockout mice will indicate conservation of gene function. We will characterize the expression of Npnt in human corneas and examine its potential function using in vitro culture of corneal cells. All proposed studies are supplemented with micromanipulation of embryonic eyes, in vitro culture and molecular biology techniques, and pharmacological inhibition of gene function. The proposed studies will reveal novel functions of Cxcl12 and Npnt in cellular processes required for normal development, function, and repair of the cornea. Successful completion of these studies will broaden our understanding of the molecular mechanisms involved in neurovascular development and demonstrate for the first time that Npnt is required for proper corneal development, homeostasis, and wound healing.
Congenital defects during corneal development and defects caused by disease and trauma are associated with morbidity and deficiencies that compromise vision or result in blindness. Little is known about the molecular mechanisms underlying these defects. The proposed studies are founded on novel observations during ocular development and aim to elucidate the mechanisms underlying cell migration, proliferation, and differentiation during ocular development and wound healing.
