Neural Crest and the Cornea: Transformation of the transcriptome and the role of Nephronectin during ocular development
Lwigale, Peter; Carson, Daniel
Doctor of Philosophy
Corneal development is an intricate process that involves signaling and interactions between the periocular neural crest cells (pNC)s, presumptive corneal epithelium, lens, and the extracellular matrix. Cross-talk between these divisions are critical in regulating the migration, proliferation, and differentiation of the periocular neural crest to form the cornea. Major signaling pathways (retinoic acid, TGFβ, and Wnt) and cell-matrix interactions are essential for corneal formation, but the mechanisms of how they influence pNC behavior are not well understood. In this study, I use RNA-sequencing to establish the transformation in the transcriptomic profile from the pNC to the formation of the cornea. With this data, I establish the pNC identity and analyze how differential regulation of the major signaling pathways regulate pNC behavior and contribute to the corneal cell fate. In addition, I take note of the expression of key extracellular matrix, matrix-remodeling, and cell-matrix receptor genes and link their differential regulation to essential corneal developmental events. Within this niche, I focused on studying the role of a particular strongly expressed matrix protein, nephronectin (Npnt). Npnt has been linked to many embryonic developmental roles including the kidney, bone, heart, tooth, and lungs. However, a role for it has yet to be described in the cornea. Using both the chick and mouse as my model organisms, I found that both Npnt and its cellular receptor integrin α8β1 were strongly expressed in the migrating pNC. This led to the hypothesis that Npnt/Itgα8β1 has a role in regulating pNC behavior during corneal development. Using functional studies by knocking down or overexpressing Npnt in the chick, I show that loss or gain of Npnt correlates to a decrease or increase of corneal thickness and cell counts, respectively. This result was further confirmed by knocking down Itga8 and see corresponding decrease in corneal thickness, suggesting that Npnt/Itgα8β1 has a role in cell migration and/or proliferation. In clarifying respective assays. I found that cell proliferation was not increased by overexpressing Npnt and that Npnt-mediated signaling, which is a stronger inducer of periocular neural crest migration in vitro, is abrogated by inhibition of Itgα8β1. Altogether, in this thesis, I analyzed the transcriptomic profile in the developing cornea and characterize a previously unknown ECM-receptor interaction (Npnt/Itgα8β1), which may be a novel regulator of pNC migration and corneal development. Identifying a functional role for Npnt also validates the potential of the transcriptomic study as a valuable resource to identify novel genes essential for corneal development and potential targets for corneal therapies.
Neural Crest Cell; Cornea; Nephronectin; integrins; ECM