Mutations in genes that are involved in periocular neural crest cell (PNC) development cause ocular defects and loss of vision. Despite the abundant contribution of PNC to the cornea, the molecular mechanisms and developmental cues underlying their differentiation into corneal cells are still not well understood. The goal of this proposal is to provide a complete profile of genes that are differentially expressed at various stages of PNC contribution to the cornea, and to establish the gene pathways involved in this process. Specifically, we will: (1) identify novel genes involved in PNC differentiation into corneal endothelium and keratocytes; (2) establish the lens' involvement in the induction of PNC expression of corneal genes; and (3) determine whether the expression of developmental genes is recapitulated during PNC regeneration of the corneal stroma. To test the hypothesis that PNC differentiation into corneal cells is regulated by spatiotemporal expression of a critical set of genes in response to patterning signals from surrounding ocular tissues, we will take advantage of the differences between mouse and chick corneal development and screen for genes that are differentially expressed during PNC differentiation into corneal endothelium and keratocytes. These screens will provide a comprehensive list of genes that can be further grouped into pathways that represent corneal endothelium and keratocyte formation. The expression of promising genes will be validated by in situ hybridization of eyes at critical stages of ocular development. Since signals from the lens play a critical role in corneal development, we will determine the set of corneal genes that are induced by the lens by performing lens ablation and in vitro co- culture of lens vesicle with PNC. Analysis of gene induction will be examined by conventional PCR, qPCR, in situ hybridization, and immunohistochemistry. The stem cell potential of isolated PNC will be determined by grafting of quail cells into chick corneal stroma. Differentiation of grafted PNC into keratocytes will be evaluated in host corneas and in isolated keratocytes using a quail-specific nuclear antibody (QCPN) and keratocyte markers.
The Specific Aims are: (1) To identify the gene expression profile during neural crest differentiation into corneal endothelium and keratocytes. (2) To determine the effect of the lens on corneal gene expression. (3) To examine the stem cell potential and gene expression profile of neural crest cells grafted into the corneal stroma. The proposed studies will reveal the genes that drive PNC commitment to endothelial and keratocyte lineages and provide novel insights into the molecular pathways involved in corneal development and regeneration. A better understanding of these pathways is important not only for the management of patients with anterior segment dysgenesis (ASD), but also for the development of regenerative therapy for diseased and injured corneas.
Neural crest cells are stem cell-like embryonic cell population that give rise to multiple derivatives including the corneal endothelium and stromal keratocytes, which comprise more than 90% of the adult cornea. Mutations in genes that affect neural crest cell migration, proliferation, and differentiation cause defects such as Peters anomaly and Axenfeld-Rieger syndrome that affect genesis and function of the cornea. The proposed study will generate the expression profile of genes involved in neural crest differentiation into corneal cells and determine how environmental cues from the lens vesicle and corneal stroma influence their expression.