Analysis of genes involved in neural crest cell fate decisions during corneal development.

参与眼周神经嵴细胞（PNC）发育的基因突变导致眼部缺陷 和视力丧失尽管PNC对角膜有丰富的贡献，但其分子机制和 它们分化成角膜细胞的发育线索仍然没有很好的理解。的目标 该建议提供了在PNC的不同阶段差异表达的基因的完整谱 这是一个重要的研究课题，目的是研究对角膜的贡献，并建立参与这一过程的基因通路。具体而言，我们将：（1） 鉴定参与PNC分化为角膜内皮细胞和角膜细胞的新基因;（2）建立PNC分化为角膜内皮细胞和角膜细胞的新基因。 透镜参与角膜基因的PNC表达的诱导;和（3）确定 发育基因的表达在角膜基质的PNC再生过程中重现。测试 PNC分化为角膜细胞的假说是由一个临界集的时空表达调控的 的基因响应模式信号从周围的眼组织，我们将利用的优势， 小鼠和鸡角膜发育之间的差异，并筛选差异 在PNC分化成角膜内皮和角膜细胞期间表达。这些屏幕将提供一个 可以进一步分组为代表角膜内皮的通路的基因的全面列表， 角膜细胞形成有希望的基因的表达将通过眼睛的原位杂交来验证， 眼睛发育的关键阶段。由于来自透镜的信号在角膜发育中起着关键作用，我们 将通过进行透镜消融和体外共刺激来确定由透镜诱导的一组角膜基因 用PNC培养透镜泡。基因诱导的分析将通过常规PCR、qPCR、 原位杂交和免疫组织化学。分离的PNC的干细胞潜能将通过以下测定： 将鹌鹑细胞移植到鸡角膜基质中。移植的PNC向角膜基质细胞的分化将是 使用鹌鹑特异性核抗体（QCPN）在宿主角膜和分离的角膜细胞中进行评价， 角膜细胞标志物。本研究的具体目的是：（1）确定神经嵴发育过程中的基因表达谱 分化成角膜内皮和角膜细胞。(2)确定透镜对角膜的影响 基因表达。(3)检测神经嵴细胞移植后的干细胞潜能和基因表达谱 进入角膜基质。拟议的研究将揭示驱动PNC向内皮细胞转化的基因。 和角膜细胞谱系，并提供了新的见解，参与角膜的分子途径， 发展和再生。更好地了解这些途径不仅对 治疗眼前节发育不全（ASD）患者，也可用于再生性 治疗患病和受伤的角膜。

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.