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

与眼周神经脊细胞(PNC)发育相关的基因突变导致眼部缺陷 和失明。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不是很清楚。的目标是 这一建议是为了提供在PNC不同阶段差异表达的完整基因图谱 并建立参与这一过程的基因通路。具体来说，我们会：(1) 寻找与PNC向角膜内皮和角膜基质细胞分化有关的新基因；(2)建立 晶状体在诱导角膜基因的PNC表达中的作用；以及(3)决定是否 在角膜基质的PNC再生过程中，发育基因的表达被重述。要测试 PNC向角膜细胞分化受临界点时空表达调控的假说 对来自周围眼睛组织的图案信号做出反应的基因，我们将利用 小鼠和小鸡角膜发育的差异及差异基因的筛选 在PNC分化为角膜内皮细胞和角质形成细胞的过程中表达。这些屏幕将提供 可进一步归类为代表角膜内皮和 角质形成细胞形成。有希望的基因的表达将通过眼睛的原位杂交进行验证 眼睛发育的关键阶段。由于来自晶状体的信号在角膜发育中起着关键作用，我们 将通过进行晶状体消融和体外联合实验确定由晶状体诱导的一组角膜基因。 含PNC的晶状体囊泡培养。基因诱导的分析将通过常规的聚合酶链式反应、定量聚合酶链式反应、 原位杂交和免疫组织化学。分离的PNC的干细胞潜能将由以下因素决定 雏鸡角膜基质内移植鹌鹑细胞的研究。将移植的PNC分化为角质形成细胞 在宿主角膜和分离的角质形成细胞中使用鹌鹑特异性核抗体(QCPN)和 角质形成细胞标志物。具体目标是：(1)确定神经峰期的基因表达谱 分化为角膜内皮细胞和角质形成细胞。(2)确定晶状体对角膜的影响 基因表达。(3)检测移植的神经脊细胞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对内皮细胞承诺的基因 和角质形成细胞谱系，并提供了对参与角膜分子通路的新见解 发展和再生。更好地了解这些途径不仅对 眼前段发育不全(ASD)患者的管理，也是为了再生的发展 眼角膜病变和损伤的治疗。

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.