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

分析角膜发育过程中参与神经嵴细胞命运决定的基因。

• 批准号: 9166279
• 负责人: Peter Y Lwigale
• 金额: $ 22.84万
• 依托单位: RICE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2016
• 资助国家: 美国
• 起止时间: 2016-08-01 至 2018-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9166279
• 关键词: Ablation; Adult; Affect; Anterior; Antibodies; Axenfeld-Rieger syndrome; Binding; Bioinformatics; Blindness; Cell Differentiation process; Cells; Coculture Techniques; Cornea; Corneal Endothelium; Corneal Opacity; Corneal Stroma; Cues; Data; Defect; Development; Developmental Gene; Disease; Embryo; Endothelial Cells; Environment; Etiology; Event; Eye; Eye Development; Gene Expression; Gene Expression Profile; Genes; Genetic Transcription; Goals; Immunohistochemistry; In Situ Hybridization; In Vitro; Irido-corneo-trabecular dysgenesis; Location; Molecular; Molecular Analysis; Molecular Profiling; Mus; Mutation; N-Cadherin; Natural regeneration; Neural Crest; Neural Crest Cell; Nuclear; Organism; Pathway interactions; Patients; Pattern; Play; Population; Process; Quail; Role; Series; Signal Transduction; Staging; Stem cells; Testing; Time; Tissues; Vesicle; base; blastomere structure; candidate marker; cell motility; cell type; corneal epithelium; differential expression; gene induction; in vitro Assay; in vivo; injured; insight; lens; malformation; migration; novel; regenerative therapy; response; spatiotemporal; transcriptome sequencing

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.

参与眼周神经rest细胞（PNC）发育的基因突变导致眼部缺陷 和视力丧失。尽管PNC对角膜有很多贡献，但分子机制和 其分化为角膜细胞的发展线索仍未得到充分了解。目标 该建议是提供在PNC各个阶段差异表达的基因的完整概况 对角膜的贡献，并建立涉及此过程的基因途径。具体来说，我们将：（1） 鉴定与PNC分化为角膜内皮和角膜细胞的新型基因； （2）建立 镜头参与角膜基因的PNC表达； （3）确定是否 在角膜基质的PNC再生过程中，概括了发育基因的表达。测试 假设PNC分化为角膜细胞，由临界集的时空表达调节 基因响应于周围眼组织的模式信号，我们将利用 小鼠和雏鸡角膜发育与筛选的差异是差异的基因 在PNC分化为角膜内皮和角膜细胞时表达。这些屏幕将提供 可以进一步分为代表角膜内皮和的途径的全面基因清单 角膜细胞形成。有望基因的表达将通过在现场杂交的眼睛验证 眼部发展的关键阶段。由于镜头的信号在角膜发育中起着至关重要的作用，我们 将通过晶状体消融和体外共同体来确定晶状体诱导的角膜基因集 用PNC的晶状体培养。基因诱导的分析将通过常规PCR，qPCR进行检查 原位杂交和免疫组织化学。分离的PNC的干细胞电位将由 将鹌鹑细胞嫁接到雏鸡角膜基质中。将移植的PNC分化为角膜细胞将是 使用鹌鹑特异性核抗体（QCPN）在宿主角膜和孤立的角膜细胞中评估 角膜细胞标记。具体目的是：（1）识别神经rest期间的基因表达谱 分化为角膜内皮和角膜细胞。 （2）确定镜头对角膜的影响 基因表达。 （3）检查移植的神经rest细胞的干细胞电位和基因表达谱 进入角膜基质。拟议的研究将揭示驱动PNC承诺的基因 和角膜细胞谱系，并为参与角膜的分子途径提供新颖的见解 发展与再生。对这些途径的更好理解不仅对 治疗前部段失调（ASD）的患者，也用于发展再生 治疗患病和受伤的角膜。