Mechanisms of Embryonic Lens Determination

胚胎晶状体测定机制

• 批准号: 8622201
• 负责人: Robert M Grainger
• 金额: $ 37.87万
• 依托单位: UNIVERSITY OF VIRGINIA
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-03-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622201
• 关键词: Address; Affect; Animal Model; Aniridia; Appearance; Biological; Biological Assay; Candidate Disease Gene; Cells; ChIP-seq; Child; Chromatin; Collaborations; Commit; Complement; Crystalline Lens; Data; Defect; Development; Developmental Cell Biology; Developmental Gene; Disease; Down-Regulation; Ectoderm; Embryo; Embryonic Development; Enhancers; Epigenetic Process; Event; Eye; Gene Activation; Gene Expression; Gene Expression Regulation; Genes; Genetic; Genome; Genomics; Goals; Growth; Hereditary Disease; Histones; Homeobox Genes; Homologous Gene; Iris; Knowledge; Laboratories; Lead; Lesion; Link; Messenger RNA; Modeling; Modification; Mutation; Operative Surgical Procedures; Organ; Pathway interactions; Play; Process; Rana; Recording of previous events; Regenerative Medicine; Regulation; Regulator Genes; Regulatory Element; Resources; Role; Sampling; Signal Pathway; Signal Transduction; Staging; Stem cells; System; Testing; Time; Tissues; Transcriptional Regulation; Transgenic Organisms; Ursidae Family; Visual system structure; Work; Xenopus; Xenopus laevis; base; cell determination; eye formation; gene function; gene repression; genetic resource; histone modification; human disease; insight; lens; neural plate; novel; pluripotency; promoter; public health relevance; research study; response; signal processing; stem; stem cell biology; tool; transcriptome sequencing; xenopus development

DESCRIPTION (provided by applicant): The goal of this project is to clarify the mechanisms leading to the commitment, or determination, of particular regions of the embryo to form a tissue or organ. This proposal focuses on lens formation in the frog Xenopus. While a great deal of information has accrued about genes that are critical for organ formation, still very little is knon about the integrative mechanisms by which commitment occurs. In the case of the developing lens the long history of study, accessibility of presumptive lens ectoderm (PLE) at all stages, and the many technical advantages of the Xenopus system provide unique assets for addressing the question of embryonic determination. My laboratory has characterized the numerous steps in the lens commitment process and here are described experiments to distinguish the last steps in this process: specification and determination. The first aim of the project is to address the specific cellular mechanisms leading to determination, initially by examining the hypothesis that inhibition of responsiveness to the Wnt signaling pathway is responsible for determination. Examination of gene expression by RNA-Seq during commitment has revealed a number of candidate genes that could be key regulators of this process; for example, down regulation of Oct91, the Xenopus homologue of the stem cell gene Oct4, will be examined as a potential regulatory step in the determination process. The second aim is the development of an epigenetic resource to greatly enhance our studies of gene regulation by undertaking a set of ChIP-Seq analyses to identify key chromatin marks (e.g. histone modifications) associated with active enhancers in the PLE at three stages in the commitment process. These analyses will provide a key resource for identifying putative enhancers in regulatory genes and to assess the temporal association of the appearance of histone marks with gene activation and repression, and with the commitment process. The final aim is to examine the "spatial commitment" of a particular region of ectoderm toward lens formation at the neural plate stage. Four genes (pax6, six3, mab21l1 and mab21l2) have expression in a small zone defining the PLE region at this very early stage. Identification and analysis of the regulatory elements controlling this highly regulated expression (using information generated from the second aim) will provide new insights about how the spatial definition of the lens is established. Overall, this work will provide key information for understanding the ontogeny of the visual system. In addition it bears on important questions in stem cell biology which depend on understanding of the intimately related, but poorly understood mechanisms of commitment during early development. In turn this work bears on related issues of regenerative medicine involving cell fate reprogramming, and human diseases involving genetic lesions in key developmental genes, e.g. Aniridia in the case of eye formation.

描述（由申请人提供）：该项目的目的是阐明导致胚胎特定区域的承诺或确定的机制，以形成组织或器官。该提案着重于青蛙爪蟾中的晶状体形成。尽管有关器官形成至关重要的基因的大量信息已经计算出来，但关于承诺发生的综合机制仍然很少。在发展镜头的悠久历史上，在各个阶段，假定晶状体（PLE）的可及性，而Xenopus系统的许多技术优势为解决胚胎确定问题提供了独特的资产。我的实验室表征了镜头承诺过程中的众多步骤，此处描述了实验以区分此过程的最后一步：规范和确定。该项目的第一个目的是通过研究抑制对Wnt信号传导途径的响应能力的假设来解决导致确定的特定细胞机制。在承诺过程中，RNA-Seq对基因表达的检查表明，许多候选基因可能是该过程的关键调节因子。例如，将对OCT91（干细胞基因Oct4的异爪蟾）同源物的下调节，将被检查为确定过程中潜在的调节步骤。第二个目的是开发表观遗传资源，通过进行一系列CHIP-SEQ分析来大大增强我们对基因调节的研究，以鉴定与PLE在承诺过程中三个阶段的活性增强子相关的关键染色质标记（例如组蛋白修饰）。这些分析将为识别调节基因中的推定增强子提供关键资源，并评估组蛋白标记与基因激活和抑制以及承诺过程的时间关联。最终的目的是检查神经板阶段的外胚层对晶状体形成的特定区域的“空间承诺”。四个基因（PAX6，SIX3，MAB21L1和MAB21L2）在这个早期阶段定义PLE区域的小区域中表达。对控制这种高度调节表达的调节元素的识别和分析（使用第二个目标产生的信息）将提供有关如何建立镜头空间定义的新见解。总体而言，这项工作将提供关键信息，以了解视觉系统的个体发育。此外，它依靠干细胞生物学中的重要问题，这些问题取决于对早期发展过程中承诺的紧密相关但知之甚少的理解。反过来，这项工作涉及涉及细胞命运重编程的再生医学的相关问题，以及涉及关键发育基因遗传病变的人类疾病，例如在眼睛形成的情况下。