Mechanisms of Embryonic Lens Determination

胚胎晶状体测定机制

• 批准号: 8422247
• 负责人: Robert M Grainger
• 金额: $ 39.42万
• 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/8422247
• 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; computerized data processing; 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; 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(由申请者提供)：该项目的目标是阐明导致胚胎特定区域承诺或决定形成组织或器官的机制。这项研究的重点是非洲爪蛙的晶状体形成。虽然已经积累了大量关于器官形成的关键基因的信息，但对于承诺发生的综合机制仍然知之甚少。就发展中的晶状体而言，悠久的研究历史、推测晶状体外胚层(PLE)在所有阶段的可及性以及非洲爪哇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。我的实验室已经确定了镜片承诺过程中的许多步骤，这里描述了区分这一过程中最后几个步骤的实验：指定和确定。该项目的第一个目标是解决导致确定的特定细胞机制，最初是通过检验对Wnt信号通路的反应抑制负责确定的假设。RNA-Seq对承诺过程中的基因表达进行了检测，发现了一些可能是这一过程的关键调控基因；例如，下调干细胞基因Oct4的非洲爪哇同源物Oct91的表达将被视为确定过程中潜在的调控步骤。第二个目标是开发表观遗传学资源，通过进行一系列芯片序列分析来识别与承诺过程中三个阶段的PLE中的活性增强子相关的关键染色质标记(例如组蛋白修饰)，从而极大地加强我们对基因调控的研究。这些分析将为确定调控基因中可能的增强子提供关键资源，并评估组蛋白标记的出现与基因激活和抑制以及承诺过程的时间关联。最终目的是研究外胚层特定区域在神经板阶段对晶状体形成的“空间承诺”。4个基因(Pax6、Six3、mab21l1和mab21l2)在这个非常早期的阶段就在一个定义PLE区域的小区域中表达。识别和分析控制这种高度调控的表达的调控元件(使用第二个目标产生的信息)将为如何建立晶状体的空间定义提供新的见解。总体而言，这项工作将为理解视觉系统的个体发育提供关键信息。此外，它还涉及干细胞生物学中的重要问题，这些问题依赖于对早期发育过程中密切相关但知之甚少的承诺机制的理解。反过来，这项工作涉及再生医学的相关问题，涉及细胞命运重新编程，以及涉及关键发育基因的遗传损伤的人类疾病，例如眼睛形成时的虹膜缺失。