Mechanisms of Embryonic Lens Determination

描述（由申请人提供）：该项目的目标是阐明导致胚胎特定区域形成组织或器官的承诺或决定的机制。这个建议的重点是非洲爪蟾的晶状体形成。虽然关于器官形成的关键基因的大量信息已经积累起来，但关于承诺发生的综合机制仍然知之甚少。在晶状体发育的情况下，长期的研究历史，在所有阶段推定晶状体外胚层（PLE）的可及性，以及非洲爪蟾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。我的实验室已经描述了镜头承诺过程中的许多步骤，这里有描述的实验来区分这个过程中的最后几个步骤：规范和确定。该项目的第一个目标是解决导致决定的特定细胞机制，最初通过检查抑制对Wnt信号通路的反应性是负责决定的假设。通过RNA-Seq检测承育过程中的基因表达，揭示了一些可能是这一过程的关键调节因子的候选基因；例如，Oct91 （Xenopus干细胞基因Oct4的同系物）的下调将作为测定过程中的一个潜在调控步骤进行研究。第二个目标是开发表观遗传资源，通过开展一组ChIP-Seq分析来识别在承诺过程的三个阶段与PLE中活性增强子相关的关键染色质标记（例如组蛋白修饰），从而大大增强我们对基因调控的研究。这些分析将为识别调节基因中假定的增强子，以及评估组蛋白标记的出现与基因激活和抑制以及与承诺过程的时间关联提供关键资源。最终目的是检查外胚层特定区域在神经板阶段对晶状体形成的“空间承诺”。四个基因（pax6, six3， mab21l1和mab21l2）在这个非常早期的阶段在一个定义PLE区域的小区域中表达。识别和分析控制这种高度调控表达的调控元件（使用从第二个目标产生的信息）将为如何建立透镜的空间定义提供新的见解。总的来说，这项工作将为理解视觉系统的个体发生提供关键信息。此外，它还涉及干细胞生物学中的重要问题，这些问题取决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，这项工作涉及到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无虹膜。

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.