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机制。这项建议的重点是透镜形成的青蛙爪蟾。虽然关于对器官形成至关重要的基因已经积累了大量的信息，但关于承诺发生的整合机制仍然知之甚少。在发展中的透镜的情况下，研究的悠久历史，推定的透镜外胚层（PLE）在所有阶段的可及性，以及爪蟾系统的许多技术优势，为解决胚胎决定的问题提供了独特的资产。我的实验室已经表征了在透镜承诺过程中的众多步骤，这里描述的实验，以区分在这个过程中的最后几个步骤：质量标准和测定。该项目的第一个目的是解决特定的细胞机制，导致确定，最初通过检查的假设，抑制Wnt信号通路的反应是负责确定。在提交过程中通过RNA-Seq对基因表达进行的检查揭示了许多候选基因，这些基因可能是该过程的关键调节因子;例如，Oct 91（干细胞基因Oct 4的非洲爪蟾同源物）的下调将作为确定过程中的潜在调节步骤进行检查。第二个目标是开发一种表观遗传资源，通过进行一组ChIP-Seq分析来识别与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），从而大大增强我们对基因调控的研究。这些分析将提供一个关键的资源，以确定公认的增强子在调控基因和评估的时间关联的组蛋白标记的外观与基因激活和抑制，并与承诺的过程。最后的目的是检查“空间承诺”的一个特定区域的外胚层对透镜形成在神经板阶段。四个基因（pax 6，six 3，mab 21 l1和mab 21 l2）在这个非常早期的阶段在一个小的区域定义的PLE区域的表达。控制这种高度调节的表达的调节元件的鉴定和分析（使用从第二目的产生的信息）将提供关于如何建立透镜的空间定义的新见解。总的来说，这项工作将提供关键信息，了解视觉系统的个体发育。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，这项工作涉及再生医学的相关问题，涉及细胞命运重编程，以及涉及关键发育基因遗传病变的人类疾病，例如眼睛形成中的无虹膜。

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.