Specification and differentiation of endoderm in C. elegans

描述（由申请人提供）：该研究的主要目标是阐明从规范到分化的控制机制，并以秀丽隐杆线虫内胚层的发育为模型系统，研究驱动细胞从多能性到定向分化状态的开关。调控早期胚胎内胚层发育的调控级联包括两个冗余的中胚层特异性GATA型转录因子及其靶点，内胚层特异性END-1/3 GATA因子和三个下游GATA因子，它们都在肠道分化和功能中表现出功能需求。最初的研究揭示了这些因素之间的遗传相互作用，并表明一些因素可能具有内胚层规范和分化的共同功能。当广泛表达时，END-1和-3可以将非内胚层细胞重编程为内胚层分化途径；然而，细胞被重新编程的能力暂时局限于胚胎发生的早期阶段，在此之后，细胞似乎从多能性转变为承诺分化。功能基因组学筛选早期胚胎和成年种系中这种转换所需的基因，涉及细胞命运调节转录因子、染色质修饰和指导分化细胞的细胞间通讯。使胚胎细胞和发育中的种系细胞抵抗end依赖性重编程的机制将被研究。在第一个目标中，假设内胚层分化是由GATA转录因子之间连续冗余递归的前馈相互作用引导的模型将通过分析途径中选择的突变组合来测试内胚层细胞分化和行为的协同效应。反馈抑制通路和其他内胚层特异性转录因子的参与将被检查。在第二个目标中，激活和维持分化状态对于防止细胞进行重编程和反分化是必要的假设将被测试。我们将评估细胞间通讯在细胞向特定分化状态的承诺中的作用，以及Notch信号在胚胎发生过程中多能性AE承诺开关中的作用。在第三个目标中，基于rnai的筛选将被执行，以确定在早期胚胎和发育中的成年种系细胞中调节多能性和承诺的成分。鉴定的基因将被检查细胞类型和时间特异性调节能力的重编程。总的来说，这些研究将揭示逐步限制分化细胞发育潜力的机制步骤。这些研究的结果有望促进我们对正常和缺陷动物发育的理解，并可能指导从通常发育潜力有限的细胞中创造新的多潜能前体细胞的方法，这些前体细胞可用于产生新的组织和器官。公共卫生相关性：该项目分析了神经元和肠道细胞等特化细胞如何被阻止成为其他类型的细胞，提供了干细胞如何产生许多不同类型细胞的信息。它还将揭示细胞在发育过程中如何在适当的时间和地点变成正确的类型，这是避免人类出生缺陷的关键。

DESCRIPTION (provided by applicant): The major objectives of the proposed research are to illuminate the mechanisms controlling the transition from specification to differentiation and to investigate the switches that drive cells from pluripotent to committed differentiated states, using the development of the C. elegans endoderm as a model system. A regulatory cascade that specifies endoderm development in the early embryo includes two redundant mesendoderm- specifying GATA-type transcription factors, their targets, the endoderm-specifying END-1/3 GATA factors, and three downstream GATA factors, all of which show functional requirements in gut differentiation and function. Initial studies revealed substantial genetic interactions between these factors and suggest that some may share functions common to both specification and differentiation of the endoderm. When broadly expressed, END-1 and -3 can reprogram non-endodermal cells into an endoderm differentiation pathway; however, the ability of cells to be thus reprogrammed is temporally confined to early stages of embryogenesis, beyond which cells appear to switch from pluripotency to committed differentiation. Functional genomics screens for genes required for this switch in early embryos and in the adult germline implicate cell fate regulating transcription factors, chromatin modification, and cell-cell communication in directing commitment of differentiating cells. The mechanisms that make cells in the embryo and developing germline refractory to END-dependent reprogramming will be investigated. In the first aim, a model positing that endoderm differentiation is directed by a sequentially redundant recursive series of feedforward interactions between the GATA transcription factors will tested by analyzing selected combinations of mutations in the pathway for synergistic effects on differentiation and behavior of endoderm cells. Feedback inhibition in the pathway and involvement of other endoderm-specific transcription factors will be examined. In the second aim, the hypothesis that activation and maintenance of a differentiated state is necessary to prevent cells from undergoing reprogramming and trans- differentiation will be tested. The role of intercellular communication in commitment of cells to specific differentiated states and the action of Notch signaling in the pluripotency AE commitment switch during embryogenesis will be evaluated. In the third aim, RNAi-based screens will be performed to identify components that regulate pluripotency and commitment both in the early embryo and in developing adult germline cells. The identified genes will be examined for cell type and temporal specificity in regulating competence for reprogramming. Collectively, these studies will reveal mechanistic steps that progressively restrict the developmental potential of differentiating cells. Results of these studies promise to advance our understanding of normal and defective animal development and may guide methods for creating new multipotential precursor cells from cells that are otherwise normally restricted in developmental potential, which can be used for generating new tissue and organs. PUBLIC HEALTH RELEVANCE: This project analyzes how specialized cells, such as neurons and gut cells, are prevented from becoming other types of cells, providing information about how stem cells can produce many different cell types. It will also shed light on how cells become the right type at the proper time and place during development, which is key to avoiding human birth defects.