Specification and differentiation of endoderm in C. elegans

描述（由申请人提供）：拟议研究的主要目标是阐明控制从特化到分化的转变的机制，并利用C. elegans内胚层作为模型系统。在早期胚胎中指定内胚层发育的调节级联包括两个冗余的中内胚层指定的GATA型转录因子、它们的靶标、内胚层指定的END-1/3加塔因子和三个下游加塔因子，所有这些都显示出在肠道分化和功能中的功能要求。初步研究揭示了这些因子之间的大量遗传相互作用，并表明其中一些因子可能具有内胚层的特化和分化所共有的功能。当广泛表达时，END-1和END-3可以将非内胚层细胞重编程为内胚层分化途径;然而，细胞被重编程的能力暂时限于胚胎发生的早期阶段，超过该阶段，细胞似乎从多能性转变为定向分化。功能基因组学筛选在早期胚胎和成年生殖系中这种开关所需的基因，涉及细胞命运调节转录因子，染色质修饰和细胞间通讯，指导分化细胞的承诺。将研究使胚胎中的细胞和发育中的生殖细胞对END依赖性重编程难治的机制。在第一个目标中，假设内胚层分化由加塔转录因子之间的前馈相互作用的顺序冗余递归系列指导的模型将通过分析所选择的突变组合来测试，以获得对内胚层细胞的分化和行为的协同效应。反馈抑制的途径和其他内胚层特异性转录因子的参与将进行检查。在第二个目标中，将测试分化状态的激活和维持对于防止细胞经历重编程和转分化是必要的这一假设。将评价细胞间通讯在细胞定型为特定分化状态中的作用以及Notch信号传导在胚胎发生期间多能性AE定型转换中的作用。在第三个目标中，将进行基于RNAi的筛选，以确定在早期胚胎和发育中的成年生殖细胞中调节多能性和定型的组分。将检查鉴定的基因的细胞类型和调节重编程能力的时间特异性。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有缺陷的动物发育的理解，并可能指导从发育潜力通常受限的细胞中创造新的多能前体细胞的方法，这些细胞可用于产生新的组织和器官。公共卫生相关性：该项目分析了专门的细胞，如神经元和肠道细胞，如何被阻止成为其他类型的细胞，提供有关干细胞如何产生许多不同细胞类型的信息。它还将揭示细胞如何在发育过程中的适当时间和地点成为正确的类型，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.