Specification and differentiation of endoderm in C. elegans

描述(申请人提供)：本研究的主要目的是阐明控制从规范到分化的转变的机制，并以线虫内胚层的发育为模型系统，研究驱动细胞从多能性到承诺分化状态的开关。一个调控早期胚胎内胚层发育的级联调控系统包括两个冗余的中胚层特异性GATA型转录因子及其靶标、内胚层特异性的end-1/3 GATA因子和三个下游的GATA因子，所有这些都显示出肠道分化和功能的功能要求。初步研究揭示了这些因子之间的大量遗传交互作用，并表明其中一些可能共享内胚层指定和分化的共同功能。当广泛表达时，End-1和End-3可以将非内胚层细胞重新编程为内胚层分化途径；然而，细胞被重新编程的能力在时间上仅限于胚胎发育的早期阶段，超过这一阶段后，细胞似乎从多能性转变为定向分化。功能基因组学在早期胚胎和成年生殖系中筛选这种转换所需的基因，涉及调控转录因子、染色质修饰和指导分化细胞承诺的细胞-细胞通讯的细胞命运。将对胚胎和发育中的生殖系中的细胞对末端依赖的重新编程产生困难的机制进行研究。在第一个目标中，假设内胚层分化是由GATA转录因子之间的一系列冗余的递归前馈相互作用所引导的模型，将通过分析通路中选定的突变组合来检验对内胚层细胞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被检查。在第二个目标中，将检验这样的假设，即激活和维持分化状态对于防止细胞经历重新编程和转分化是必要的。将评估细胞间通讯在细胞对特定分化状态的承诺中的作用，以及Notch信号在胚胎发育过程中多潜能AE承诺开关中的作用。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承诺的成分。已识别的基因将被检查细胞类型和时间特异性，以调节重新编程的能力。总而言之，这些研究将揭示逐渐限制分化细胞发展潜力的机械步骤。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，并可能指导从正常情况下受限于发育潜力的细胞创造新的多潜能前体细胞的方法，这些细胞可以用于产生新的组织和器官。

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.