Fate Mapping and Analysis of the Definitive Endoderm

描述（由申请人提供）： 本研究的目的是阐明导致定形内胚层分化的分子和形态学机制。定形内胚层是原肠胚形成过程中产生的三个初级胚层之一，它以上皮片层的形式出现，排列在有胚前体小鼠胚胎的整个腹面。这一上皮层的细胞被内化并形成消化道和相关的消化器官，这一过程涉及与下层中胚层的诱导相互作用。虽然许多研究都集中在器官发生后的内胚层，但对这一分化阶段之前的内胚层知之甚少。这一建议将阐明导致器官发生的初始过程，利用早期定形内胚层在整个胚胎培养系统的可及性。首先，将通过用荧光染料手动标记上皮片中的组或个体细胞，通过器官发生培养胚胎并通过整体和切片荧光确定后代的贡献来产生早期内胚层的命运图。将进行第二组实验以了解器官发生中涉及的诱导组织相互作用。具体而言，将使用组织学和命运映射技术来研究物理上构成并因此潜在地影响前肝和胰腺域的动态中胚层相互作用。最后，将利用基因敲低和信号通路抑制剂作为评估基因冗余和功能初步筛选的新方法，利用胚胎培养系统中早期内胚层的暴露。这些研究将共同提供对导致正常消化系统形成的过程的更透彻的理解，从而促进疾病状态的治疗。

DESCRIPTION (provided by applicant): The goal of this proposal is to elucidate the molecular and morphological mechanisms leading to the differentiation of the definitive endoderm. The definitive endoderm, one of the three primary germ layers produced during gastrulation, emerges as an epithelial sheet that lines the entire ventral surface of the presomitic mouse embryo. The cells of this epithelial sheet become internalized and form the digestive tract and associated digestive organs, a process that involves inductive interactions with the underlying mesoderm. Although many studies have focused on the endoderm after the onset of organogenesis, very little is known about the endoderm prior to this stage of differentiation. This proposal will elucidate the initial processes leading to organogenesis by exploiting the accessibility of the early definitive endoderm in a whole embryo culture system. First, a fate map of the early endoderm will be produced by manually labeling groups or individual cells in the epithelial sheet with a fluorescent dye, culturing the embryos through organogenesis and determining the contribution of the descendants by whole mount and section fluorescence. A second set of experiments will be performed to understand the inductive tissue interactions involved in organogenesis. Specifically, histological and fate mapping techniques will be used to study the dynamic mesodermal interactions that physically underlie and thus potentially influence the pre-liver and pancreas domains. Finally, the exposure of the early endoderm in the embryo culture system will be exploited by using gene knockdown and signal pathway inhibitors as novel approaches to assessing gene redundancy and initial screens of function. Together these studies will provide for a more thorough understanding of the processes leading to the formation of a normal digestive system, allowing for the advancement of therapies for the diseased state.