Fate Mapping and Analysis of the Definitive Endoderm

描述(由申请人提供)： 这一建议的目的是阐明导致最终内胚层分化的分子和形态机制。最后的内胚层是原肠发育过程中产生的三个主要胚层之一，以一层上皮膜的形式出现，排列在小鼠成熟前胚胎的整个腹面。这种上皮层的细胞内化并形成消化道和相关的消化器官，这一过程涉及到与底层中胚层的诱导相互作用。虽然许多研究集中在器官发生开始后的内胚层，但在此分化阶段之前对内胚层知之甚少。这一建议将通过利用整个胚胎培养系统中早期最终内胚层的可及性来阐明导致器官发生的最初过程。首先，用荧光染料手动标记上皮片上的群体或单个细胞，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，从而生成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性组织相互作用。具体地说，组织学和命运映射技术将被用于研究动态的中胚层相互作用，这些相互作用是物理上基础的，因此可能影响前肝和胰腺区域。最后，将利用基因敲除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，来开发胚胎培养系统中早期内胚层的暴露。这些研究将有助于更彻底地了解导致正常消化系统形成的过程，从而推进疾病状态的治疗。

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.