Morphogenesis of mammalian gut endoderm

描述（由申请人提供）：肠道内胚层产生了许多内脏器官的主要细胞类型，包括甲状腺、胸腺、肺、胃、肝、胰腺、肠和膀胱。对正常肠道内胚层形态发生的严格理解，包括对产生肠道内胚层及其衍生组织的细胞的起源、承诺、规范和分化的了解，应该支持理解疾病进展的逻辑努力，并为这些重要器官系统设计新的治疗策略。关于哺乳动物胚胎胚层形成的普遍观点是，外胚层、中胚层和肠道内胚层仅来自原肠胚形成过程中的外胚层，而胚外组织与外胚层相互作用以建立体轴，它们仅参与胚外结构，如卵黄囊和胎盘。肠道内胚层是产生呼吸道和消化道以及肺、肝和胰腺等相关器官的组织，其形态发生背后的细胞运动是复杂的，尚未得到很好的理解。利用实时成像结合基因标记，我们已经开始研究内脏内胚层（一种假定的胚胎外组织）在小鼠肠道内胚层形成中的作用（图1）。我们的初步研究得出了三个关键发现。(1)建立了小鼠肠道内胚层形态发生的新模型，该模型包含了一种新的形态发生机制，导致胚胎细胞和胚胎外细胞大量混合。(2)胚外（内脏）内胚层细胞在原胚期胚胎信号中心周围的典型聚集。(3)作为哺乳动物发育的基本特征，胚胎外组织和胚胎组织的分离可能并不像人们认为的那样严格，而内脏内胚层——一个被定义为完全胚胎外的谱系——可能为胚胎原体提供细胞。这些问题将在以下三个具体目标中探讨。图1：结合小鼠活体成像和遗传学研究哺乳动物肠道内胚层形成。小鼠胚胎内脏内胚层及其衍生物的实时成像和基因标记。在胚胎日(E) 6.5时，整个内脏内胚层用绿色荧光蛋白（GFP）报告基因标记。GFP蛋白显示，在E7.5时，胚胎区存在分散的内脏内胚层来源的细胞群，到E8.75时，这些细胞被纳入胚胎的肠管（a，B）。目的1：阐明介导肠道内胚层形态发生的细胞行为。我们将使用现有的和新的报告菌株来可视化野生型胚胎和内胚层缺陷突变体的细胞动力学。特定目的2：确定导致胚胎外（内脏）内胚层细胞在小鼠原肠原胚信号中心周围组织的事件序列，并验证这种排列对这些信号中心功能至关重要的假设。这将通过实时成像和分析不能产生中线结构的突变体来实现。特异性目的3：确定胎儿和成年小鼠内脏内胚层和肠道内胚层组织之间是否存在谱系关系。我们将确定内脏内胚层是胚胎和成人肠道内胚层衍生组织的短暂或稳定成分。我们将使用基因诱导的命运定位方法来验证胚胎外（内脏）内胚层不是胚胎外组织的假设。

DESCRIPTION (provided by applicant): The gut endoderm gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to understand disease progression and design new therapeutic strategies for these vital organ systems. The prevailing view of germ layer formation in mammalian embryos is that ectoderm, mesoderm and gut endoderm derive solely from the epiblast during gastrulation and while extraembryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extraembryonic structures, such as the yolk sac and placenta. The cell movements underlying the morphogenesis of the gut endoderm, the tissue that gives rise to the respiratory and digestive tracts and associated organs such as the lungs, liver and pancreas, are complex and not well understood. Using live imaging combined with genetic labeling, we have begun to investigate the role of the visceral endoderm, a presumed extraembryonic tissue, in gut endoderm formation in the mouse (Figure 1). Three key findings arise from our preliminary studies. (1) A new model for gut endoderm morphogenesis in the mouse, incorporating a novel morphogenetic mechanism resulting in extensive mixing of embryonic and extraembryonic cells. (2) The stereotypical congregation of extraembryonic (visceral) endoderm cells around signaling centers of the gastrula stage embryo. (3) The segregation of extraembryonic and embryonic tissues, a fundamental feature of mammalian development, may not be as strict as believed, and the visceral endoderm, a lineage defined as exclusively extraembryonic, may contribute cells to the embryo-proper. These questions will be explored in the following three Specific Aims. Figure 1: Combining live imaging and genetics in mice to investigate gut endoderm formation in mammals. Live imaging and genetic labeling of the visceral endoderm, and its derivatives in mouse embryos. At embryonic day (E) 6.5 the entire visceral endoderm is labeled with a green fluorescent protein (GFP) reporter. GFP protein reveals a dispersed population of visceral endoderm-derived cells present in the embryonic region of the conceptus at E7.5, which by E8.75 become incorporated into the gut tube of the embryo (A,B). Specific Aim 1: To elucidate the cell behaviors that mediate gut endoderm morphogenesis. We will use both existing and new reporter strains to visualize cell dynamics in wild type embryos and mutants that exhibit endoderm defects. Specific Aim 2: To determine the sequence of events leading to the organization of extraembryonic (visceral) endoderm cells around signaling centers of the mouse gastrula, and test the hypothesis that this arrangement is central to the function of these signaling centers. This will be achieved using live imaging and the analysis of mutants that fail to generate midline structures. Specific Aim 3: To determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. We will establish if the visceral endoderm is a transient or stable component of embryonic and adult gut endoderm derived tissues. We will test the hypothesis that extraembryonic (visceral) endoderm is not an exclusively extraembryonic tissue using a genetic induced fate mapping approach. PUBLIC HEALTH RELEVANCE: The gut endoderm gives rise to the major cell types of many internal organs, including the thyroid, thymus, lung, stomach, liver, pancreas, intestine and bladder. A rigorous understanding of normal gut endoderm morphogenesis, including knowledge of the origin, commitment, specification and differentiation of cells generating gut endoderm and its derivative tissues, should underpin logical efforts to design new therapeutic strategies for this vital organ system. The prevailing view of germ layer formation in mammalian embryos is that ectoderm, mesoderm and gut endoderm derive solely from the pluripotent epiblast during gastrulation, and while extraembryonic tissues interact with the epiblast to establish the body axes, they contribute solely to extraembryonic structures, such as the yolk sac and placenta. Our preliminary studies challenge this view, and inform the hypotheses to be tested through the Specific Aims of this proposal. Three key findings arise from our preliminary studies in mice. (1) A new model for gut endoderm formation in the mouse, incorporating a novel morphogenetic mechanism resulting in extensive mixing of embryonic and extraembryonic cells. (2) The stereotypical congregation of extraembryonic (visceral) endoderm cells around signaling centers of the gastrula stage embryo. (3) The segregation of extraembryonic and embryonic tissues, a fundamental feature of mammalian development, may not be as strict as believed, and the visceral endoderm, a lineage defined as exclusively extraembryonic, may contribute cells to the embryo-proper. The broad aim of this project is to use a combination of molecular, embryological and live imaging techniques to investigate the role played by the visceral endoderm, a presumed extraembryonic lineage, in the morphogenesis of the mammalian gut endoderm, the tissue that will give rise to the respiratory and digestive tracts, and associated organs such as lungs, liver and pancreas. In Aim 1, we will define the cell behaviors that mediate gut endoderm morphogenesis in the embryo. We will use both existing and new reporter strains to visualize cell dynamics in wild type embryos and mutants that exhibit endoderm defects. In Aim 2, we will determine the sequence of events leading to the organization of extraembryonic (visceral) endoderm cells around signaling centers of the mouse gastrula, and test the hypothesis that this arrangement is central to the function of these signaling centers. This will be achieved using live imaging and the analysis of mutants that fail to generate midline structures. In Aim 3, we will determine if a lineage relationship exists between the visceral endoderm and the gut endoderm tissues of the fetus and adult mouse. We will establish if the visceral endoderm is a transient or stable component of embryonic and adult gut endoderm derived tissues, and will test the hypothesis that extraembryonic (visceral) endoderm is not an exclusively extraembryonic tissue using a genetic induced fate mapping approach.