Morphogenesis of mammalian gut endoderm

描述(申请人提供)：肠道内胚层产生许多内脏的主要细胞类型，包括甲状腺、胸腺、肺、胃、肝、胰腺、肠和膀胱。对正常的肠道内皮细胞形态发生的严格理解，包括产生肠道内皮细胞及其衍生组织的起源、承诺、规范和分化的知识，应该是理解疾病进展和为这些重要器官系统设计新的治疗策略的逻辑努力的基础。哺乳动物胚胎中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形成过程中的上胚层，而胚外组织与上胚层相互作用以建立体轴，它们只对胚外结构起作用，如卵黄囊和胎盘。肠道内胚层是形成呼吸道和消化道以及相关器官，如肺、肝和胰腺的组织，其形态发生的基础细胞运动是复杂的，而且还没有被很好地了解。利用实时成像和基因标记相结合的方法，我们已经开始研究内脏内胚层在小鼠肠道内胚层形成中的作用，内脏内胚层被认为是胚外组织(图1)。我们的初步研究得出了三个关键发现。(1)一种新的小鼠肠道内胚层形态发生模型，结合了一种新的形态发生机制，导致胚胎和胚胎外细胞的广泛混合。(2)原肠胚期胚胎信号中心周围典型的胚外(内脏)内胚层细胞聚集。(3)胚胎外组织和胚胎组织的分离，这是哺乳动物发育的一个基本特征，可能并不像人们认为的那样严格，而内脏内胚层，一个被定义为完全是胚胎外的谱系，可能会为胚胎本身贡献细胞。这些问题将在以下三个具体目标中加以探讨。图1：结合小鼠的活体成像和遗传学研究哺乳动物的肠道内胚层形成。小鼠胚胎内脏内胚层及其衍生物的实时成像和遗传标记。在胚胎第6.5天，整个内脏内胚层被绿色荧光蛋白(GFP)标记。GFP蛋白显示内脏内胚层来源的细胞在胚胎7.5时存在于胚胎区域，到8.75时这些细胞进入胚胎的肠管(A，B)。具体目的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表现内胚层缺陷的野生型胚胎和突变体的细胞动力学。具体目标2：确定导致胚胎外(内脏)内胚层细胞围绕原肠胚信号中心组织的事件序列，并检验这种排列对这些信号中心的功能至关重要的假设。这将使用实时成像和对未能产生中线结构的突变的分析来实现。具体目的3：确定胚胎和成年小鼠的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来源组织的瞬时成分还是稳定成分。我们将使用基因诱导命运图谱的方法来检验胚胎外(内脏)内胚层不是唯一的胚胎外组织这一假设。

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.