Endoderm patterning in the mouse embryo

最后的内胚层(DE)是胚胎中的主要胚层之一，产生呼吸道和胃肠道的器官，包括肺、胃、结肠、肠、肝和胰腺的上皮细胞。考虑到这些组织是健康问题的主要部位，包括先天性缺陷、癌症和器官功能障碍，我们对DE的早期胚胎发育知之甚少，令人惊讶。这主要是因为缺乏可以用来评估胚胎模式的特定的、区域性表达的基因，以及缺乏一组强大的Cre驱动鼠系，可以用来评估DE特定区域的基因功能。*我们的团队一直在使用转录组学来识别DE中表达的新基因，以更好地了解DE被模式化为器官结构域的机制。然而，这些研究是在大量组织中进行的，这使得深入研究细胞多样性变得不可能。*指导该项目的假设是，单细胞RNA测序(scRNA-seq)与单分子原位杂交相结合，将定义细胞与细胞之间的相互作用，以及与原肠形成小鼠胚胎特定区域模式有关的相关生长因子。我们有两个具体的目标。*目标1：确定肠道内胚层发育中的细胞多样性和分化格局。DE的复杂性和调节其模式的途径仍然知之甚少。我们将使用scRNA-seq来检测信号通路、生长因子和转录因子，这些信号通路、生长因子和转录因子对DE从原肠形成到早期器官发生形成模式。*目标2：评估中肠内胚祖细胞的NEPN-CreERT2驱动。我们发现的一个基因，称为Nephrocan(NEPN)，在小鼠胚胎的中肠中唯一表达，开始于原肠形成。我们已经建立了一个小鼠模型，其中CreERT2在NEPN基因座上表达。我们将表征NEPN表达细胞在小鼠胚胎中的命运，以评估中肠内胚层的命运，并开发一种新的Cre驱动程序，用于DE中表达的基因的功能评估。*通过这些实验，我们将深入了解中肠DE前体细胞是如何以及在哪里被决定的，这些细胞在小鼠胚胎中的命运以及控制中肠形成的分子机制。了解胚胎中组织发育的机制将有助于深入了解潜在的胃肠道干细胞的特性。总而言之，这里提出的实验将开始解决涉及小鼠中肠发育的机制。

The definitive endoderm (DE), one of the primary germ layers in the embryo, gives rise to the organs of the respiratory and gastrointestinal tracts, including the epithelial cells of the lungs, stomach, colon, intestine, liver, and pancreas. Considering that these tissues are major sites of health problems, including congenital defects, cancers, and organ malfunctions, we know surprisingly little about the early embryonic development of the DE. This is primarily due to a lack of specific, regionally-expressed genes that can be used to evaluate patterning in the embryo, and the lack of a robust set of CRE driver mouse lines that can used to evaluate functions of genes in specific regions of the DE. ***Our group has been using transcriptomics to identify novel genes expressed in the DE to better understand the mechanisms through which the DE is patterned into the organ domains. However, these studies have been done in bulk tissues, which does not permit in-depth studies of cell diversity. ***The hypothesis guiding this project is that single cell RNA sequencing (scRNA-seq) combined with single molecule in situ hybridization will define cell-cell interactions and the associated growth factors involved in patterning specific regions in the gastrulating mouse embryo. We have two specific aims.***Aim 1: Define the cellular diversity and differentiation landscape in gut endoderm development. The complexity of the DE and the pathways that regulate its patterning are still poorly understood. We will use scRNA-seq to examine the signaling pathways, growth factors and transcription factors that pattern the DE from gastrulation through to early organogenesis.***Aim 2: Evaluate a Nepn-CreERT2 driver for midgut endoderm progenitors. One gene that we identified, known as Nephrocan (Nepn), is exclusively expressed in the midgut of the mouse embryo beginning at gastrulation. We have generated a mouse model in which CreERT2 is expressed in the Nepn locus. We will characterize the fate of Nepn expressing cells in the mouse embryo to evaluate the fate of midgut endoderm and to develop a novel CRE driver for functional evaluation of genes expressed in the DE.***Through these experiments we will gain insight into how and where midgut DE progenitor cells are determined, the fate of these cells in the mouse embryo and the molecular mechanisms controlling midgut formation. Knowledge of the mechanisms through which tissues develop in the embryo will provide insight into the properties of potential stem cells of the gastrointestinal tract. Together, the experiments presented here will begin to address the mechanisms involved in the development of the midgut in the mouse.**