Endoderm patterning in the mouse embryo

小鼠胚胎中的内胚层模式

• 批准号: RGPIN-2018-05018
• 负责人: Hoodless, Pamela
• 金额: $ 3.64万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2020
• 资助国家: 加拿大
• 起止时间: 2020-01-01 至 2021-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=713236
• 关键词: Endoderm; patterning; mouse; embryo

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.

定形内胚层（DE）是胚胎中的初级胚层之一，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。这主要是由于缺乏可用于评估胚胎中模式化的特异性区域表达基因，以及缺乏可用于评估DE特定区域中基因功能的一组稳健的CRE驱动小鼠品系。 我们的研究小组一直在使用转录组学来鉴定DE中表达的新基因，以更好地理解DE被模式化到器官结构域中的机制。然而，这些研究是在大量组织中进行的，这不允许对细胞多样性进行深入研究。 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，将定义细胞间相互作用和参与原肠形成小鼠胚胎中特定区域图案化的相关生长因子。我们有两个具体目标。 目的1：确定肠道内胚层发育中的细胞多样性和分化景观。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来检查从原肠胚形成到早期器官发生的DE模式的信号通路、生长因子和转录因子。 目的2：评估用于中肠内胚层祖细胞的Nepn-CreERT 2驱动器。我们发现的一个基因，称为Nephrocan（Nepn），只在原肠胚形成时开始的小鼠胚胎中肠中表达。我们已经产生了小鼠模型，其中CreERT 2在Nepn基因座中表达。我们将描述小鼠胚胎中Nepn表达细胞的命运，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祖细胞是如何以及在哪里确定的，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胃肠道潜在干细胞的特性。总之，这里提出的实验将开始，以解决在小鼠中肠的发展所涉及的机制。