Investigation of a neuromesendodermal progenitor population in the posterior avian endoderm

项目总结/摘要 早期胚胎从一个看似杂乱无章的干细胞球转化为复杂的干细胞， 精确模式化的成体形式需要通过生物物理和生物化学的定型调节细胞行为 线索使用鸡胚，我的研究计划的长期目标是从一个高度了解 从定量的角度来看，这是如何编排的。大约一半的实验室专注于机械生物学， 胚胎发育，试图了解在胚胎中形成组织和器官的力量， 指定这些力的线索。实验室的其余部分研究细胞命运的决定， 早期大脑发育或发育中的肠道。本申请源于后者。 在胚胎发育过程中，谱系分离的第一个主要步骤之一是原肠胚形成，此时， 单个全能细胞层分成三个胚层，即内胚层、中胚层和外胚层。而 传统上，长期以来一直认为，每个胚层的所有祖细胞都是在发育过程中产生的。 最近的研究对此提出了挑战，确定了一个多能祖细胞群体， 在尾芽中原肠胚形成结束后。在这群被称为神经中胚层祖细胞的细胞中， 细胞可以产生对脊索（中胚层）和神经管（外胚层）都有贡献后代。而 在研究鸡胚中肠管的形成时，我们最近发现了一群内胚层细胞， 腹侧表面的尾芽，并似乎经历了广泛的和以前未报告的上皮到- 间充质转化（EMT）。这些细胞侵入邻近的尾芽组织，远达背侧 tailbud的表面。重要的是，可以看到细胞分散在神经中胚层的整个区域， 已知祖细胞存在于神经管和脊索中。这表明这些 内胚层细胞代表了神经中胚层祖细胞的一个未被重视的来源，如果它们保留了神经中胚层祖细胞， 产生内胚层组织的能力也是非常惊人的。本申请结合了 经典的胚胎学方法，如基因错误表达的命运作图和嫁接实验， 单细胞RNA测序，试图弄清楚简单但令人困惑的观察，内胚层 细胞经历局部EMT，并在原肠胚形成完成时侵入尾芽。该提案 目的是确定这一潜在的祖细胞群体所贡献的组织，并构建一个 详细的单细胞转录组图谱的这一人口，从身份，调节电路，并提出了 可以推断谱系轨迹。在拟议的工作完成后，我们将建立一个新的 体内模型，研究内胚层EMT，并可能确定了一种新的干细胞群体居住在 内胚层，能够产生中胚层和外胚层细胞。这与长期以来 在胚胎发育过程中的谱系分离的概念，因此有可能是 对于发育生物学和干细胞重编程领域具有变革性意义。

PROJECT SUMMARY/ABSTRACT Transformation of the early embryo from a seemingly disorganized ball of stem cells to the complex and precisely patterned adult form requires stereotyped regulation cell behaviors via biophysical and biochemical cues. Using the chick embryo, the long term goals of my research program are to understand from a highly quantitative perspective how this is orchestrated. Approximately half of the lab focuses on mechanobiology of embryonic development, seeking to understand the forces that shape tissues and organs in the embryo and the cues that specify those forces. The remainder of the lab studies cell fate determination, either in the context of early brain development or in the developing gut. The present application stems from the latter. During embryonic development, one of the first major steps in lineage segregation is gastrulation, when a single totipotent layer of cells separates into three germ layers, endoderm, mesoderm, and ectoderm. While conventionally, it has long been accepted that all progenitors of each germ layer are generated only during gastrulation, recent studies have challenged this, identifying a multipotent progenitor population that persists after gastrulation ends in the tailbud. Within this population, known as neuromesodermal progenitors, a single cell can give rise to progeny that contribute to both notochord (mesoderm) and neural tube (ectoderm). While studying gut tube formation in the chick embryo, we recently identified a population of endoderm cells that line the ventral surface of the tailbud, and appear to undergo a widespread and previously unreported epithelial-to- mesenchymal transition (EMT). These cells invade the neighboring tailbud tissue, reaching as far as the dorsal surface of the tailbud. Importantly, cells can be seen dispersed throughout the region where neuromesodermal progenitors are known to reside, as well as in the neural tube and notochord. This suggests that these endodermal cells represent an unappreciated source of neuromesodermal progenitors, which, if they retain the ability to generate endodermal tissues as well, would be remarkable. The present application combines classical embryology approaches such as fate mapping and grafting experiments with gene misexpression and single-cell RNA Sequencing in an effort to make sense of the simple yet puzzling observation that endoderm cells undergo a localized EMT and invade the tailbud at a time when gastrulation is complete. The proposal aims to identify the tissues to which this prospective progenitor population contributes, and to construct a detailed single-cell transcriptomic atlas of this population, from which identity, regulatory circuits, and proposed lineage trajectories can be inferred. At the completion of the proposed work, we will have established a new in vivo model to study endodermal EMT, and potentially identified a novel stem cell population residing in the endoderm, with the ability to give rise to mesodermal and ectodermal cells. This stands in contrast to long-held notions of lineage segregation during embryonic development, and therefore has the potential to be transformative for the fields of developmental biology and stem cell reprogramming.