Investigation of a neuromesendodermal progenitor population in the posterior avian endoderm

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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.