Grant: Discovery Grants Program - Individual

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.