Cellular and transcriptional analysis of endoderm patterning and organogenesis.

摘要

Utilizing embryonic stem (ES) cells for regenerative therapy in endodermal organs holds immense promise. Directing differentiation of ES cells requires knowledge of embryonic endoderm development, which is poorly understood. I have increased understanding of how cells specified as endoderm become patterned and specified to organ domains in the mouse embryo.;I have shed light on genes that guide mouse endoderm patterning and organogenesis. I identified a cell surface protein, EpCAM, which allows flow cytometric isolation of endoderm, and performed microarray analysis on embryonic day 8.25 (E8.25) definitive and extraembryonic endoderm. I have developed a gene expression signature for definitive and vextraembryonic endoderm. Microarray analysis was also performed on early endodermal organ domains, identifying key transcription factors involved in organogenesis. This global analysis was followed up by dynamic immunofluorescence analysis. One key early patterning event involves converging waves of Sox2 and Cdx2 sweeping through the endoderm and meeting at the prospective stomach-intestine border at E8.5. Genes involved in endodermal organ development are activated overlapping this border in progressively refined domains, suggesting a model of sequential axial patterning events and gradual refinement of organ fate potential.;I have identified two signaling pathways, Wnt and retinoic acid (RA), which are involved in early patterning of the endoderm. Wnt signaling dominantly induces an intestinal gene program and represses anterior fates, additionally controlling subdivision of the large and small intestine. RA acts as a posteriorizing agent for non-intestinal endoderm, inducing the earliest genes necessary for formation of endodermal organs at high doses and patterning the pharyngeal endoderm at lower doses. Both of these pathways induce high efficiency differentiation of ES cell-derived endoderm with similar gene expression changes as in embryonic endoderm.;Finally, I have developed an assay that allows direct investigation of the behavior of early pancreatic progenitors. When injected into pancreatic mesenchyme, single pancreatic progenitors are capable of constructing correctly patterned lobes of the pancreas, and this activity is dependent on the origin of both endoderm and mesenchyme. This work raises the prospect of creating transplantable organs in vitro from ES cell-derived endodermal organ progenitors.