Relatively little is known about the early events that result in specification of lung progenitor cells from theforegut endoderm. In vitro differentiation of embryonic stem cells (ESC) offers an unprecedented opportunityto investigate the mechanisms that control lineage specification and differentiation of a variety of cell types.In contrast to animal models, ESC culture systems allow easy access to cells at early developmental stageswhen lineage commitment decisions are taking place. With regard to lung epithelial lineage specification, thisproposal presents a novel ex vivo system where undifferentiated ES cells are directed through a controlledsequence of differentiation events that model in vivo developmental milestones. Through three specific aimsthis system is employed to recapitulate the specification of Ttf1+ lung progenitors from multipotentendoderm.The overall approach is to employ a novel mouse ESC line with GFP cDNA targeted to thebrachyury locus and a human CD4 reporter targeted to the Foxa2 locus in order purify cells resembling aprimitive streak-like stage. These cells are then differentiated in cells with multipotent endoderm capacity.We propose to develop a novel methodology for the purification of Ttf1+ lung progenitors as they arespecified from these multipotent endoderm-stage cells. Finally, the system is applied in order to define thethe roles of FGF signaling, canonical Wnt signaling, and chromatin remodeling mechanisms in early lunglineage specification. The ultimate goal of these studies is to develop an in vitro system that can be appliedto study the complete genetic and epigenetic programs responsible for lung epithelial development.
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