Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive, or extraembryonic, endoderm that is specified in the blastocyst and the definitive endoderm that forms later, at gastrulation, as one of the germ layers of the embryo proper. Fate mapping evidence suggests that definitive endoderm occurs in two waves, which potentially reflect two distinct populations. Early primitive ectoderm-like (EPL) cells have been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during pluripotent cell differentiation. The outer cell layer of embryoid bodies (EBs) derived from EPL cells (EPLEBs) has been shown to comprise almost exclusively definitive endoderm similar to that formed in the E7.5 embryo. We have used EPLEBs to characterise the intracellular pathways required for definitive endoderm formation. Analysis of the outcomes from EPLEBs differentiated in Activin A, BMP4 or serum and an inhibitor of p38 mitogen-activated protein kinase (MAPK) showed that the formation of definitive endoderm was dependent on p38 MAPK activity. Further characterisation has shown that EPL cell-derived definitive endoderm comprises two distinct populations formed in response to alternate signalling environments and potentially from distinct progenitor populations. Mapping of these populations, identified in vitro, onto the embryo has shown the definitive endoderm comprises two genetically and spatially distinct populations, proximal and distal definitive endoderm. Positional specification may restrict the developmental potential of pluripotent cell-derived definitive endoderm and characterisation of the derivatives of proximal and distal definitive endoderm populations in the embryo and in culture will allow differentiation protocols to be tailored to ensure enrichment of the appropriate definitive endoderm for subsequent differentiation.
CY and HNG have contributed equally to this work.