The formation of proximal and distal definitive endoderm populations in culture requires p38 MAPK activity.

Endoderm formation in the mammal is a complex process with two lineages forming during the first weeks of development, the primitive (or extraembryonic) endoderm, which 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 the definitive endoderm arises as two waves, which potentially reflect two distinct cell populations. Early primitive ectoderm-like (EPL) cell differentiation has been used successfully to identify and characterise mechanisms regulating molecular gastrulation and lineage choice during differentiation. The roles of the p38 MAPK family in the formation of definitive endoderm were investigated using EPL cells and chemical inhibitors of p38 MAPK activity. These approaches define a role for p38 MAPK activity in the formation of the primitive streak and a second role in the formation of the definitive endoderm. Characterisation of the definitive endoderm populations formed from EPL cells demonstrates the formation of two distinct populations, defined by gene expression and ontogeny, that were analogous to the proximal and distal definitive endoderm populations of the embryo. Formation of the proximal definitive endoderm was found to require p38 MAPK activity and is correlated with molecular gastrulation, defined by the expression of brachyury (T). Distal definitive endoderm formation also requires p38 MAPK activity but can form when T expression is inhibited. Understanding lineage complexity will be a prerequisite for the generation of endoderm derivatives for commercial and clinical use.

Regulation of pluripotent cell differentiation by a small molecule, staurosporine.

Research in the embryo and in culture has resulted in a sophisticated understanding of many regulators of pluripotent cell differentiation. As a consequence, protocols for the differentiation of pluripotent cells generally rely on a combination of exogenous growth factors and endogenous signalling. Little consideration has been given to manipulating other pathways to achieve pluripotent cell differentiation. The integrity of cell:cell contacts has been shown to influence lineage choice during pluripotent cell differentiation, with disruption of cell:cell contacts promoting mesendoderm formation and maintenance of cell:cell contacts resulting in the preferential formation of neurectoderm. Staurosporine is a broad spectrum inhibitor of serine/threonine kinases which has several effects on cell function, including interruption of cell:cell contacts, decreasing focal contact size, inducing epithelial to mesenchyme transition (EMT) and promoting cell differentiation. The possibility that staurosporine could influence lineage choice from pluripotent cells in culture was investigated. The addition of staurosporine to differentiating mouse EPL resulted in preferential formation of mesendoderm and mesoderm populations, and inhibited the formation of neurectoderm. Addition of staurosporine to human ES cells similarly induced primitive streak marker gene expression. These data demonstrate the ability of staurosporine to influence lineage choice during pluripotent cell differentiation and to mimic the effect of disrupting cell:cell contacts. Staurosporine induced mesendoderm in the absence of known inducers of formation, such as serum and BMP4. Staurosporine induced the expression of mesendoderm markers, including markers that were not induced by BMP4, suggesting it acted as a broad spectrum inducer of molecular gastrulation. This approach has identified a small molecule regulator of lineage choice with potential applications in the commercial development of ES cell derivatives, specifically as a method for forming mesendoderm progenitors or as a culture adjunct to prevent the formation of ectoderm progenitors during pluripotent cell differentiation.

Transcriptional control of pluripotency: decisions in early development.

The pathways controlling the maintenance and loss of pluripotency in cells of the early embryo regulate the formation of the tissues that will support development. Several transcription factors have been identified as being integral to the establishment and/or maintenance of pluripotency, coordinately regulating the expression of genes within pluripotent cells and acting as gene targets of these same processes. Recent advances in understanding the transcriptional regulation of these factors have revealed differences in the transcriptional complexes present within sub-populations of the pluripotent lineage and in the mechanisms regulating the loss of pluripotency on differentiation.