Patterning of mouse embryonic stem cell-derived pan-mesoderm by Activin A/Nodal and Bmp4 signaling requires Fibroblast Growth Factor activity.

Embryonic stem (ES) cells have the potential to differentiate into all cell types of the adult body, and could allow regeneration of damaged tissues. The challenge is to alter differentiation toward functional cell types or tissues by directing ES cells to a specific fate. Efforts have been made to understand the molecular mechanisms that are required for the formation of the different germ layers and tissues from ES cells, and these mechanisms appear to be very similar in the mouse embryo. Differentiation toward mesoderm and mesoderm derivatives such as cardiac tissue or hemangioblasts has been demonstrated; however, the roles of Activin A/Nodal, bone morphogenetic protein (BMP), and fibroblast growth factor (FGF) signaling in the early patterning of ES cell-derived pan-mesoderm and anterior visceral endoderm (aVE) have not been reported yet. We therefore analyzed the roles of Activin A/Nodal, BMP, and FGF signaling in the patterning of ES cell-derived mesoderm as well as specification of the aVE by using a dual ES cell differentiation system combining a loss-of-function with a gain-of-function approach. We found that Activin A or Nodal directed the nascent mesoderm toward axial mesoderm and mesendoderm, while Bmp4 was inducing posterior and extraembryonic mesoderm at the expense of anterior primitive streak cells. FGF signaling appeared to have an important role in mesoderm differentiation by allowing an epithelial-to-mesenchymal transition of the newly formed mesoderm cells that would lead to their further patterning. Moreover, inhibition of FGF signaling resulted in increased expression of axial mesoderm markers. Additionally, we revealed that the formation of aVE cells from ES cells requires FGF-dependent Activin A/Nodal signaling and the attenuation of Bmp4 signaling.