Retinoic acid synthesis promotes development of neural progenitors from mouse embryonic stem cells by suppressing endogenous, Wnt-dependent nodal signaling.

Embryonic stem (ES) cells differentiate spontaneously toward a neuroectodermal fate in serum-free, adherent monocultures. Here, we show that this spontaneous neural fate requires retinoic acid (RA) synthesis. We monitor ES cells containing reporter genes for markers of the early neural plate as well as the primitive streak and its progeny to determine the cell fates induced when RA signaling is perturbed. We demonstrate that the spontaneous neural commitment of mouse ES cells requires endogenous RA production from vitamin A (vitA) in the medium. Formation of neural progenitors is inhibited by removing vitA from the medium, by inhibiting the enzymes that catalyze the synthesis of RA, or by inhibiting RA receptors. We show that subnanomolar concentrations of RA restore neuroectodermal differentiation when RA synthesis is blocked. We demonstrate that a neural to mesodermal fate change occurring when RA signaling is inhibited is dependent on Nodal-, Wnt-, and fibroblast growth factor-signaling. We show that Nodal suppresses neural development in a Wnt-dependent manner and that Wnt-mediated inhibition of neural development is reversed by inhibition of Nodal signaling. Together, our results show that neural induction in ES cells requires RA at subnanomolar levels to suppress Nodal signaling and suggest that the mechanism by which Wnt signaling suppresses neural development is through facilitation of Nodal signaling.

A late requirement for Wnt and FGF signaling during activin-induced formation of foregut endoderm from mouse embryonic stem cells.

Here we examine how BMP, Wnt, and FGF signaling modulate activin-induced mesendodermal differentiation of mouse ES cells grown under defined conditions in adherent monoculture. We monitor ES cells containing reporter genes for markers of primitive streak (PS) and its progeny and extend previous findings on the ability of increasing concentrations of activin to progressively induce more ES cell progeny to anterior PS and endodermal fates. We find that the number of Sox17- and Gsc-expressing cells increases with increasing activin concentration while the highest number of T-expressing cells is found at the lowest activin concentration. The expression of Gsc and other anterior markers induced by activin is prevented by treatment with BMP4, which induces T expression and subsequent mesodermal development. We show that canonical Wnt signaling is required only during late stages of activin-induced development of Sox17-expressing endodermal cells. Furthermore, Dkk1 treatment is less effective in reducing development of Sox17(+) endodermal cells in adherent culture than in aggregate culture and appears to inhibit nodal-mediated induction of Sox17(+) cells more effectively than activin-mediated induction. Notably, activin induction of Gsc-GFP(+) cells appears refractory to inhibition of canonical Wnt signaling but shows a dependence on early as well as late FGF signaling. Additionally, we find a late dependence on FGF signaling during induction of Sox17(+) cells by activin while BMP4-induced T expression requires FGF signaling in adherent but not aggregate culture. Lastly, we demonstrate that activin-induced definitive endoderm derived from mouse ES cells can incorporate into the developing foregut endoderm in vivo and adopt a mostly anterior foregut character after further culture in vitro.