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

Abstract

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.

Functional evaluation of ES cell-derived endodermal populations reveals differences between Nodal and Activin A-guided differentiation

“…Nodal and Activin A promote the differentiation of highly similar definitive endoderm profiles in vitro Although some reports have included brief mention of Nodal in the induction of mesendoderm and endoderm (Hansson et al, 2009), current protocols for differentiating ES cells into endoderm have predominantly involved the use of Activin A (D'Amour et al, 2005;Gadue et al, 2006;Kubo et al, 2004;Yasunaga et al, 2005;Nostro et al, 2011). Nodal, however, is an essential signal during vertebrate gastrulation and specification of the DE (Ding et al, 1998;Lowe et al, 2001;Vincent et al, 2003).…”

A Functional Assay for Putative Mouse and Human Definitive Endoderm using Chick Whole-Embryo Cultures

“…The in vivo functionality of ESC-derived DE has previously been investigated in some studies using mouse or chick embryos [19,48]. Here, we demonstrate a detailed description of an in vivo functional assay for putative DE.…”

“…It has been used to assess the proliferation, differentiation, migration capacity, and/or function of rodent and human undifferentiated and differentiated ESCs [12][13][14][15][16][17][18]. Previously, we have reported the integration and differentiation of mouse ESCderived DE after grafting to the endoderm of developing chick embryos [19]. Here, we extend previous findings and present a functional assay where putative DE, derived from mouse ESCs (mESC) or human ESCs (hESC), is grafted to the endoderm of the chick embryo.…”

A factor(s) secreted from MIN-6 β-cells stimulates differentiation of definitive endoderm enriched embryonic stem cells towards a pancreatic lineage

“…for nodal signalling [17][18][19]. Thus, when ES cells (CGR8) were cultured as embryoid bodies (EBs), activin A at 30 ng/ml and 100 ng/ml increased the levels of the definitive endoderm markers Sox17, Foxa2 and CXCR4, while decreasing the expression of the mesoderm marker Bry (Fig.…”