Figure 4. Wnt induces intestinal differentiation from mouse and human ES cell-derived endoderm.

(A–D) Immunofluorescence analysis of mouse ES cells treated with Activin A from days 0–5 (A–B) or Activin A from days 2–5, GSK3iXV from day 2–3 and Dorsomorphin from days 3–5 (C–D) before fixation and stained for Sox17 (green) and Foxa2 (red). Nuclei are stained with Hoechst 33342 (B, D). (E–J) Immunofluorescence analysis of mouse ES-derived endoderm treated with DMSO (E–F) or 50 nM GSK3iXV (G–H) or 1 µg/mL Wnt3a (I–J) for 24 hours before fixation and stained for Cdx2 (white in E, G, I, green in F, H, J) and Foxa2 (red in F, H, J). (K) Graph comparing the microarray foldchange induced by 24 hours of GSK3iXV treatment in ES-derived endoderm (X-axis) and E8.25 endoderm (Y-axis). Only genes significantly affected by GSK3iXV in E8.25 endoderm are displayed, and the correlation coefficient (r) is shown. (L–M) Venn diagrams displaying the fraction of genes induced (L) or downregulated (M) by GSK3iXV treatment (red) and Cdx2 induction (blue) of ES-derived endoderm as determined by microarray analysis. (N) Graph displaying the microarray foldchange in certain genes induced by 24 hours of GSK3iXV treatment (blue) or 24 hours of Cdx2 induction (red). (O–R) Immunofluorescence analysis of HUES8 human ES-derived endoderm treated with DMSO (O–P) or 125 nM GSK3iXV (Q–R) for 24 hours before fixation and stained for Cdx2 (white in O, Q, green in P, R) and Foxa2 (red in P, R).Scale bar equals 100 µm in B and D, 50 µm in F, H, J, P, and R.