a, Micropatterned hESC colonies (1,000-μm and 500-μm diameter) stimulated with WNT3A + Activin, fixed 24 h after stimulation and stained for GSC and BRA. Note that, as previously observed1 for BMP4 induction, shrinking the colony size results in removal of the central micropattern fate region, resulting here in a higher proportion of GSC-expressing cells. This experiment was repeated at least three times independently with similar results. b, Quantification of a. The radial profile represents the mean ± s.d. of n = 25 colonies. c, Scatter plot of single-cell expression of GSC versus BRA. Note that at 24 h most cells co-express BRA and GSC, but by 48 h GSC expression is increased and BRA expression is decreased. We therefore grafted micropatterns at 24 h as well as at 48 h post-stimulation, reasoning that earlier coexpression of BRA and GSC would result in increased graft contribution to axial mesoderm structures. d, qPCR of additional organizer markers. RNA samples were collected from 500-μm-diameter micropatterns stimulated with BMP4, WNT3A, WNT3A + SB, or WNT3A + Activin for 24 or 48 h. With the exception of NOG, the characteristic organizer-secreted inhibitors DKK1, CER1, CHRD, LEFTY1 and LEFTY2, are all most highly expressed in the WNT3A + Activin condition. The high induction of NOG by BMP4 in hESCs has been noted before20, and may represent a species difference between human and mouse. NODAL, which in mouse is restricted to the organizer later in gastrulation, is also most highly expressed in WNT3A + Activin conditions. Data are mean ± s.d. of three biologically independent replicates. Source Data