Extended Data Figure 4. Further organizer characterization.

(a) 1000μm and 500μm diameter micropatterned colonies stimulated with WNT3A+ACTIVIN and fixed and stained for GSC and BRA at 24 hours. Note that as observed by Warmflash et al.,¹ for BMP induction, shrinking the colony size results in removal of center micropattern fate region, thus resulting here in a higher proportion of GSC expressing cells. This experiment was repeated at least n=3 times independently with similar results.

(b) Quantification of (a). The radial profile represents the average of n=25 colonies and errors bars represent the standard deviation.

(c) Scatterplot of single-cell expression of GSC vs BRA. Note that at 24 hours most cells co-express BRA and GSC, but that by 48 hours GSC expression is increased and BRA expression is decreased. Because of this we grafted micropatterns at 24 hours as well as at 48 hours post-stimulation, reasoning that earlier coexpression of BRA and GSC would result in greater graft contribution to axial mesoderm structures.

(d) qPCRs of additional organizer markers, taken from RNA collected from 500μm diameter micropatterns stimulated with either BMP4, WNT3A, WNT3A+SB, or WNT3A+ACTIVIN for 24 or 48 hrs. With the exception of NOGGIN, the characteristic organizer secreted inhibitors DKK1, CER1, CHORDIN, LEFTY1, and LEFTY2, are all most highly expressed in WNT3A+ACTIVIN conditions. The high NOGGIN induction by BMP4 in hESCs has been noted before²⁰, and may represent a human-mouse species difference. NODAL, which in mouse is restricted to the organizer later in gastrulation, is also most highly expressed in WNT3A+ACTIVIN conditions. Error bars represent the standard deviation of n=3 biologically independent replicates and the measure of the center represents the mean.