a, Snapshots of HES7-Achilles;MESP2-mCherry double-reporter cells on days 2–5 of differentiation in CLFBR medium at 0, 20, 45 and 60 h. Cultures treated with DMSO (control), 25 μM DAPT, 2 μM XAV and 250 nM PD03 are shown. n = 10 independent experiments. Scale bar, 100 μm. b, Time of onset of MESP2-mCherry expression in PSM cells derived from human iPS cells treated with vehicle control (DMSO), 2 μM XAV, 250 nM PD17 or 100 nM, 250 nM or 500 nM PD03. Onset of expression is defined by a threshold of 25 AU. Mean ± s.d. One-way ANOVA: control versus XAV, P = 4.6 × 10−15; control versus 100 nM PD03, P = 5.1−17; control versus 250 nM PD03, P = 1.3 × 10−17; control versus 500 nM PD03, P = 1.4 × 10−18; 100 nM versus 250 nM PD03, P = 2.6 × 10−15; 100 nM versus 500 nM PD03, P = 7.7 × 10−16; 250 nM versus 500 nM PD03, P = 6.9 × 10−5. n = 10 independent experiments. c, HES7–Achilles and MESP2–mCherry fluorescence intensity profiles in small ROIs within PSM cultures derived from human iPS cells, treated with 25 μM DAPT on days 2–5 of differentiation in CLFBR medium. Mean ± s.d. Dotted line denotes the threshold for MESP2 activation (25 AU). n = 15 independent experiments. d, qRT–PCR for the genes HES7, LFNG, MSGN1, TBX6, DUSP6, FOXC2, MESP2 and RIPPLY2 in PSM cultures derived from human iPS cells, treated for 24 h with vehicle control (DMSO) or 250 nM PD03 in CLFBR medium. Relative expression is shown as the fold change relative to iPS cells at day 0. Mean ± s.d. n = 3 biological replicates. e, Immunofluorescence staining for TBX6 on day 3 of differentiation (CLFBR medium) in cells treated with DMSO or PD03 (250 nM). n = 4 independent experiments. Scale bar, 100 μm.