Pmg0034

Gastruloids were generated as previously described (Baillie-Johnson et al., 2015 ). Briefly, 300–700 mESCs were plated in 40 μL N2B27 in 96-well Clear Round Bottom Ultra-Low Attachment Microplates (7007, Corning). After 48 h, 150 μL of N2B27 containing 3 μM Chi were added to each well. After 72 h, medium was changed with N2B27. Starting from 96 h, the protocol was optimized as described in Figure S1A. At 96 h, gastruloids were transferred in Ultra-Low Attachment 24-well Plates (3473, Corning) in 100 μL of medium, plus 700 μL of fresh N2B27 containing 30ng ml⁻¹ bFGF (PMG0034, GIBCO), 5ng ml⁻¹ VEGF 165 (PHC9394, GIBCO) and 0.5mM L-ascorbic acid phosphate (013–12061, Wako) (N2B27+++) and cultured on an orbital shaker placed at 37°C, 5%CO₂ at 100rpm (VWR mini shaker). From 120 h onward, half medium was changed daily. Unless differently specified, N2B27+++ was applied from 96 to 144 h, while from 144 h to 168 h N2B27 was used for medium change. To generate Hcn4-GFP::Tbx1Cre-RFP gastruloids, 800–1200 cells were employed, due to initial difficulties in cell aggregation and extreme susceptibility to Chi treatment. For the same reason, Chi pulse for this line was done with 1 μM Chi, with the exact same modalities described for the other lines.