Combinatorial interpretation of BMP and WNT allows BMP to act as a morphogen in time but not in concentration

We performed bulk RNA sequencing of cells exposed to either 300ng/ml of the ligand WNT3A for 6 or 18 hours, or 8μM of the GSK3b inhibitor CHIR99021 for 6 hours, which is commonly used as a substitute for WNT in differentiation. We observed transcriptional activation of the canonical WNT genes WNT3 and WNT10B confirming that Wnt signaling induces canonical WNTs. hPSCs were seeded at the density 200k per well in a 12-well plate 24 hours before treatment. hPSCs were grown under 4 experimental conditions: untreated pluripotent cells grown in mTeSR1, hPSCs treated with WNT3a (300ng/ml) for 6 hours, hPSCs treated with WNT3a (300ng/ml) for 18 hours or hPSCs treated with CHIR (8uM) for 6 hours. Total RNA was collected with the Invitrogen RNAqueous-Micro Total RNA Isolation Kit. Processed RNA was stored at −80 ̊C, and RNA integrity was checked by Nanodrop, agarose gel electrophoresis, and qPCR and the Agilent 2100 system. Sequencing was performed by Novogene Co. using the Illumina paired-end 150 platform (Novaseq 6000). Another biological repeat was performed using the same protocol, but also adding a new experimental condition: hPSCs treated with CHIR (8uM) for 18 hours, with two replicates.