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. Overall design: 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.