Fig. 6. YAP levels and dynamics differentially control cellular differentiation and proliferation.

a Here we investigated how the steady-state levels of YAP control specification of the mesoderm, endoderm, and ectoderm lineages. mESCs were grown under spontenous differentiation conditions (FBS-based or N2B27, see Methods), and cell types were determined by lineage markers from IF images as shown in Supplementary Fig. 9b, c. YAP expression was induced by titration of doxcycycline at indicated concentrations (see Supplementary Fig. 3c, e). Data is normalized to doxycycline treated WT cells. Mesendoderm but not ectoderm respond to steady-state increases of YAP. Shown are mean ± SEM, N = 4 independent experiments. Two-sided Student’s t test comparing 0 ng/ml dox vs. 100 ng/ml dox, P = 0.02 (endoderm), P = 0.0004 (mesoderm), P = n.s. (ectoderm). b Quantification of cell number as a function of steady-state YAP levels. Spontaneous differentiation in FBS-based media, YAP induction conditions are the same as described for (a). mESC proliferation shows no correlation to YAP levels under these conditions. Shown are mean ± SEM, N = 4 independent experiments. Two-sided Student’s t test comparing 0 ng/ml dox vs. 100 ng/ml dox: P = n.s. c, d Quantification of differentiation induction (c) and cell number (d) of LEXY-YAP cells upon chronic and pulsed light illumination in FBS-based differentiation media. Light pulses induce constant YAP export durations of 4 h and varying import duration ranging from 15 min to 4 h as indicated. Persistent YAP export (chronic light) induces Otx2 expression (line graph, blue vs gray line) more efficiently than pulsed light conditions (bar graph inset). In contrast, proliferation is most efficiently induced upon dynamic illumination with short (15-60 min) YAP import cycles (d). Gray shadings in d indicate pulse durations (lower to upper quartile) of YAP dynamics observed during pluripotency exit (see Fig. 1f). Shown are mean ± SEM, N = 8 independent experiments. p values shown in c, d are from two-sided unpaired Student’s t test comparing lit vs. dark condition (c) and LEXY-YAP vs. YAP for each illumination condition (d). t-tests are not corrected for multiple comparison.