Fig. 6: SOX2 perturbation reduces amnion-like differentiation in agreement with a mathematical model.

A Plot of the change in each GFP::SOX2 curve in Supplementary Fig. 5H over the first 16 hours of differentiation against SMAD4 signaling integral in that time. SMAD4 integral is determined in each condition based on data in Fig. 4. A dashed line indicates a linear fit of the data. B Measured (thick, solid lines) and simulated (thin, semi-transparent lines) GFP::SOX2 dynamics over the course of 42 hours of differentiation with indicated treatments applied for 42 (left) or 32 (right) hours. C Measured (left) and simulated (right) ISL1 level as a function of SMAD4 integral for the conditions in B. Data presented as mean +/- SD across N = 4 images. D Equations used to model the regulation of SOX2 and ISL1 by BMP-SMAD4 signaling, and their mutual inhibition. E–H Example IF data (E) and quantification showing ISL1 (F), SOX2 (G), and NANOG (H) expression after 42 hours of differentiation in standard culture with 50 ng/mL BMP4 + WNTi, +/- doxycycline from 12 to 24 hours. Violin plots show expression distributions over n = 2867 cells (-DOX) and n = 3081 cells (+DOX) in one of two independent experiments. The box plot within the violin shows the 25%, 50% (median), and 75% quartiles, with whiskers to the maximum and minimum values in the data. I–K Example IF data (I) and radial expression profiles (J, K) in micropatterned colonies after 42 h of differentiation with 50 ng/mL BMP4 + WNTi, without (J) or with (K) the addition of doxycycline from 12 to 24 hours. Scale bars 50um. Source data are provided in a Source Data file.