Fig. 2: A pipeline relating single-cell signaling history to fate.

A Schematic of the experimental procedure to collect data on signaling histories and cell fate. B Example images showing two tracked cells 0, 2, and 42 hours after treatment with BMP4 + WNTi in the SMAD4 and H2B channels. Nuclear centroids are marked with a solid blue circle, tracks are indicated by a solid and dashed line. C Nuclei with overlaid classification of cells as non-dividing (yellow) or dividing (magenta). D Iterative immunofluorescence (IF) data showing an initial stain for ISL1, SOX2, and NANOG (top), followed by a restain for OCT4, HAND1, and GATA3 (middle), in the same field of view as the live data in B. Bottom panel shows the DAPI image from the first round of fixed imaging. E Two-color overlay of the live (sparsely labeled) H2B image at 42 hours in B (green) and the DAPI image in D (magenta). The two cells for which tracks are shown in B are circled in white. F Heatmap of all single-cell signaling histories collected in a single experiment (N = 369 histories), sorted by mean signaling level. Signaling histories of the two cells tracked in B are marked by white lines. G Signaling histories of the two cells tracked in B. H Signaling histories of the two cells tracked in B, colored for fate after matching nuclei from the live imaging time lapse to nuclei in the fixed IF data. I Scatterplot of log-transformed ISL1 and NANOG intensity in single cells, colored for local density, showing a bimodal distribution of ISL1+ amnion-like cells and NANOG+ pluripotent cells. Scale bar 50um. Source data are provided in a Source Data file.