Figure 3.

pESCs cytonemes are non-selective and cannot facilitate stable interactions with TSCs. (A) Frames from time-lapse imaging of ESCs (blue, top) or pESCs (orange, bottom) interacting with TSCs that express GFP. Examples of reactive interactions (left) or non-reactive interactions (right) are shown. Time in minutes. Arrowheads (yellow) indicate initial contact through cytonemes. Scale bars, 20 µm. (B) Percentage of reactive interactions between ESCs (blue) or pESCs (orange) and TSCs in different conditions. Where indicated, TSCs were pretreated with 2 µM IWP2 for 24 h. n ≥ 71 cells pooled from three experiments. Statistical significance calculated by multiple Fisher’s exact two-sided tests: *, P < 0.05; ***, P < 0.001. CNTRL, control. (C) Frames from time-lapse imaging of ESCs (blue, top) or pESCs (orange, bottom) expressing the F-actin reporter Ftractin-mRuby3 (presented as inverted grayscale), interacting with Wnt beads. Reactive (left) and non-reactive (right) interactions are shown. Time in minutes. Wnt beads are highlighted by yellow dashed circle. Inserts are magnified and contrast-enhanced for clarity. Scale bars, 20 µm for larger images or 5 µm for inserts. (D) Percentage of reactive interactions between ESCs (blue, top) or pESCs (orange, bottom) and different types of beads. n ≥ 40 cells pooled from ≥3 experiments. Statistical significance calculated by multiple Fisher’s exact two-sided tests: ns, nonsignificant, P > 0.05; ***, P < 0.001. (E and F) Box and whiskers plots describing the distribution of the Wnt3a bead retention time (E) and time between initial Wnt3a bead contact and recruitment (F) for ESCs (blue) or pESCs (orange). For details on the measurement, see Fig. S3 B. Whiskers are 5–95% of data, middle line is median, and dots are data outside range. n ≥ 40 cells pooled from ≥3 experiments. Statistical significance calculated by unpaired two-sided t tests: **, P < 0.01; ***, P < 0.001.