Fig. 2-S1

Ror2 binds to Wnt8a and Frizzled7a.

(A) 3D confocal image of a cell clone at 50% epiboly. The embryo was injected with memCherry and Wnt8a-GFP. Cytonemes feature Wnt8a-GFP clusters at their tip (arrows). (B) Single confocal planes of embryos injected with Wnt5b-GFP and memCherry or Ror2-mCherry. (C) Intensity profiles along 15 µm of cell membrane containing Wnt5b-GFP spots, showing spatial intensity correlations with Ror2-mCherry. (D) Schematic depiction of the principle of line scanning fluorescence correlation spectroscopy (FCS) measurements, which allows us to remove fluorescence fluctuations that result from membrane motion. Red and green emitting laser foci are scanned perpendicularly through the plasma membrane. The emission is recorded as a function of time and corrected for membrane fluctuations. The autocorrelation functions of Ror2-mCherry (red excitation) and Wnt8a-GFP (green excitation) are computed from the intensity time traces and yield local concentrations (i.e., area densities) and diffusional correlation times. The cross-correlation between red and green is a measure of joint intensity fluctuations in the two channels caused by Wnt8a-GFP bound to Ror2-mCherry diffusing together through the excitation focus. (E) Diagram of confocal planes of embryos injected with Wnt8a-GFP, Ror2-mCherry and Fzd7a-CFP and the corresponding intensity correlation. (F, G) Pearson correlation coefficients of pairs of fluorescent proteins in a dual-color image (as indicated by the black boxes below the bar graph). The result is +1 for perfect correlation, 0 for no correlation. (F) Analysis of confocal images of embryos at 50% epiboly injected with the indicated constructs, as determined by Imaris software. (G) A Pearson co-localization analysis of the indicated fluorescent constructs in a tissue volume of 40 × 40 × 60 µm³ of eight different embryos. ***=P<0.001.