Fig. 3.

ESC cytonemes exhibit glutamate receptor activity upon contact with a Wnt source. (A) Representative frames from time-lapse imaging of an ESC (Left, magenta; Right, grayscale) expressing the Ca²⁺ reporter GCaMP6s and Ftractin-mRuby3, where a cytoneme contacts an eGFP-expressing TSC (green, dashed black line). (Top) interaction between ESC and TSCs, (Scale bar, 10 μm); (Middle) generation of Ca²⁺ transients upon cytoneme-TSC contact; with fluorescence signal represented by heat-map colors using the Fire look-up table in Fiji; (Bottom) position of the cytoneme in the interaction (yellow arrowhead points to the tip of the cytoneme). Time is expressed in minutes and seconds. (Scale bar, 5 μm.) (B) Representative frames from time-lapse imaging of an ESC expressing GCaMP6s, where a cytoneme contacts a Wnt3a bead. (Top) generation of Ca²⁺ transients upon cytoneme−Wnt3a bead contact; (Bottom) absence of Ca²⁺ transients, with the bead shown as a black sphere in the DIC panels, outlined by a yellow dashed circle. Time is expressed in minutes and seconds. (Scale bars, 10 μm.) DIC, differential interference contrast. (C) Quantification of the percentage of cells with Ca²⁺ transients in the cytonemes, in different ESC cell lines, in the presence of Wnt3a or iWnt3a beads or cells treated with CNQX (10 μM), MK801 (20 μM), or EGTA (2 mM). (D) Quantification of the percentage of reactive (green) or nonreactive (red) interactions between ESCs and Wnt3a beads in the presence of 5-μM TG, 2-mM EGTA, 10-μM CNQX, 20-μM MK801, or control treatment water or dimethyl sulfoxide (DMSO; CNTRL). Water or DMSO treatment yielded similar percentages of interactions. n ≥ 30 cells from three independent experiments. (E) Quantification of the percentage of reactive (green) or nonreactive (red) interactions between ESCs and TSCs in the presence of 10-μM CNQX. n = 55 cells from three independent experiments. Asterisks indicate statistical significance calculated by Fisher’s exact test for C, D, and E: ns, not significant; *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.