Supplementary Figure 7: Localized Wnt/Fz7-signalling defines the spatial pattern of deep cell cohesion within the blastoderm and validation of the CellFIT3D method for estimating in vivo TST.

a, Exemplary confocal image of WT marginal cells marked by membrane-GFP (green) and transplanted in the blastoderm centre of a WT host embryo marked by membrane-RFP (grey) at the onset of doming. Interstitial fluid is labelled by dextran (blue). Bar plot on the right of cell-cell contact time at dome stage between WT central cells, WT host central cells and WT donor marginal cells (WT centre, n = 20 contacts; WT host centre, n = 17 contacts; WT margin donor, n = 25 contacts). b, Schematic illustration of WT homotypic (WT marginal cells to WT margin), WT heterotypic (WT central cells to WT margin), Fz7a overexpression (oe) WT heterotypic (Fz7a oe central cells to WT margin) and Fz7a oe slb heterotypic (Fz7a oe central cells to slb margin) deep cell transplantations. c, Exemplary confocal images of transplanted donor and host deep cells for the conditions shown in (b) at the onset of doming. Transplanted donor cells are marked by membrane-GFP pseudo-coloured in green (WT homotypic), red (WT heterotypic) and by Fz7a-Neon pseudo-coloured in cyan (Fz7a oe heterotypic), host cells by membrane-RFP (grey for WT, yellow for slb) and interstitial fluid by dextran (blue). d, Bar plot of normalized cell-cell contact time between transplanted donor cells at dome stage (WT homotypic n = 71; WT heterotypic n = 107; Fz7a oe WT heterotypic n = 44; Fz7a oe slb heterotypic n = 32). e, Bright-field/fluorescent images of exemplary deep cell aspirations in the transplantations described in (b). Black arrowheads indicate how far the cells have flown into the micropipette upon application of the same pressure. Bar plot on the right shows tissue viscosity of the transplanted area at the onset of doming calculated from the aspiration experiments (WT homotypic, n = 10 transplants; WT heterotypic, n = 17 transplants; Fz7a oe WT heterotypic, n = 15 transplants; Fz7a oe slb heterotypic, n = 8 transplants). f, Exemplary single plane fluorescence/bright-field images of the transplanted embryos for WT homotypic (WT marginal cells to WT margin), WT heterotypic (WT central cells to WT margin) and Fz7a oe WT heterotypic (Fz7a oe central cells to WT margin) at the onset and end of doming. Transplanted cells are fluorescently marked by H2B-GFP pseudo-coloured in green (WT marginal cells), red (WT central cells) and cyan (Fz7a oe central cells), respectively. Dashed lines indicate the BYI angle at the margin-to-centre transition zone. Schematic diagram and bar plot on the right shows the BYI angle formed at the transition zone between the marginal and central regions at dome stage (indicated by the dashed lines) for the different transplantations described above (WT homotypic n = 28 transplants; WT heterotypic n = 32 transplants; Fz7a oe WT heterotypic n = 12 transplants). g, Representative CellFIT-3D analysis of TST from fluorescent confocal z-stacks of central and marginal cells in an in vivo (left panel) versus ex vivo (right panel) context. For the in vivo context, marginal donor cells (marked by membrane-GFP in green) were transplanted in the central blastoderm of WT host embryos (host cells were marked by membrane-RFP in red and the surrounding interstitial fluid by dextran in blue). For the ex vivo context, central (red) and marginal (green) cells were isolated and cultured as described in the Methods section. The white dashed rectangles show close-ups of the angles measured (yellow marks) at the triplet edges using CellFIT-3D software. h, Plot of the relative TST calculated by the CellFIT-3D software based on the measured angles (see Methods) with marginal cells displaying increased TST compared to central cells ex vivo, but not in vivo (centre in vivo, n = 41 interfaces; margin in vivo, n = 120 interfaces; centre ex vivo, n = 50 interfaces; margin ex vivo, n = 48 interfaces). i, Representative micropipette aspiration (MPA) analysis of TST from aspirated central and marginal cells. Plot on the right shows the calculated TST from the aspiration experiments with marginal cells displaying higher TST than central cells at their interface to the buffer in the pipette (centre, n = 30 embryos; margin, n = 22 embryos). The observation that 3D-VFM and MPA reveal similar tension distributions at comparable interfaces (cell-to-buffer for MPA and cell-to-culture medium for 3D-VFM) suggests that 3D-VFM is a valid method for analysing interfacial tensions both in vivo and ex vivo. Images are representative of 3 embryos each (c) or at least 5 embryos/cultures (g) and 2 (a), 4 (e, f), 5 (h) and 9 (i) biological replicates (different embryos in a, c; independent embryo batches in e, f, h, i). Data are mean ± s.e.m; Kruskal-Wallis test (a, d-g); two-tailed Mann-Whitney test (h, i). Scale bars: (a, c) 20 µm; (e, f) 100 µm; (g, close up) 10 μm.