Fig. 6.

Integrin signaling cooperates with FGFR function during submandibular gland branching. (A) Submandibular gland (SMG) explants from various Fgfr1 and Fgfr2 compound mutants (n=18; E14.5) were cultured for 40 h in presence of Mn²⁺ (integrin signaling activation) and compared with untreated controls. Mutants were analyzed in more than 20 separate experiments. Tissues were subjected to immunofluorescence on eight separate occasions, and data were compiled from six separate experiments. Explants were imaged during branching at 18 h and 40 h of culture. The curvature of the epithelial surface is indicated by dashed red lines. Median average curvatures (error bar represent the 95% confidence interval, CI) and average change in bud numbers (error bar represent s.d.) were measured for each genotype and are shown in the bar graphs. Robust branching was observed upon integrin signaling activation in all mutants except for Fgfr1^cKO/cKO; Fgfr2^cKO/cKO. Scale bars: 100 µm. ****P<0.0001; ns, P<0.05 (two-way ANOVA using Bonferroni multiple comparisons test, two tailed). (B) SMG explants (n=14; E14.5) were treated with an integrin β1-blocking antibody from various Fgfr1 and Fgfr2 compound mutants. Mutants were analyzed in more than 20 separate experiments. Tissues were subjected to immunofluorescence on eight separate occasions, and data were compiled from six separate experiments. Epithelial branching was assessed at 18 h and 40 h during culture (median average curvature is plotted as bar graphs in Fig. S6). Neither control Fgfr1^+/cKO; Fgfr2^+/cKO nor Fgfr1^cKO/cKO; Fgfr2^cKO/cKO compound mutant explants showed robust changes in branching upon integrin signaling inhibition. However, several GFP⁺ double mutant cells extruded from the explants (yellow arrowheads). Scale bars: 100 µm. (C) Upon integrin signaling inhibition for 40 h, explants were immunostained for ECAD to mark epithelial cells. Compared with Fgfr1^+/cKO; Fgfr2^+/cKO controls, several GFP⁺ cells in Fgfr1^cKO/cKO; Fgfr2^cKO/cKO explants extruded out of the ECAD⁺ epithelial domain into the surrounding mesenchyme (yellow arrowheads). Scale bars: 200 µm.