Fig. 4.

Defects in the basement membrane in Fgfr1/2 mutants. (A) E-cadherin immunofluorescence was used to analyze submandibular gland (SMG) epithelial branching between E13.5 and E15.5 in control SMGs (n=6). The epithelial surface (epi) sheet is enclosed by a basement membrane (bm) that undergoes dramatic folding within the mesenchyme (mes), due to recurrent clefting and outgrowth. The median of average curvature of the epithelial surface is represented as violin plots (A′) from E13.5 to E15.5. The horizontal darker dashed bar represents the median. The lighter bars represent the first and third quartiles. Scale bar: 200 µm. (B) The basement membrane (BM) of the SMG was labeled using laminin immunofluorescence in Fgfr1/2 compound mutant sections at E15.5 to estimate the extent of branching. Numerous invaginations (yellow arrowheads) that represent independent clefting events, were detected in control Fgfr1^+/cKO (n=14) and Fgfr2^+/cKO and Fgfr1^cKO/cKO; Fgfr2^+/cKO mutant (n=9) SMGs. A significant reduction in clefting was observed in Fgfr1^+/cKO; Fgfr2^cKO/cKO mutants and Fgfr1^cKO/cKO; Fgfr2^cKO/cKO compound mutant SMGs. Median average curvature for the laminin surface is shown as violin plots for controls and Fgfr1^cKO/cKO; Fgfr2^cKO/cKO mutants (B′). Scale bars: 50 µm. (C) Compound Fgfr1/2 mutant SMGs were analyzed at E14.5 and E15.5. GFP⁺ Fgfr1^cKO/cKO; Fgfr2^cKO/cKO (n=3 at E14.5; n=4 at E15.5) K14 lineage cells were restricted to the inner ECAD⁺ epithelial domain compared with controls (n=7 at E14.5; n=12 at E15.5) at E14.5. Very few GFP⁺ cells could be detected in the peripheral border region (yellow arrowheads) in Fgfr1^cKO/cKO; Fgfr2^cKO/cKO mutants at E14.5. Scale bars: 50 µm. ****P<0.0001; ns, P<0.05 (A′, one-way ANOVA using Bonferroni multiple comparisons test, two tailed; B′, a two-tailed Student's t-test).