Figure 5: Fat4 signalling is required for the integrity of cell junctions and the localization of Amotl1.

(a) Immunodetection of cell junctions (arrowheads), marked by N-cadherin (Cadh2) and Plakophilin2 (Pkp2), in E18.5 hearts. They are disorganized in the absence of Fat4. (b) Transmission electron micrographs of Fat4^+/+ and Fat4^−/− hearts at P0. The electron-dense material of desmosomes (arrows) in the intercalated discs is abnormally spread in mutant hearts. (c) Immunodetection of Amotl1 and Yap1 (white arrowheads) at similar positions near the cell membrane, marked by vinculin (Vcl), of cardiomyocytes, marked by α-actinin (Actn2) in P0 control hearts. (d) Amotl1 is relocalized to cardiomyocyte nuclei (arrowheads) in Fat4^−/− hearts at E18.5. *, non-cardiomyocyte nuclei. (e) Quantification of nuclear to cytoplasmic localization of Amotl1 in Fat4^+/+ (n=3), Fat4^+/− (n=3) or Fat4^−/− (n=3) hearts. *P<0.05 (analysis of variance (ANOVA)). (f) The percentage of proliferating Ki67-positive cardiomyocytes, counted by flow cytometry from primary cultures of neonatal rat cardiomyocytes, indicates a genetic rescue of Fat4 siRNA by Amotl1 siRNA and lower proliferation with Amotl1 siRNA (n=20 ctrl, 20 Fat4, 16 Amotl1 and 16 Fat4+Amotl1 siRNA cultures). **P<0.01, ***P<0.001 (ANOVA). Ctrl, control; ns, no significant difference. Scale bars: 0.2 μm in b, 5 μm in c, 10 μm elsewhere.