Figure 4: Fat4 modulates non-canonical Hippo signalling.
From: Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth
(a) Histological sections of control Fat4Flox/+, conditional mutant Fat4Flox/−;Mesp1Cre/+ and rescued Fat4Flox/−;Yap1Flox/+;Mesp1Cre/+ hearts at P0. (b) Corresponding quantification of the ventricular thickening (n=6 ctrl, 6 mutant, 6 rescued hearts). ***P<0.001 (analysis of variance, (ANOVA)). (c) Quantification of the percentage of mitotic cardiomyocytes positive for PH3 in Fat4Flox/+ (controls, n=5), Fat4Flox/−;Mesp1Cre/+ (mutants, n=5) and Fat4Flox/−;Yap1Flox/+;Mesp1Cre/+ (rescue, n=4) hearts at P0. *P<0.05, **P<0.01 (ANOVA). (d) Percentage of cardiomyocytes per total number of cells, analysed by ImageStream, in Fat4Flox/+ (controls, n=7), Fat4Flox/−;Mesp1Cre/+ (mutants, n=6) and Fat4Flox/−;Yap1Flox/+;Mesp1Cre/+ (rescue, n=3) hearts at P14. *P<0.05 (ANOVA). (e) Western blot showing normal Yap1 phosphorylation at the Hippo kinase target site. (f) Corresponding quantification, from the blot shown in Supplementary Fig. 3b, in Fat4+/+ (n=4), Fat4+/− (n=6) or Fat4−/− (n=4) hearts at P0. (g) Immunodetection of the localization of phospho-resistant Yap1 (Yap5SA) in primary cultures of cardiomyocytes treated with the indicated siRNA. (h) Quantification of its nuclear to cytoplasmic localization, which is increased when Fat4 is downregulated (n=6 cultures) relative to the control (n=6). *P<0.05 (Student test). Ctrl, control; ns, no significant difference. Scale bars: 500 μm in a, 10 μm elsewhere.
