Figure 2: Fat4 restricts cell proliferation and hypertrophy.
From: Amotl1 mediates sequestration of the Hippo effector Yap1 downstream of Fat4 to restrict heart growth
Immunodetection (a) and quantification (b) of the percentage of mitotic cardiomyocytes positive for phosphorylated histone H3 (PH3) (arrowheads) in Fat4+/+ (n=6), Fat4+/− (n=6) and Fat4−/− (n=5) hearts at P0. **P<0.01 (analysis of variance (ANOVA)). (c) Percentage of cardiomyocytes per total number of cells, analysed by ImageStream, in Fat4Flox/+ (controls, n=7) and Fat4Flox/−;Mesp1Cre/+ (mutants, n=6) hearts at P14. *P<0.05 (Student test). (d) Relative transcript levels of cell cycle (Aurkb, Ccna2, Cdc20), cycle exit (Cdkn1b) or survival (Birc2/5) genes in Fat4+/+ (n=5), Fat4+/− (n=5) or Fat4−/− (n=7) hearts at P0. *P<0.05, **P<0.01, ***P<0.001 (ANOVA). (e) Increased percentage of proliferating Ki67-positive cardiomyocytes, as counted by flow cytometry from primary cultures of neonatal rat cardiomyocytes treated with Fat4 siRNA (n=20) compared with control (ctrl) cultures (n=20). ***P<0.001 (Student test). (f) Immunodetection of cardiomyocyte cross-sectional area, with an antibody to Caveolin3. (g) Quantification of this in the interventricular septum indicates cell hypertrophy in Fat4−/− (n=6) compared with Fat4+/+ (n=9) hearts at P0. **P<0.01 (Student test). (h) Expression of positive (Nppb and Acta1 (skeletal actin)) and negative (Myh6 (α myosin heavy chain)) hypertrophy markers and of a marker of wall stress (Nppa) quantified by RT–qPCR in Fat4+/+ (n=5), Fat4+/− (n=5) or Fat4−/− (n=7) hearts at P0. *P<0.05, **P<0.01 (ANOVA). ns, no significant difference. Scale bars: 10 μm.
