FIGURE 3

Myocardial expression of Ruvbl2 rescues cardiomyocyte hyperproliferation in ruvbl2 ^?/? mutants. (A?B?) Confocal single plane optical sections of endogenous ruvbl2 expression (white signal) relative to myocardium (green signal) processed by RNAscope fluorescent in situ hybridization and immunohistochemistry for GFP in wild-type (A, A?; n = 4) or ruvbl2 ^?/? (B, B?; n = 4) Tg (cmlc2:nucGFP) embryos with DAPI nuclei counterstaining (blue signal). Boxed regions in A and B are shown at higher magnifications in A? and B?, respectively. The black asterix in A? denotes myocardial ruvbl2 expression, while yellow astrices mark presumptive endocardial ruvbl2 expression. (C) Schematic of the transgene generated to achieve constitutive ruvbl2 overexpression in the myocardium (OEmyo). (D) Confocal single plane optical section of the ruvbl2 expression domain (white signal) relative to myocardium (green signal) processed by RNAscope fluorescent in situ hybridization and immunohistochemistry for GFP in Tg (cmlc2:ruvbl2) Tg (cmlc2:nucGFP) double transgenic embryos (n = 4) with DAPI nuclei counterstaining (blue signal). Boxed region in D is shown at higher magnifications in D?. Black asterices in (D?) denote myocardial ruvbl2 expression, which when compared to wildtype (A,A?) is substantially higher. (E?H?) Single plane confocal images of wildtype control (E,E?), OEmyo (F,F?), ruvbl2 ^?/? (G,G?), ruvbl2 ^?/? with OEmyo (H,H?) hearts double immunostained to detect cycling (BrdU+) cardiomyocytes (MF20+) at 72 hpf. Boxed regions in (E?H) are magnified in (E??H?) with white arrows highlighting double positive cells. Sample sizes are n = 6 for all groups. (I) Quantification of fold change in cardiomyocyte proliferation indices between cohorts. Means ± s.d are shown. ns; not significant; *p < 0.05, **p < 0.01, ***p < 0.001. Scale bars: 25 ?m.