FIGURE 2

Ruvbl2 suppresses cardiomyocyte proliferation during embryonic heart growth. (A) Schematic of the ruvbl2 locus deletion (ruvbl2 ^Δ ) targeting strategy by CRISPR-Cas9 genome editing. gRNAs (blue arrowheads) were designed that target genomic regions outside of the ruvbl2 5′ and 3′ UTR. Location of PCR primers used to detect the wild-type (WT; turquoise arrows) and mutant (Δ; orange arrows) alleles. (B) Image of a DNA agarose gel showing amplicons for the wild-type (+) and mutant (Δ) alleles. (C,D) and (K,L) Brightfield images of wild-type (C,K), ruvbl2 ^Δ/Δ (D), ruvbl2 ^lik/Δ (L) embryos at 72 hpf. Anterior left, lateral view. (E,F) and (M,N) Confocal projections of fluorescent cardiomyocyte nuclei in hearts of 72 hpf CTRL (F; n = 6 and M; n = 6), ruvbl2 ^Δ/Δ (G; n = 6), and ruvbl2 ^lik/Δ (N; n = 6) Tg (cmlc2:nucGFP) embryos. (G,O) Quantification of fold change in ventricular cardiomyocyte number in 72 hpf CTRL and ruvbl2 ^Δ/Δ (G) or CTRL and ruvbl2 ^lik/Δ (O) hearts. Means ± s.d are shown. ***p < 0.001; **p < 0.01. (H–I’,P–Q′) Single plane confocal images of CTRL (H; n = 6 and P; n = 6), ruvbl2 ^Δ/Δ (N; n = 6), and ruvbl2 ^lik/Δ (Q; n = 6) hearts double immunostained to detect cycling (BrdU+) cardiomyocytes (MF20+) at 72 hpf. Boxed regions in (H,I,P,Q) are magnified in (H′,I′,P′,Q′) with white arrows highlighting double positive cells. (J,R) Quantification of cardiomyocyte proliferation indices in CTRL and ruvbl2 ^Δ/Δ (J) or CTRL and ruvbl2 ^lik/Δ (R) ventricles. Means ± s.d are shown. *p < 0.05. Scale bars: 25 μm.