Fig. S3

Regulation of Cardiomyocyte Proliferation and Heart Regeneration by

Vitamin D (Related to Figure 3)

(A) Lateral view of cyp24a1mGFP expression in 4 dpf wild-type (WT) and cmlc2:dn-vdra

sibling embryos. The image acquisition exposure was increased for both genotypes to

better visualize cardiac expression (arrowheads). Scale bar, 500 μm.

(B) Lateral view of cyp24a1mGFP expression in 4 dpf WT and cmlc2:ca-vdra sibling

embryos. Images were merged with dim brightfield images to indicate whole embryos.

Arrowheads indicate hearts. Scale bar, 500 μm.

(C) Representative maximum projection images of dissected hearts from 4 dpf

cmlc2:H2A-EGFP larvae in WT, cmlc2:dn-vdra and cmlc2:ca-vdra backgrounds. Scale

bar, 50 μm.

(D) Quantification of CM numbers from hearts of WT, cmlc2:dn-vdra and cmlc2:ca-vdra

embryos. ****p < 0.0001. n = 15 for all groups.

(E) Maximum intensity projection images of cTnT expression in 1 mpf WT and cmlc2:cavdra

hearts. Boxed regions were enlarged in inset or on the right side. Note the massive

growth of both ventricular and atrial cardiomyoctes in cmlc2:ca-vdra hearts.

(F) Quantification of 2 mpf WT and cmlc2:dn-vdra heart-to-body ratio. The ratio was

calculated as area of ventricle (mm2) divides body length (mm). ****p < 0.0001. n = 11,

12 respectively.

(G) Lateral view of cyp24a1mGFP expression in 4 dpf WT, fabp10a:dn-vdra, and

fabp10a:ca-vdra embryos. Note the images were merged with brightfield images to

indicate whole embryos. Arrowheads indicate livers. Scale bar, 500 μm.

(H) Maximum intensity projection images of dissected 4 dpf cmlc2:FUCCI hearts from

fabp10a:dn-vdra or fabp10a:ca-vdra and their wild-type siblings. Scale bar, 50 μm.

(I) Quantification of FUCCI+ CMs for experiments in b. n=15, 14, 14, 12, respectively. ns,

not significant.

(J) Maximum intensity projection images of uninjured and 7 dpa hearts of cyp24a1mGFP

reporter. Arrowheads indicate expression in cardiomyocytes.

(K) Maximum intensity projection images of MHC staining for cyp24a1mGFP reporter

crossed with Z-CAT fish at 7 days post tamoxifen treatment (dpi). Arrowhead indicates

expression in cardiomyocytes and asterisks indicate expression in endocardial cells.

(L) Representative images and quantification of Mef2+PCNA+ nuclei versus total Mef2+

nuclei from wild-type or hsp70:dn-vdra zebrafish at 7 dpa after heat-shock induction

protocols, indicating reduced CM proliferation during heart regeneration. Dashed lines

represent amputation planes. n = 10, 8, respectively. ****p < 0.0001. Scale bar, 100 μm.

(M) Representative images and quantification of proliferation of early postnatal (P7)

mouse CMs cultured and treated with vehicle or calcitriol for 72 hours. cTnT is a marker

of CMs and Ki67 is a nuclear marker of cell proliferation. Arrowheads indicate Ki67+ CM

nuclei. Three biological replicate experiments were performed with three technical

repeats for each concentration. ns, not significant; **p < 0.01.

(N) Confocal images of injured ventricles stained for cTnT (CM) expression from wildtype

(N’) or hsp70:dn-vdra (N’’) zebrafish at 50 dpa following daily heat-shocks

beginning at 6 dpa. n = 8, 11, respectively. Magenta numbers at the bottom right

corners are regeneration scores for Figure 3J.