IMAGE

Fig. S3

ID
ZDB-IMAGE-190716-7
Source
Figures for Han et al., 2019
Image
Figure Caption

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.

Acknowledgments
This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image.

Reprinted from Developmental Cell, 48(6), Han, Y., Chen, A., Umansky, K.B., Oonk, K.A., Choi, W.Y., Dickson, A.L., Ou, J., Cigliola, V., Yifa, O., Cao, J., Tornini, V.A., Cox, B.D., Tzahor, E., Poss, K.D., Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish, 853-863.e5, Copyright (2019) with permission from Elsevier. Full text @ Dev. Cell