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Han et al., 2019 - Vitamin D Stimulates Cardiomyocyte Proliferation and Controls Organ Size and Regeneration in Zebrafish. Developmental Cell   48(6):853-863.e5 Full text @ Dev. Cell
ADDITIONAL FIGURES
EXPRESSION / LABELING:
Genes:
Antibodies:
Fish:
Conditions:
Anatomical Terms:
Stage Range: Day 4 to Adult
PHENOTYPE:
Fish:
Conditions:
Observed In:
Stage Range: Day 4 to Adult

Fig. 3

Tissue-Intrinsic Effects of Vitamin D Signaling and Impact on Heart Regeneration

(A) MIP images of cyp24a1mGFP expression in dissected hearts. Scale bar, 50 μm.

(B) MIP images of cyp24a1mGFP expression in dissected hearts. Scale bar, 50 μm.

(C) MIP images of dissected cmlc2:FUCCI hearts in indicated transgenic backgrounds. Scale bar, 50 μm.

(D) Quantification of FUCCI+ CMs in (C). Mean ± SD, Mann-Whitney test and Student's t test. ∗∗∗p 0.001; ∗∗∗∗p < 0.0001. n = 9–10.

(E) Examples of 1 mpf cmlc2:ca-vdra and WT zebrafish. Red arrow indicates gross pericardial enlargement caused by cardiomegaly in cmlc2:ca-vdra zebrafish. Scale bar, 2 mm.

(F) Darkfield images of dissected 1 mpf hearts. a, atrium; ba, bulbus arteriosus; v, ventricle. Scale bar, 500 μm.

(G) Darkfield images of dissected livers (l) and hearts (h) from 1 mpf zebrafish. Scale bar, 500 μm.

(H) Examples of Mef2/PCNA staining of 7 dpa hearts after a single injection. Dashed lines represent amputation planes. Scale bar, 100 μm.

(I) Quantification of CM proliferation indices in (H). Mean ± SD, Mann-Whitney test. p < 0.05. n = 9.

(J) Quantified heart regeneration scores of WT or hsp70:dn-vdra(dn-vdra) zebrafish given daily heat shocks from 6 to 50 dpa. Regeneration scores (reg scores) indicate vigorous regeneration (1), partial regeneration (2), or blocked regeneration (3), as shown in Figure S3N. Two-tailed Fisher’s exact test. ∗∗∗p < 0.001. n = 8–11.

(K) Representative images of cTnT staining at 50 dpa, after daily heat shock from 6 dpa. Scale bar, 100 μm.

(L) Representative images of AFOG stains to indicate fibrin (red) and collagen (blue) within the same hearts in (K). Arrowheadindicates scar tissue.

Gene Expression Details
Gene Antibody Fish Conditions Stage Anatomy Assay
GFP cyp24a1pd272Tg/+ control Day 4 cardiac ventricle IFL
cyp24a1pd272Tg/+ ; pd301Tg/+ control Day 4 cardiac ventricle IFL
pd302Tg/+ ; cyp24a1pd272Tg/+ control Day 4 atrium IFL
Day 4 cardiac ventricle IFL
Day 4 heart IFL
mCherry pd57Tg/+ ; pd302Tg/+ ; pd58Tg/+ control Day 4 cardiac muscle cell IFL
pd57Tg/+; pd58Tg/+ control Day 4 cardiac muscle cell IFL
pd58Tg/+ ; pd57Tg/+ ; pd301Tg/+ control Day 4 cardiac muscle cell IFL
TagBFP cyp24a1pd272Tg/+ ; pd301Tg/+ control Day 4 cardiac ventricle IFL
pd302Tg/+ ; cyp24a1pd272Tg/+ control Day 4 cardiac ventricle IFL
Venus pd57Tg/+ ; pd302Tg/+ ; pd58Tg/+ control Day 4 cardiac muscle cell IFL
pd57Tg/+; pd58Tg/+ control Day 4 cardiac muscle cell IFL
pd58Tg/+ ; pd57Tg/+ ; pd301Tg/+ control Day 4 cardiac muscle cell IFL
Antibody Labeling Details
Antibody Assay Fish Conditions Stage Anatomy
Ab1-mef2 IHC WT resection: cardiac ventricle Adult cardiac muscle cell
IHC WT chemical treatment by injection: alfacalcidol, resection: cardiac ventricle Adult cardiac muscle cell
Ab2-pcna IHC WT resection: cardiac ventricle Adult cardiac ventricle regenerating tissue
IHC WT chemical treatment by injection: alfacalcidol, resection: cardiac ventricle Adult cardiac ventricle regenerating tissue
Phenotype Details
Fish Conditions Stage Phenotype
WT chemical treatment by injection: alfacalcidol, resection: cardiac ventricle Adult regenerating tissue cardiac muscle cell proliferation increased occurrence, abnormal
cyp24a1pd272Tg/+ ; pd301Tg/+ control Day 4 cardiac ventricle GFP expression decreased amount, abnormal
pd276Tg/+ resection: cardiac ventricle, heat shock Adult cardiac muscle tissue regeneration decreased occurrence, abnormal
Adult cardiac muscle tissue regeneration decreased rate, abnormal
pd302Tg/+ control Days 30-44 heart increased size, abnormal
pd302Tg/+ ; cyp24a1pd272Tg/+ control Day 4 atrium GFP expression increased amount, abnormal
Day 4 cardiac ventricle GFP expression increased amount, abnormal
Day 4 heart GFP expression increased amount, abnormal
Day 4 heart GFP expression increased distribution, abnormal
Day 4 heart hyperplastic, abnormal
Day 4 heart increased size, abnormal
pd304Tg/+ control Days 30-44 heart size, normal
Days 30-44 liver increased size, abnormal
pd57Tg/+ ; pd302Tg/+ ; pd58Tg/+ control Day 4 cardiac ventricle cardiac muscle cell proliferation increased occurrence, abnormal
pd58Tg/+ ; pd57Tg/+ ; pd301Tg/+ control Day 4 cardiac ventricle cardiac muscle cell proliferation decreased occurrence, abnormal
Acknowledgments:
ZFIN wishes to thank the journal Developmental Cell for permission to reproduce figures from this article. Please note that this material may be protected by copyright.

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