Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2011 Mar 15;20(3):397-404.
doi: 10.1016/j.devcel.2011.01.010.

Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration

Kazu Kikuchi  1 , Jennifer E Holdway, Robert J Major, Nicola Blum, Randall D Dahn, Gerrit Begemann, Kenneth D Poss
Affiliations

Retinoic acid production by endocardium and epicardium is an injury response essential for zebrafish heart regeneration

Kazu Kikuchi et al. Dev Cell. .

Abstract

Zebrafish heart regeneration occurs through the activation of cardiomyocyte proliferation in areas of trauma. Here, we show that within 3 hr of ventricular injury, the entire endocardium undergoes morphological changes and induces expression of the retinoic acid (RA)-synthesizing enzyme raldh2. By one day posttrauma, raldh2 expression becomes localized to endocardial cells at the injury site, an area that is supplemented with raldh2-expressing epicardial cells as cardiogenesis begins. Induced transgenic inhibition of RA receptors or expression of an RA-degrading enzyme blocked regenerative cardiomyocyte proliferation. Injured hearts of the ancient fish Polypterus senegalus also induced and maintained robust endocardial and epicardial raldh2 expression coincident with cardiomyocyte proliferation, whereas poorly regenerative infarcted murine hearts did not. Our findings reveal that the endocardium is a dynamic, injury-responsive source of RA in zebrafish, and indicate key roles for endocardial and epicardial cells in targeting RA synthesis to damaged heart tissue and promoting cardiomyocyte proliferation.

PubMed Disclaimer

Figures

Figure 1
Figure 1. Resection of the Ventricular Apex Stimulates Immediate, Organ-wide Morphological and Molecular Changes in Endocardium
(A–D) Transmission electron microscope (TEM) analyses of endocardium in uninjured (A) and injured ventricles (B–D). Arrowheads, endocardial nuclei. Arrows, endocardial cell bodies. M, cardiac muscle. Asterisk, red blood cell. Scale bar, 2 µm. (E–K) raldh2 expression assessed by in situ hybridization (E–J) and Raldh2 immunostaining (K) in uninjured (E) and injured (F–K) ventricles. Brackets in (I–K), injury site. Arrows in (J, K), epicardial cells. Scale bar (E–Q), 100 µm. (L–N) Confocal images of altered endocardial cell shape, and enhanced flk1 driven DsRed2 fluorescence in the injury site (A, brackets) in a 7 dpa cmlc2:EGFP; flk1:DsRed2 double transgenic ventricle. Arrowheads, endocardial nuclei. Arrows, endocardial lining of myofiber. An antibody against DsRed was used. DAPI (4'-6-Diamidino-2-phenylindole) stains nuclei. (O–Q) Sections of 7 dpa fli1:EGFP (O), hand2:EGFP (P), or gata5:EGFP (Q) transgenic ventricles. Brackets, injury sites. Arrowheads in inset, Raldh2+/EGFP+ endocardial nuclei with rounded morphology (G–I).
Figure 2
Figure 2. Induction of raldh2 Expression in Various Injury Models
(A,B) Stab injuries into the ventricular apex assessed for raldh2 induction (A) and fli1:EGFP expression (B) at 7 days post-stab (dps). Arrows, needle entry site. Scale bar, 100 µm. (C) Confocal image of Raldh2 immunofluorescence in fli1:EGFP+ endocardial cells with rounded morphology at the injury site (arrowheads). Scale bar, 20 µm. (D–H) raldh2 induction after intraperitoneal LPS or vehicle (PBS) injection. Scale bar, 100 µm (D–L). (I–L) Endocardial raldh2 (J), hand2 (K), and gata5 (L) expression surrounding spontaneous infarcts (asterisks) within cultured ventricular explants. Dead cardiac tissue was identifiable by the absence of cell nuclei (I).
Figure 3
Figure 3. Transgenic Inhibition of RA Signaling Blocks Cardiomyocyte Proliferation during Regeneration
(A) Assessment of Mef2+PCNA+ cells (arrows) in wild-type (wt) and hsp70:dn-zrar transgenic fish at 7 dpa, after a single heat-shock at 6 dpa. Maximum projection images of 10 µm z-stacks are shown. Insets, high-magnification images of the rectangle. Arrowheads, proliferating epicardial cells. Brackets, injury site. Scale bar, 100 µm (A, C). (B) Quantification of CM proliferation in wt and hsp70:dn-zrar transgenic fish at 7 dpa. Data are mean ± SEM from 6 animals each (3097 wt and 2482 transgenic CMs analyzed). *p < 3 × 10−5, Student's t-test. (C) Assessment of Mef2/PCNA double-positive cells (arrows) in wt and hsp70:cyp26a1 transgenic fish at 7 dpa, after a single heat-shock at 6 dpa. Maximum projection images of 10 µm z-stacks are shown. (D) Quantification of CM proliferation in wt and hsp70:cyp26a1 transgenic fish at 7 dpa. Data are mean ± SEM from 4 wt and 6 transgenic animals (3888 wt and 4760 transgenic CMs analyzed). *p < 2 × 10−4, Student's t-test.
Figure 4
Figure 4. Cardiac Injury Responses in Polypterus, Mouse, and Zebrafish
(A–D) raldh2 (p. raldh2) expression by in situ hybridization in uninjured (A) and injured (B–D) polypterus ventricles. Arrowheads in (A), pigment cells. Brackets in (B–D), injury site. Insets in (B–D), lateral ventricular wall including epicardium (ep). Arrows in (D), p. raldh2-expressing epicardial cells. Scale bar, 100 µm (A–H). (E–G) Assessment of Mef2+PCNA+ cells (arrows) in uninjured (E) and injured (F, G) polpyterus ventricles. Brackets in (F), injury site. Insets, high-zoom images of the rectangle. Arrowhead in (G), entry site of glass needle. Arrows in (F, G), proliferating CMs. (H) In situ hybridization of p. raldh2 after stab injury (arrowhead). (I–L) Raldh2 (m. Raldh2) expression by in situ hybridization at various timepoints post-ligation (pl). Insets in (J–L), high-zoom images of the rectangle. LVL, left ventricular lumen; ep, epicardium; en, endocardium. Scale bar, 200 µm. (M) Summary of injury responses observed in polypterus, zebrafish, and mouse hearts.
See this image and copyright information in PMC

References

    1. Brade T, Kumar S, Cunningham TJ, Chatzi C, Zhao X, Cavallero S, Li P, Sucov HM, Ruiz-Lozano P, Duester G. Retinoic acid stimulates myocardial expansion by induction of hepatic erythropoietin which activates epicardial Igf2. Development (Cambridge, England) 2011;138:139–148. - PMC - PubMed
    1. Chen J, Kubalak SW, Chien KR. Ventricular muscle-restricted targeting of the RXRalpha gene reveals a non-cell-autonomous requirement in cardiac chamber morphogenesis. Development (Cambridge, England) 1998;125:1943–1949. - PubMed
    1. Curcio A, Noma T, Naga Prasad SV, Wolf MJ, Lemaire A, Perrino C, Mao L, Rockman HA. Competitive displacement of phosphoinositide 3-kinase from beta-adrenergic receptor kinase-1 improves postinfarction adverse myocardial remodeling. American journal of physiology. 2006;291:H1754–H1760. - PubMed
    1. Gittenberger-de Groot AC, Winter EM, Poelmann RE. Epicardium-derived cells (EPDCs) in development, cardiac disease and repair of ischemia. J Cell Mol Med. 2010;14:1056–1060. - PMC - PubMed
    1. Hoover LL, Burton EG, Brooks BA, Kubalak SW. The expanding role for retinoid signaling in heart development. The Scientific World Journal. 2008;8:194–211. - PMC - PubMed

Publication types

MeSH terms

Associated data