Epicardium-derived progenitor cells require beta-catenin for coronary artery formation - PubMed (original) (raw)

Epicardium-derived progenitor cells require beta-catenin for coronary artery formation

Mónica Zamora et al. Proc Natl Acad Sci U S A. 2007.

Abstract

We have previously identified several members of the Wnt/beta-catenin pathway that are differentially expressed in a mouse model with deficient coronary vessel formation. Systemic ablation of beta-catenin expression affects mouse development at gastrulation with failure of both mesoderm development and axis formation. To circumvent this early embryonic lethality and study the specific role of beta-catenin in coronary arteriogenesis, we have generated conditional beta-catenin-deletion mutant animals in the proepicardium by interbreeding with a Cre-expressing mouse that targets coronary progenitor cells in the proepicardium and its derivatives. Ablation of beta-catenin in the proepicardium results in lethality between embryonic day 15 and birth. Mutant mice display impaired coronary artery formation, whereas the venous system and microvasculature are normal. Analysis of proepicardial beta-catenin mutant cells in the context of an epicardial tracer mouse reveals that the formation of the proepicardium, the migration of proepicardial cells to the heart, and the formation of the primitive epicardium are unaffected. However, subsequent processes of epicardial development are dramatically impaired in epicardial-beta-catenin mutant mice, including failed expansion of the subepicardial space, blunted invasion of the myocardium, and impaired differentiation of epicardium-derived mesenchymal cells into coronary smooth muscle cells. Our data demonstrate a functional role of the epicardial beta-catenin pathway in coronary arteriogenesis.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Fig. 1.

Ablation of β-catenin protein in proepicardial and epicardial cells. Immunohistochemical staining of β-catenin protein in sections from embryonic hearts. (A–B′) WT E9.5 (A and A′) and epicardial β-catenin mutants E9.5 (epiBC-KO) (B and B′) show the specific ablation of β-catenin in the proepicardial mutant cells, compared with the WT control littermate. Dotted circles in A′ and B′ indicate the localization of the proepicardium in the embryo E9.5. A and B are magnifications of the proepicardia in WT (A) and mutant (B). (C and D) In the epicardium of older embryos (E13.5) β-catenin appears in the WT epicardial cells (C), whereas its expression is ablated in the mutant epicardial cells (D). (C′ and D′) Zoom image that visualizes details of epicardial β-catenin expression. β-Catenin signal is detected by peroxide staining (brown) mainly in the cell membrane of WT embryos and is absent in the mutant epicardium (arrows). Nuclei are counterstained with hematoxylin staining (blue). V, ventricle; Atr, atrium; OFT, outflow track; PE, proepicardium; epi, epicardium; myo, cardiac myocytes. (Magnifications: A and B, ×630; A′ and B′, ×100; C and D, ×400; C′ and D′, ×630.)

Fig. 2.

Fig. 2.

Cardiac growth defects in epicardium-restricted β-catenin mutant mice. (A–C) Gross morphological comparison of cardiac size between E12.5 (A), E15.5 (B), and E18.5 (C) WT hearts (left hearts) and epiBC-KO hearts (right hearts). (D–I) H&E staining of E13.5 WT mice (D, F, and H) and epiBC-KO mice (E, G, and I). As indicated by the boxes, F–I are magnifications of images in D and E. (J–M) BrdUra immunostaining of paraffin sections from E12.5 (J and K), and E15.5 (L and M) hearts. BrdUra staining is brown, and nuclear counterstaining with hematoxylin is blue. Atr, atrium; RV, right ventricle; LV, left ventricle; ivs, interventricular septum; cz, compact zone myocardium; epi, epicardium. Arrowheads point to the interventricular sulcus. (N) Quantification of BrdUra-positive nuclei in cardiac samples at different ages of development shows hypoproliferation of epiBC-KO hearts after E13.5. Data are expressed as percentage of the mean ± SE relative to control and compared by using two-tailed Student's t analysis. Significant differences were defined as P < 0.05. (Magnifications: A–C, ×20; D and E, ×100; F–I, ×200; J–M, ×400.)

Fig. 3.

Fig. 3.

β-Galactosidase staining of proepicardial derivatives using Wilms' tumor transgenic mouse shows deficient expansion of the subepicardial space and impaired EMT in the epiBC-KO mice. (A and B) Proepicardial derivatives (blue) in E13.5 wt LacZ (A) and E13.5 epiBC-KOLacZ counterstained with nuclear fast red (B). Observe the lack of subepicardial space (arrows) and lack of subepicardial vascularization (arrowheads) in the mutant hearts. (C and D) In vivo EMT as measured by coexpression of Wilm's tumor and vimentin in the epicardium. Epithelial cells activated to EMT coexpress both epithelial (Wilm's tumor, blue) and mesenchymal markers (vimentin, brown). Coexpression (arrowheads) is not detected in epiBC-KO epicardium. (E–H) For migration analysis, tenascin/LacZ costaining was performed in hearts of E13.5 WTLacZ (E), E13.5 epiBC-KOLacZ (F), E14.5 WTLacZ (G), and E14.5 epiBC-KOLacZ (H) mice. Red arrows point to migrating cells. (Magnifications: A and B, ×200; E–H, ×400; C and D, ×630; Insets A, B, G, and H, ×100.)

Fig. 4.

Fig. 4.

Epicardium-specific β-catenin mutation leads to coronary artery defects as shown by whole-mount PECAM-1 immunostaining and α-SMA. (A–B′) Ventral view of the intact whole-mount PECAM-1 staining of WT (A and A′) and epiBC-KO (B and B′) mice shows the absence of coronary arterial vasculature (red arrows in computerized image A′) in the epiBC-KO mice at E18.5. (C–D′) Dorsal views depict coronary veins vasculature in WT (C and C′) and epiBC-KO (D and D′) embryos. Note that the coronary veins are well developed in epiBC-KO mice (blue in computerized image D′), although some nonvascular areas and numerous blood-filled cysts are detected in the subepicardial space (B′, blue circles). (E and F) Paraffin sections from E18.5 hearts were immunostained with α-SMA antibody in WT (E) and mutant (F) mice to analyze the smooth muscle component of the coronary vessels (arrows). (G and H) H&E stain (G) or Van Gieson stain (H) were used for histological analysis of the smooth muscle layer on the vessels. White arrowheads point to elastic fibers. (Magnifications: A–D′, ×20; E and F, ×200; G, H, and Insets E and F, ×630.)

Fig. 5.

Fig. 5.

EMT and SMC differentiation of epicardium cultured cells. (A) EMT activation upon treatment with FBS and FGF2 in epicardium explanted cells from WT (Left) or epiBC-KO (Right) E12.5 embryo hearts. (B) SMC differentiation of WT epicardium cultured cells upon stimulation with vehicle (Upper Left), Wnt3A (Upper Right), or TGF-β1 (Lower Left). Stimulation with TGF-β1 was also tested in epicardium cells from epiBC-KO (Lower Right). Phalloidin (red) shows cell morphology, DAPI (blue) indicates nuclear staining, and α-SMA expression by immunostaining is green. (Magnifications: ×200.)

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