Cardiovascular development and survival require Mef2c function in the myocardial but not the endothelial lineage

Abstract

MEF2C is a member of the highly conserved MEF2 family of transcription factors and is a key regulator of cardiovascular development. In mice, Mef2c is expressed in the developing heart and vasculature, including the endothelium. Loss of Mef2c function in germline knockout mice leads to early embryonic demise and profound developmental abnormalities in the cardiovascular system. Previous attempts to uncover the cause of embryonic lethality by specifically disrupting Mef2c function in the heart or vasculature failed to recapitulate the global Mef2c knockout phenotype and instead resulted in relatively minor defects that did not compromise viability or result in significant cardiovascular defects. However, previous studies examined the requirement of Mef2c in the myocardial and endothelial lineages using Cre lines that begin to be expressed after the expression of Mef2c has already commenced. Here, we tested the requirement of Mef2c in the myocardial and endothelial lineages using conditional knockout approaches in mice with Cre lines that deleted Mef2c prior to onset of its expression in embryonic development. We found that deletion of Mef2c in the early myocardial lineage using Nkx2-5^Cre resulted in cardiac and vascular abnormalities that were indistinguishable from the defects in the global Mef2c knockout. In contrast, early deletion of Mef2c in the vascular endothelium using an Etv2::Cre line active prior to the onset of Mef2c expression resulted in viable offspring that were indistinguishable from wild type controls with no overt defects in vascular development, despite nearly complete early deletion of Mef2c in the vascular endothelium. Thus, these studies support the idea that the requirement of MEF2C for vascular development is secondary to its requirement in the heart and suggest that the observed failure in vascular remodeling in Mef2c knockout mice results from defective heart function.

Keywords: Endothelial cells; Endothelium; Heart development; MEF2C; Morphogenesis; Mouse; Vascular development; Vascular remodeling.

Fig. 1.

Cardiac-specific deletion of Mef2c, using Nkx2–5^Cre/+, recapitulates the global Mef2c knockout phenotype. Embryos were harvested at embryonic day 9.5, inspected for gross appearance (A, C, E, G) or sectioned and hematoxylin and eosin (H&E) stained (B, D, F, H). Embryos with global or cardiacspecific loss of Mef2c function (C, G) were noticeably smaller than control littermates (A, E). The main blood vessels in the trunk were present in cross sections of control embryos (red dashed circles in B, F), but absent in global or conditional knockouts (red dashed circles in D, H). Immunohistochemical staining with anti-CD31 antibody in whole mount embryos (I, K, M, O) or yolk sacs (J, L, N, P) shows that the blood vasculature initially forms in all embryos (unlabeled arrows in I, K, M, O indicate intersomitic vessels), but fails to remodel in global and Nkx2–5^Cre cardiac-specific knockout embryos. Yolk sacs of control embryos show arborization (arrowheads in J, N), but it remained as an unremodeled plexus in global and Nkx2–5^Cre conditional knockouts (arrowheads in L, P). cv, cardinal vein; da, dorsal aorta; hrt, heart; lv, left ventricle; nt, neural tube; rv, right ventricle; ys, yolk sac. Scale bars, 100 μm.

Fig. 2.

Genetic fate mapping of Etv2::Cre shows that Cre activity is restricted to hematopoietic and endothelial cells and/or their precursors. Etv2::Cre^Tg/0 mice were crossed to Rosa26^lacZ/lacZ mice, and embryos were collected at E7.0 (A), E8.5 (B), or E 9.5 (C–G), and stained with either Salmon-gal (S-gal) or X-gal to detect ß-galactosidase activity from the Rosa26^lacZ/lacZ reporter. Embryos and yolk sacs were analyzed either as whole mounts (A–C) or sectioned and counterstained with neutral fast red (D– G). Arrowheads in (A, B) indicate staining in extra-embryonic blood and blood vessel-forming regions. At E9.5, the reporter was active exclusively in endocardium (D), vascular endothelial cells of the embryo and yolk sac (arrows in F, G), and blood cells contained within patent vessels. (H–J) ßgalactosidase expression (red) overlapped with CD31 expression (green) in vascular endothelium (asterisks in H, I). (J) MF20 antibody detects myosin heavy chain (Myosin 4), expressed in cardiomyocytes (red) and did not colocalize with endocardial reporter expression (green). Asterisk in (J) marks Etv2::Cre ß-gal activity in the endocardium. cv, cardinal vein; da, dorsal aorta; ec, endocardium; fg, foregut; hrt, heart; lv, left ventricle; nt, neural tube; rv, right ventricle; ys, yolk sac. Scale bars, 100 μm.

Fig. 3.

Complete endothelium-specific deletion of Mef2c prior to its expression in the vasculature does not impair formation of the cardiovascular system. (A-H, J, K) Wild type or Etv2::Cre endothelial-specific Mef2c knockout embryos were harvested at E9.5 (A–D, J, K), E12.5 (E, F), or E14.5 (G, H) and examined for gross appearance or sectioned and H&E stained (C, D). In all cases and at all time points Etv2::Cre endothelial-specific Mef2c knockout embryos were indistinguishable from control littermates. Asterisks mark the carotid artery at E12.5 (E, F). (I) Quantification of exons 2 and 3 in genomic DNA by quantitative PCR revealed efficient excision of exon 2 in endothelial cells by E8.5 in Mef2c^flox/–; Etv2::Cre^Tg/0 conditional knockout embryos. P-values were calculated by Student’s t-test; p=0.0069; n=5. (J, K) In situ hybridization for exon 2 in the Mef2c transcript shows loss of detectable Mef2c transcript in the vasculature at E9.5 in Etv2::Cre endothelial-specific Mef2c knockout embryos; transcripts were still clearly evident in the heart. (L) Exon 2-containing Mef2c transcripts were reduced by 97% at E9.5 in Etv2::Cre endothelial-specific Mef2c knockout embryos compared to controls. Pvalues were calculated by Student’s t-test; p=0.0003; n=6. cv, cardinal vein; da, dorsal aorta; fg, foregut; hrt, heart; nt, neural tube; ys, yolk sac. Scale bars, 100 μm.