Zebrafish Mef2ca and Mef2cb are essential for both first and second heart field cardiomyocyte differentiation

Abstract

Summary Mef2 transcription factors have been strongly linked with early heart development. D-mef2 is required for heart formation in Drosophila, but whether Mef2 is essential for vertebrate cardiomyocyte (CM) differentiation is unclear. In mice, although Mef2c is expressed in all CMs, targeted deletion of Mef2c causes lethal loss of second heart field (SHF) derivatives and failure of cardiac looping, but first heart field CMs can differentiate. Here we examine Mef2 function in early heart development in zebrafish. Two Mef2c genes exist in zebrafish, mef2ca and mef2cb. Both are expressed similarly in the bilateral heart fields but mef2cb is strongly expressed in the heart poles at the primitive heart tube stage. By using fish mutants for mef2ca and mef2cb and antisense morpholinos to knock down either or both Mef2cs, we show that Mef2ca and Mef2cb have essential but redundant roles in myocardial differentiation. Loss of both Mef2ca and Mef2cb function does not interfere with early cardiogenic markers such as nkx2.5, gata4 and hand2 but results in a dramatic loss of expression of sarcomeric genes and myocardial markers such as bmp4, nppa, smyd1b and late nkx2.5 mRNA. Rare residual CMs observed in mef2ca;mef2cb double mutants are ablated by a morpholino capable of knocking down other Mef2s. Mef2cb over-expression activates bmp4 within the cardiogenic region, but no ectopic CMs are formed. Surprisingly, anterior mesoderm and other tissues become skeletal muscle. Mef2ca single mutants have delayed heart development, but form an apparently normal heart. Mef2cb single mutants have a functional heart and are viable adults. Our results show that the key role of Mef2c in myocardial differentiation is conserved throughout the vertebrate heart.

Hand2 elevates cardiomyocyte production during zebrafish heart development and regeneration

“…At the same time, hand2-overexpressing embryos exhibited expanded expression of mef2cb (Fig. 7G,H), which is found in myocardial progenitor cells at this stage (Hinits et al, 2012). However, not all myocardial progenitor markers are similarly expanded, as we found that hand2-overexpressing embryos exhibit normal expression of nkx2.5 at 12 somites (Fig.…”

“…As overexpression of Hand1 has been shown to enhance proliferation within the outflow tract in mice (Risebro et al, 2006), we were especially interested in determining the origins of the elongated outflow tract in hand2-overexpressing embryos. First, we examined the expression of mef2cb at 36 hpf; at this stage, mef2cb marks a population of SHF-derived cells that contributes to the outflow tract (Lazic and Scott, 2011;Hinits et al, 2012). We found excess mef2cb expression at the arterial pole of the heart in hand2-overexpressing embryos (Fig.…”

Glypican4 promotes cardiac specification and differentiation by attenuating canonical Wnt and Bmp signaling

“…Previous work by various research groups has demonstrated that cells which are located in a region posterior to the arterial pole and express nkx2.5, ltbp3 and mef2cb contribute to cardiac cells of the arterial pole (Lazic and Scott, 2011;Zhou et al, 2011;Hinits et al, 2012). We therefore analyzed the expression of these genes and found that they are still expressed in kny/gpc4 mutant embryos (supplementary material Fig.…”

“…Using a developmental timing assay and expression of a photoconvertible protein, we demonstrated that the observed increase in myl7 + cardiomyocytes is due to the continuous differentiation of cardiomyocytes at the poles of the heart tube, thus explaining the increase in both atrial and ventricular cardiomyocytes (de Pater et al, 2009). A distinct population of progenitor cells that express nkx2.5, mef2c and latent TGF-β-binding protein 3 (ltbp3) is located anterior to the arterial pole of the linear heart tube (Lazic and Scott, 2011;Zhou et al, 2011;Hinits et al, 2012). Moreover, lineage tracing of ltbp3-expressing cells demonstrated that these progenitors give rise to three cardiovascular lineages (myocardium, endocardium and smooth muscle cells) at the arterial pole (Zhou et al, 2011).…”

The AP-1 transcription factor component Fosl2 potentiates the rate of myocardial differentiation from the zebrafish second heart field

“…1A-D), suggesting preliminarily that Fosl2 supports ventricular morphogenesis and/or growth. However, because multiple studies have reported discrepancies between morphant and genetic loss-of-function phenotypes (den Broeder et al, 2009;Hinits et al, 2012;Kok et al, 2015;Rossi et al, 2015;Wright et al, 2004), we opted to isolate null alleles of fosl2 using TALEN-mediated genome editing before implicating Fosl2 in ventricular development.…”