Mef2ca and mef2cb expression during early cardiogenesis. In situ mRNA hybridisation of indicated genes, or immunodetection of Mef2ca/cb protein (G) for wild type (A–E, G–I) and Tg(myl7:EGFP) (F) in dorsal (A and B) or lateral (C–E) views of wholemount embryos or in flatmounts of dorsal views of the cardiac region, anterior to top (F–I). A and B. Mef2ca and mef2cb mRNAs accumulate in the bilateral heart fields in ALPM (black arrowheads) and adaxial cells (green arrowhead). C–E. Egr2b expression in rhombomeres 3 and 5, and ntl expression in the notochord positions the row of ventral cells in the ALPM (black arrowheads; D and E) that contain mef2ca and mef2cb, but not mef2aa mRNA (C). F. Confocal stack of Tg(myl7:EGFP) heart at 25 ss showing co-localisation of mef2cb mRNA (Fast Red) and EGFP. G. Mef2c protein in nuclei of a similar crescent of CMs spanning the midline. H and I. By 24 hpf, both mef2ca (H) and mef2cb (I) mRNAs are detected weakly in the heart tube, but mef2cb also accumulates strongly in the venous (blue arrow) and arterial (pink arrow) poles of the heart. Scale=100 μm (A–F), 20 μm (G–I). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Early cardiomyocytes fail to differentiate after loss of Mef2c function. A–F. In situ mRNA hybridisation for myl7, vmhc and smyd1b in wild type control (A), mef2ca^b1086+mef2cb MO (B), mef2ca^b1086 (C) mef2cb MO (D), mef2cb^fh288 (E) and mef2ca^b1086;mef2cb^fh288 (F) embryos, shown in wholemounts in dorsal view, anterior to top. Loss of both mef2ca and mef2cb function greatly reduces myl7, vmhc and smyd1b mRNAs in the bilateral heart fields (arrowheads; A,B and F). Mef2ca^b1086 mutant embryos have weak myl7 and smyd1b mRNAs early, but recover later, and show no change in vmhc (C). Mef2cb single morphants or mef2cb^fh288 mutant show no changes (D). G. Immunostaining for MyHC (A4.1025) in 24 ss mef2ca^b1086;mef2cb^fh288 embryos and their siblings, shown in wholemounts in dorsal view, anterior to top (left panel) and lateral view, anterior to left (right panel). No MyHC is detected in the heart, whereas somitic muscle appears normal (white arrowheads). Scale=100 μm.

Redundant and specific functions of Mef2ca and Mef2cb drive cardiomyogenesis and heart tube formation. Immunodetection of MyHC (confocal stacks, top panels) or in situ mRNA hybridisation for indicated genes (A–F, lower panels and G-J) in hearts of 24 hpf zebrafish embryos shown in a dorsal view, anterior to top. A,B and F. Loss of both mef2ca (tn213 allele, in MyHC and b1086 allele, in bmp4 and myl7+vmhc) and mef2cb function (B and F) led to lack of all markers, compared with control (A). Note the few cells expressing myl7 only (black arrowhead) or both myl7 and vmhc (white arrowhead). C.Mef2ca mutants have a normal heart. D and E.Mef2cb morphants have a shortened heart with substantial loss of both atrial and ventricular volume, yet mef2cb^fh288 mutants have a normal heart. G–I. Loss of Mef2c function with mef2d/c MO ablated all actin (acta1b, G), tnnc2 (H), mybpc1 (I) and nppa (J) mRNAs. Scale=100 μm.

Loss of Mef2c function abolishes sarcomeric gene expression. Hearts of 48–50 hpf (A–F) or 72 hpf (G) mef2ca^b1086;mef2cb^fh288 mutant embryos and their siblings in bright field (A), after immunodetection (C,D and G) or in situ mRNA hybridisation (B,E and F) shown in lateral view, anterior to left (A and G) or ventral (B–F) view, anterior to top. A.mef2ca^b1086;mef2cb^fh288 embryos had a tiny residual heart (arrow) and cardiac chamber edema. B,E and F. Double mutant embryos lacked almost all myl7, vmhc, bmp4 and nppa mRNAs as well as MyHC and Mef2 proteins in ventricle (v), atrium (a) or AV canal (red arrows in F) except for one (arrows in C,E) or two small heart structures expressing these markers. C. Confocal stack showing genotyped double mutant expressing low levels MyHC and nuclear Mef2 in a residual myocardial tissue. Sibling presented is mef2ca^+/b1086;mef2cb^+/+. D. No MyHC or atrial MyHC is detected in mef2d/c morphants. G. Immunodetection for GFP (atrium and ventricle, green) and Elastin (bulbus arteriosus, ba) in hearts of three different genotyped mef2ca^b1086;mef2cb^fh288;Tg(myl7:EGFP)twu26 showing variation in residual differentiated myocardial and bulbus tissue, compared to the normal heart of a mef2ca^+/b1086;mef2cb^+/fh288;Tg(myl7:EGFP)twu26 sibling. Scale=100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Cardiomyocytes of mef2ca;mef2cb dual loss of function are specified but developmentally arrested. In situ mRNA hybridisation for indicated genes (wholemounts in dorsal view, anterior to top, except J, ventral view). A–C. ALPM expression (arrowheads) of nkx2.5 (A), hand2 (B) and gata4 (C) at 12 ss is unaffected by lack of Mef2ca and Mef2cb. Notochord and rhombomeres 3 and 5 are marked by ntl (blue) and egr2b (red), respectively. D,E.Tbx20 and gata6 mRNAs are present but disorganised in the heart region of mef2d/c morphants and mef2ca^b1086;mef2cb^fh288 mutant embryos (lacking myl7 expression in red, E, right panel) compared to the typical ring-shape in control and sibling embryos. F. During heart cone stage (22 ss), nkx2.5 mRNA is abolished. G. hand2 mRNA is enhanced in a sheet of cells spanning the cardiac region but not elsewhere. Pharyngeal pouch expression is unchanged (white arrowheads). H, Whereas myocardial cells are undifferentiated in mef2d/c morphants and mef2ca^b1086;mef2cb^fh288 mutants (myl7, red), the endocardium is expanded and cdh5 is up-regulated. I. Endocardium (cdh5, arrow) lines the myocardium (myl7, red) in the normal looped heart of a sibling embryo. In mef2ca;mef2cb mutant embryos, little endocardial makrer is co-localised with the residual myocardium (arrows). Extra cdh5-expressing tissue is detected in the cardiac region (arrowheads). Scale=100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mef2cb overexpression induces skeletal muscle at the expense of myocardial and endothelial cells. In situ mRNA hybridisation (or immunodetection, A) for indicated genes, shown as wholemounts in dorsal (G and C) or lateral (B and D, top panel) views, or as flatmounts in dorsal view (A and D, bottom panel, E and F). A. Injection of mef2cb RNA results in many ectopic muscle cells expressing strong Mef2ca/cb and MyHC at 24 hpf (white arrowheads) anterior to the first somite (white arrow). In the heart, MyHC and Mef2ca/cb are mosaically stronger than in control embryos. Asterisks=ectopic muscle. B.Smyd1b is upregulated in CMs (white arrow), and in ectopic muscle in the head region (white arrowheads). C. Expression of bmp4 is upregulated in much of a sheet of cardiogenic cells but not elsewhere in the embryo. D. In 14 ss control embryos, myod mRNA is expressed in somites (black arrowheads) and myl7 is expressed weakly in CMs (white arrow). Embryos injected with mef2cb RNA express no ectopic myl7, but have ectopic myod mRNA in the head region (white arrowheads). E. By 24 ss, high levels of ectopic myod correlated with reduction or lack of myl7-expressing CMs (right panel) and defective brain development. F. At 24 ss, mef2cb RNA-injected embryos that had fewer myl7-expressing CMs (white arrows) also had less cdh5 expression in vascular endothelium (green arrows) and disorganised endocardium (blue arrows). G. Compared to wild type control (leftmost panel), three examples of embryos injected with mef2cb BAC DNA show ectopic myl7 mRNA either contiguous with (left panels) or detached from (right panel) the arterial (flanking panels) or venous (middle panel) poles. Scale=100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Loss of mef2cb function results in no heart phenotype. In situ mRNA hybridisation for myl7, vmhc and bmp4 (A,B, ventral view, anterior to top) and immunodetection of DM-GRASP (zn5) and Elastin (C, lateral view, anterior to left) of hearts at indicated stage of genotyped mef2cb^fh288mutants and their siblings. A,B. mef2cb^fh288 mutants had a normal looped heart and normal expression of chamber markers, and bmp4 mRNA in OFT (blue arrow), IFT (purple arrow) and AV canal (red arrows). C. Confocal stacks of genotyped mef2cb^fh288 mutants and siblings with a normal looped heart with developed chambers and bulbus arteriosus. Scale=100 μm. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

Mef2cb mRNA and protein are upregulated after mef2cb over-expression.
A-D. In situ hybridisation for mef2cb mRNA in controls or embryos injected with BAC DNA containing the mef2cb locus shown in flatmounts, lateral view, anterior to left (A), transverse section through the somites (B), wholemounts in dorsal view, anterior to top (C and D). Cells with high levels of mef2cb are indicated with arrows in muscle fibres (A,B), head vasculature (C), heart (D) and telencephalon (white arrows, C). E. Mef2 immunoreactivity is chimerically strong (yellow arrows) in somitic cells after injection of mef2cb BAC DNA. F. Mef2ca/cb and GFP immunoreactivity are ectopically strong and co-localise in fibres (white arrowhead) in the first somite (asterisk), and in cells outside the somite and in the head (white arrows) after injection of hs-mef2cb-IRES-GFP plasmid DNA followed by heat shock activation, shown in dorsal view, anterior to top. G. Ectopic muscle fibres are forming in the head region of embryos injected with 20 pg mef2cb mRNA and immunoreact with Mef2ca/cb and MyHC. Scale = 100 μm (except in E and G= 20 μm).

Mef2c protein is downregulated in mef2ca mutants.
Confocal stacks of Mef2ca/cb immunodetection (Anaspec, red in A-C) and DAPI (blue, A and C) in wildtype and mef2ca mutant embryos. A. Reaction with Mef2c antibody is strong in nuclei of differentiated slow muscle fibres in the somite (left panel), but is missing from the PSM (right panel), and from cranial ganglia indicating that the antibody is not crossreacting with Mef2d and Mef2a protein, respectively. B and C. Anti-Mef2c antibody reacts weakly in CMs of mef2cab1086 mutant embryos (B), but is absent from nuclei in the myotome of the somites (C). Scale = 50 μm (except in A=100 μm).

Mef2cb morpholinos are specific and efficient in targeting translation and splicing of mef2cb.
A and B. mef2cb ATG MO specifically blocked translation of mef2cb-GFP mRNA. Representative embryos (A) and quantification (B) of mosaic GFP accumulation in 24 hpf embryos injected with pCMV:mef2cb-GFP, with or without MOs. Embryos injected with plasmid alone or with plasmid and control myog MO, have numerous cells with strong GFP. In contrast, embryos injected with plasmid and mef2cb ATG MO had little if any GFP expression. C. Schematic diagram of 5′UTR (white) and ORF (black) of Exons 1-3 in mef2cb mRNA and RT-PCR strategy used to detect spliced mRNAs in control and mef2cb E1I1 MO-injected embryos (top left). Gel showing RT-PCR of mRNA from 24 hpf uninjected control and mef2cb E1I1 MO-injected embryos (two independent samples each, top right). Morphant cDNA had strong reduction of the normal splice form (nor; 321 bp) and the appearance of a large aberrant band (ab; 403 bp). The aberrant transcript (bottom) results in a premature stop codon. Exon (upper case) and intron sequences (lower case) are coloured (exon1, blue; intron1, orange; exon2, green; exon3, purple). Primer sequences are underlined. Amino acid sequence of the aberrant CDS is shown below the nucleotide sequence leading to a stop codon in the first half of the MADS domain. Scale = 100 μm.

Endothelial markers are little affected by Mef2 knockdown.
In situ mRNA hybridisation for mef2cb (A), kdrl (B and C), myl7 (C, red) and cdh5 (E) or immunodetection of MyHC and GFP (D) in wild type or Tg(fli1:GFP) control or injected with mef2d/c MO (except C; mef2ca^b1086;mef2cb^fh288 and sibling embryos). A. Mef2cb mRNA detected in heart (blue arrowhead), telencephalon (white arrowheads), and head vasculature (yellow arrows). B and C. Kdrl (flk-1) expression in mef2d/c morphant and double mef2ca;mef2cb mutant embryos at 21 ss is not changed compare with control and sibling embryos, respectively. D. Expression of endothelial marker fli1:GFP at 24 hpf is similar in control and mef2d/c morphants in the heart and head region (upper panels), but some defects in intersomitic vessels of morphants (lower panels) parallel the somitic muscle MyHC phenotype previously described (Hinits and Hughes, 2007). E. Mild upregulation of cdh5 mRNA in head and heart region (upper panels) and in the trunk region (lower panels) at 24 hpf. lda, lateral dorsal aorta; pmbc, primordial midbrain channel; da, dorsal aorta; pcv, posterior cardinal vein; se, intersegmental vessel. Scale = 100 μm.

Mef2ca and Mef2cb dual loss of function abolishes myocardial differentiation.
In situ mRNA hybridisation for myl7 (A), vmhc (B), bmp4 (C), smyd1b (D) and gata4,5,6 (E) in hearts of zebrafish embryos shown in a dorsal view, anterior to top (A-C and E) or in lateral view, anterior to left (D). A,B. Myl7 and vmhc expression in hearts (arrowheads) is lost after dual loss of function of Mef2ca and Mef2cb by various combinations of MOs and mutants. C. Bmp4 is dramatically reduced from heart of 22 ss mef2d/c morphants. D. Smyd1b mRNA is abolished from hearts (arrowheads) of 22 ss mef2d/c morphants. Expression in somites is slightly downregulated (arrow). E. Gata4, gata5 and gata6 appear unchanged in 22 ss mef2d/c morphants. Scale = 100 μm.

Mef2ca and Mef2cb dual loss of function abolishes myocardial differentiation.
A,B. In situ mRNA hybridisation for myl7 in hearts of 24 hpf mef2ca^b1086;mef2cb^fh288 mutant embryos and their siblings (A) or control and mef2cb^+/fh288 incross embryos non-injected or injected with mef2ca MO, shown in a dorsal view, anterior to top. Double mutants have almost no myl7 expression albeit few cells in the cardiac region (arrows, A). Mef2cb^fh288 mutant embryos injected with mef2ca MO have a thinner than normal heart tube. All other control or embryos with only one Mef2 depleted have a normal heart. C. Bright field images of mef2cbfh288 mutant embryos injected with mef2ca MO showing cardiac edema, compared with mef2ca MO injected siblings with normal heart and no edema. Scale = 100 μm.

Mef2ca and Mef2cb are expressed in hand2 knockdown embryos.
In situ mRNA hybridisation for mef2ca (A) and mef2cb (B) in hearts of control and hand2 MO zebrafish embryos, shown in dorsal view, anterior to top. A and B. Hearts of hand2morphants show cardia bifida phenotype (arrows). CMs on both sides of the midline express both mef2ca and mef2cb. Expression of mef2ca in pharyngeal arches CNC (cranial neural crest) is marked with asterisks. Scale = 100 μm.

Mef2ca mutant has a normal heart.
A and B. Double mRNA in situ hybridisation for myl7 (red, A and B), vmhc (blue, A) and bmp4 (blue, B) in genotyped 48 hpf mef2ca mutant embryos and their siblings shown in ventral view, anterior to top. Mutants have normal looping and gene expression is no different than siblings. OFT, blue arrow, IFT, purple arrow, AVC, red arrows. C. Confocal stacks of 72 hpf mef2ca^b1086;Tg(myl7:GFP) mutants and their siblings and of Tg(myl7:GFP) injected with mef2ca MO immunostained for Elastin showing no difference between morphants, mutants and siblings. D. GFP+dsRED- CMs can only be found in OFT and IFT of the heart (dotted line) of mef2ca^b1086;Tg(myl7:GFP;myl7;ndsRed) mutants and their siblings at 60 hpf. E. Heart rate (bpm) of control wild type, mef2camorphants, mef2ca^tn213 and their siblings at 25-48 hpf. Embryos were grown in 24-well plates and genotype was determined at 5 dpf according to jaw phenotype (see Piotrowski et al., 1996). No significant differences according to t-test statistics were found at any stage between morphants/mutants and control/siblings. Number of embryos per condition is shown on columns. Variation in heart rate between experiments is due to variation in temperature. Scale = 100 μm.

Mef2cb morphants have a linear defective heart and loss of bulbus arteriosus.
In situ mRNA hybridisation for indicated genes (A and B, ventral view, anterior to top) and immunodetection of EGFP, MyHC (MF20), Elastin and DAF-2DA detection (C and D, lateral view, anterior to left) of hearts at indicated stage of mef2cb morphants and controls. A and B. Mef2cb morphants have a linear unlooped hearts and no obvious AV canal as marked by bmp4 expression (red arrows). C and D. Confocal stacks of control or mef2cb MO-injected Tg(myl7:EGFP) (E) or wild-type (F) embryos at 72 hpf. Hearts are unlooped and bulbus arteriosus markers (BA, arrows) are absent in mef2cb morphants. Insets in D show YZ sections at the OFT region, indicating to the area bordering myocardial and smooth muscle cells. sh, sternohyoideus; cl, cleithrum; v, ventricle; a, atrium IFT, inflow tract; OFT, outflow tract; AVC, atrioventricular canal. Scale = 100 μm, except for C and D= 50 μm.