FIGURE SUMMARY
Title

Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle

Authors
Kula-Alwar, D., Marber, M.S., Hughes, S.M., Hinits, Y.
Source
Full text @ Dev. Biol.

Ventricular myocardium expands and thickens by CM addition. (A) Ventricular axes were measured on confocal images of live Tg(myl7:EGFP) embryonic hearts at 3 and 5 dpf. Ventral half of ventricle shown. Graph shows increase in length of each axis. (B) Myocardial thickness increased significantly from 3 to 5 dpf. (C,D) Myocardial volume (C) and surface area (D) were measured by thresholding for GFP in scans of Tg(myl7:EGFP) embryos (see materials and methods), both increased significantly by 52% between 3 and 5 dpf. (E) Trabecular length increased 500% between 3 and 5 dpf. (F)Tg(myl7:EGFP);Tg(myl7:DsRed2-NLS) were thresholded to count DsRed CM nuclei. Number of ventricular CMs increased by 26% from 3 to 5 dpf, n ​= ​5 and n ​= ​4 respectively. (G,H) Confocal section through Tg(myl7:EGFP-Has.HRAS)s883 scatter labelled with cytoplasmic mCherry (G, white arrow). Graph of average cell volume (H) measured from three hearts at 3 dpf (9 ​cells) or 5 dpf (10 ​cells). (I,J) Confocal optical sections through Tg(myl7:EGFP-Has.HRAS)s883 fish at 3 and 5 dpf with four CM perimeter measures carried out in each optical plane. Graph (J) shows cell measurements from four hearts for each time point. Values in all graphs are mean ​± ​SEM. N for each group shown on bars. Statistics used unpaired Student’s t-test. Scale bars ​= ​20 ​μm.

CM addition, hypertrophy and proliferation are involved in ventricular growth between 3 and 5 dpf. (A) Three Optical sections of 5 dpf Tg(myl7:GAL4FF)hu6531;Tg(5XUAS:RFP)zf83;Tg(UAS:h2az2a-GFP)hu6851 larval heart immunostained for GFP (green), RFP (red) and PH3 (magenta) to reveal the sole PH3+nucGFP+ CM detected in this heart (arrow). Note the additional PH3+nucGFP cells enclosed by the myocardial layer (arrowheads). (B) Number of PH3+ CMs/ventricle at 3 dpf, 4 dpf and 5 dpf. Mean ​± ​SEM. Each dot represents an animal. (C,D)Tg(myl7:GAL4FF)hu6531;Tg(UAS:h2az2a-GFP)hu6851 embryos were exposed to EdU between 4 and 5 dpf. A short confocal stack reveals EdU-labelled CMs (arrowheads, C), which were quantified as the fraction of EdU+ nuclei in H2A-GFP-labelled CMs in ventricle, atrium and whole heart (D). (E,F) Confocal images of anterior ventricle and OFT region of a Tg(nkx2.5:galFF);Tg(UAS:h2a-gfp);Tg(myl7:nucDsRed) larva imaged at 3 and 5 dpf showing a nucGFP+;nucDsRed cell population (white bracket) adjacent to the OFT region (arrow) at each stage (E). nucGFP+ cells were counted in the bracketed areas (F). (G,H) Confocal stacks of embryos of 3.5 (G) and 6 dpf (H) carrying Tg(myl7:EGFP)twu26 and Tg(–5.1myl7:DsRed2-NLS)f2. G′ and H′ show in greater detail the boxed OFT region. EGFP+ dsRed cells are clearly present at 3.5 dpf indicating recently differentiated CMs (arrowheads), whereas by 6 dpf all cells seem to be positive for both EGFP and DsRed. (I) Schematic diagram of the Kaede conversion experiment. (J) Confocal stacks of hearts 5 dpf embryos carrying Tg(myl7:GAL4FF)hu6531and Tg(UAS:Kaede)rk8 in which Kaede was converted from green to red at 3 dpf. Transmitted light of the scanned area is shown in J’. Cells expressing green Kaede but no or little red Kaede are marked by white arrow (OFT and ventricle nearby), light blue arrow (AVC), and yellow arrow (ventricle apex). Note, atrial cell expressing green Kaede only (asterisk) was found only in this embryo. Scale bars ​= ​50 ​μm, except A and E ​= ​20 ​μm.

Mef2c is required for the formation of the heart. (A) In situ mRNA hybridisation for myl7 on 1 dpf embryos from an incross of mef2ca+/;mef2cb+/ in dorsal view, anterior to top, sequence genotyped retrospectively. Images are representative of the majority of embryos of each genotype. mef2ca−/;mef2cb−/ embryos had a severe reduction in myl7 expression. mef2ca+/;mef2cb−/ also showed a reduction in myl7 expression but to a lesser extent than double homozygous mutants. (B) Confocal stack of live hearts of mef2ca−/−;mef2cb−/− and its sibling on a Tg(myl7:EGFP) background. Double homozygous mutants continue to show a severe heart defect at 3 and 5 dpf but some growth in the differentiated structure is observed. (C) Heart rate measurements at 1–3 dpf for genotyped embryos showing no differences in heart rate of mef2ca+/;mef2cb−/− embryos compared with their siblings (unpaired Student’s t-test, P ​> ​0.05). Values are mean ​± ​SEM. N for each group shown on bars. (D) Hearts of 52 hpf embryos from a cross of mef2ca+/;mef2cb+/ to mef2cb−/− shown in ventral view after a double in situ hybridisation for nppa (blue) and myl7 (red). Sibling shown is mef2ca+/+;mef2cb+/. Hearts of mef2ca+/;mef2cb−/− are smaller and underdeveloped. Scale bars ​= ​50 ​μm.

Mef2ca+/;mef2cb−/−mutant embryos show reduced ventricular size compared to siblings. (A) Confocal images of 3 dpf (top panels) or 5 dpf hearts (bottom panels) from a cross of mef2ca+/;mef2cb+/;Tg(myl7:EGFP) to either mef2ca+/+;mef2cb+/;Tg(myl7:EGFP) or mef2ca+/;mef2cb+/+;Tg(myl7:EGFP) fish. mef2ca+/;mef2cb−/ embryos have a smaller heart at 3 and 5 dpf. Scale bars ​= ​50 ​μm. (B,C) Volume, trabecular length and width were quantified for embryos from crosses in A designed to test Mef2ca (B) and Mef2cb (C) function. One-way ANOVA analysis followed by Bonferroni post hoc tests, p-values indicated on graphs. Graphs show mean ​± ​S.E.M. N for each group shown on bars.

Mef2ca+/;mef2cb−/−mutant embryos have reduced CM and changes in SHF gene expression. (A,B) Embryos from a cross of mef2ca+/;mef2cb+/;Tg(myl7:EGFP) and mef2cb+/;Tg(myl7:EGFP);Tg(myl7:nucDsRed) were fixed and immunostained with GFP (green) and RFP (red) antibodies at 1 dpf or imaged live at 3 and 5 dpf and CM nuclei counted. Confocal stacks are shown on red channel only at 1 dpf for clarity (A). Nuclei number of mef2ca+/;mef2cb−/− (red bars) was significantly lower than in siblings (blue bars) at all stages (Unpaired Student’s t-test, P-values indicated on graph). Values are mean ​± ​S.E.M. N shown on bars. (C,D) Embryos from a cross of mef2ca+/;mef2cb+/;Tg(myl7:EGFP) to mef2cb+/ at 28 hpf (C) and 3 dpf (D) hybridised for ltbp3 mRNA (Fast Red) and myl7:EGFP detected with anti-GFP antibody. Hearts of mef2ca+/;mef2cb−/− showed less differentiated myocardium (myl7:EGFP) co-expressing ltbp3 mRNA and ltbp3 levels outside the heart were also reduced (C), but by 3 dpf little difference was seen between genotypes (D). (E,F) In situ mRNA hybridisation for nkx2.5 (blue) and myl7 (red) in embryos from a cross of mef2ca+/;mef2cb+/ and mef2cb−/− at 28 hpf (E, dorsal view) and 52 hpf (F, ventral view, same embryos shown in top panel for nkx2.5 only and in bottom panel for nkx2.5 and myl7). nkx2.5 ​mRNA was upregulated slightly at 28 hpf but was clearly upregulated throughout the ventricle and down in the atrium at 52 hpf, beyond the normal strong expression in AVC (white arrowhead) and the arterial pole (yellow arrowhead). Dotted line (bottom panel, F) marks chambers. (G) Confocal stack of immunodetection of Nkx2.5 and GFP (myl7:EGFP) of hearts of mef2ca+/;mef2cb+/ and sibling at 28 hpf in dorsal view showing Nkx2.5 upregulation in the ventricular region of the heart tube (white arrow) and in the arterial pole (yellow arrowhead). Scale bars ​= ​50 ​μm.

Mef2ca+/mef2cb−/−adults are viable but small. (A) Weight and standard length (SL) of genotyped three to four month old adult fish were measured and standard weight (K) calculated. Mef2ca+/;mef2cb−/− mutant adults had a significantly lower BW and SL compared with their siblings. Graphs show mean ​± ​S.E.M. for pooled data from 2 lays. N for each group shown on bars. One-way ANOVA was performed followed by Dunnett test for multiple comparison. (BD)Mef2ca+/;mef2cb−/−, mef2cb−/− and siblings at 4 months of age were measured and their heart dissected and imaged (B). Ventricle length (VL) was measured from images from the centre of the bulbous to the apex of the heart (diagram in C). Graph shows mean ​± ​S.E.M. N for each group shown on bars. VL was plotted vs. SL (D). Note that points for mef2ca+/;mef2cb−/− mutants fall below the linear regression line. Scale bars ​= ​1 ​mm.

Acknowledgments
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Reprinted from Developmental Biology, 470, Kula-Alwar, D., Marber, M.S., Hughes, S.M., Hinits, Y., Mef2c factors are required for early but not late addition of cardiomyocytes to the ventricle, 95-107, Copyright (2020) with permission from Elsevier. Full text @ Dev. Biol.