Myc overexpression enhances epicardial contribution to the developing heart and promotes extensive expansion of the cardiomyocyte population

Figures

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  Figure 1. Enhanced myocardial contribution by Myc-overexpressing cells. (A–B’) Confocal images from histological sections of iMOSWT(WT) (A) and iMOST1-Myc(MYC) (B) hearts at E14.5 induced with Wt1-Cre. (A’,B’) show magnification of the boxed areas shown in (A,B). (C–D’) Confocal images from histological sections of P0 iMOSWT(WT) (C) and iMOST1-Myc(MYC) (D) recombined with Wt1-Cre. (C’,D’) show magnification of the boxed areas shown in (C,D). (E–G) Quantification of the percentage of recombined area detected in iMOSWT(WT) and iMOST1-Myc(MYC) at E14.5 and P0 in the RV (E), IVS (F) and LV (G). (H) Cytometer histogram plot showing Counts (X axis) versus FITC laser (EYFP detection) in iMOSWT(WT) and iMOST1-Myc(MYC) E14.5 whole digested hearts (upper panel). Lower panel shows quantification on EYFP population related to the total amount of live cells detected on the cytometer for iMOSWT(WT) and iMOST1-Myc(MYC) E14.5 hearts. Graphs in E, F, G, H show means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Bar, 200 μm in A, B, C, D and 50 in A’, B’, C’, D’ n ≥ 5 hearts for (A–G) and n ≥ 7 for (H).

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  Figure 2. Preferential colonization of the cardiomyocyte lineage by Myc-overexpressing cells. (A–B’) Confocal images from histological sections of iMOSWT (WT) (A) and iMOST1-Myc(MYC) (B) hearts at E14.5 induced with Wt1-Cre. (A’,B’) show amplified regions from boxed areas in (A,B). (A”,B”) depict amplified regions from boxed areas in (A,B) showing native EYFP and anti-GFP staining. A”, solid indicates an EYFP-negative, anti-GFP-positive cell and empty arrowhead indicates a double-positive cell. (C–E) Percentage of EYFP cells within the iMOS-positive population observed at E14.5 in whole hearts of the iMOSWT (WT) and iMOST1-Myc(MYC) mosaics in the non-cardiomyocyte fraction and in the cardiomyocyte fraction in the RV (C), IVS (D) and LV (E–G) Confocal images showing cultured cells from dissociated E14.5 hearts from iMOSWT(WT) (F) and iMOST1-Myc(MYC) embryos (G,H) Quantification of the proportion of EYFP cells within the non-cardiomyocyte fraction (left graph) and cardiomyocyte fraction (right graph) from dissociated E14.5 hearts from iMOSWT(WT) and iMOST1-Myc(MYC) embryos. (I), Quantification of proliferation in cardiomyocytes of the WT1Cre lineage. Graph shows the frequency per area unit…

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  Figure 3. Progressive overcolonization of the epicardium and epicardial derivatives by Myc-overexpressing cells. (A–B’) Confocal images from histological sections of iMOSWT(WT) (A) and iMOST1-Myc(MYC) (B) hearts at E14.5 induced with Wt1-Cre. (A’,B’) show magnification of the boxed areas in (A) and (B). Co-localization of EYFP native fluorescence and anti-GFP immunofluorescence (in red) detects both ECFP (filled arrowheads) and EYFP (open arrowheads) cells. (C) frequency of EYFP cells with respect to total iMOS-recombined cells in epicardium (E), subepicardium (SE) and myocardium (MYO). Throughout the figure WT refers to iMOSWTmosaics and MYC to iMOST1-Mycmosaics. (D,E’) 6-day old epicardial cultures and magnifications (D’,E’). (F) Quantification of the EYFP frequency within the epicardial explants. (G,H) Co-culture of E10.5 epicardium and a WT E9.0 heart. (I,J), Similar cultures as shown in (G,H) showing a maximum fluorescence intensity projection (Z = 36 μm). (*) indicates the WT heart and (E) the epicardial explant. Arrowhead shows invading EYFP-Myc epicardial-derived cells. K Quantification of epicardial cells per explant that colonize (Co) or invade (In) the WT myocardium. (L–O)…

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  Figure 4. The Wt1Cre-recombined cardiomyocytes do not derive from the epicardium. (A,B) Confocal images of histological sections from E14.5 hearts iMOSWT(WT) (A) and iMOST1-Myc(MYC) (B); Wt1-Cre ERT2. A’,B’ show a magnification of the boxed areas shown in (A,B). (C–D’) Proepicardial explants cultured for 48 h from iMOSWT(WT) (C) and iMOST1-Myc(MYC) (D) where big clusters of beating cells were detected to be positive for cadiac TnT staining (arrowheads). E Quantification of the proportion of cardiomyocytes that expressed EYFP. (F,G) Confocal images from histological sections of Mef2C-AHF-Cre-recombined iMOSWT(WT) (F) and iMOST1-Myc(MYC) (G) mosaic hearts at E14.5. Arrowheads show the front of EYFP cardiomyocyte colonization. (H,H’) Confocal images from histological sections of iMOST1-Myc(MYC) E14.5 heart induced with Tie2-Cre. H’ shows a magnification from the boxed area in (H). Graph in E shows means +SEM. *p < 0.05; **p < 0.01; ***p < 0.001. Bar, 100 μm in A,B,C,D,F,G,H and 20 μm in A’,B’,C’,D’,H’, LA left atrium, LV left ventricle.

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  Figure 5. Myc overexpression does not affect the initial pattern of recombined cardiomyocytes (A,B) Confocal images of histological sections from E9.5 hearts iMOSWT(WT) (A) and iMOST1-Myc(MYC) (B) stained for c-TnT. A’ and B’ show magnification of boxed areas in (A–C), Graphs showing the proportion of embryos showing Wt1 lineage contribution to cardiomyocytes in iMOSWT(WT) and iMOST1-Mycat stage E9.5 (left) and the distribution of Wt1 contribution to different areas of the heart in iMOSWT(WT) and iMOST1-Myc(MYC) at stage E9.5 (right). (D). Detection of Cre-recombined cardiomyocytes in the E9.5 heart following recombination induction at E8.0 in the Wt1-Cre ERT2 line. From left to right, images show MF20 expression, GFP detection and their co-localization. Panels to the right show a magnification of the boxed area. Bar is 50 μm. V, ventricle of the early cardiac tube. Graphs in (C) show means ± SEM. n ≥ 3 hearts in (A–C), n ≥ 5 in D.

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  Figure 6. Cardiomyocyte recombination does not correlate with Cre or CreERT detection in cardiomyocytes (A,B) show immunofluorescence with anti-Wt1 and anti-SMA antibodies on sections of the developing cardiac tube of wild type embryos. (B’) shows a high magnification from the boxed area in (B). Arrowheads indicate pericardial Wt1+ cells. (C,D) show immunofluorescence with anti-Cre and anti-SMA antibodies on sections of the developing heart tube of Wt1Cre embryos. (D’) shows specific areas magnified from (D). Solid arrowheads indicate Cre-positive epicardial cells; empty arrowheads indicate Cre-positive pro-epicardial cells. (E,F) show immunofluorescence with anti-ER and anti-SMA antibodies on sections of the developing heart tube of Wt1CreERT2 embryos. (F’) shows specific areas magnified from F. Solid arrowheads indicate CreERT2-positive epicardial cells. Bar is 50 μm in (A,B) and 100 μm in (C–F).