Transient Hypertrophy and Polyploidy in Regenerating Epicardial Cells

(A) Schematic for epicardial ablation and regeneration in vivo. White arrows indicate the direction of regeneration. OFT, outflow tract; V, ventricle. The frames with letters indicate regions shown in (C) to (E).

(B) Flattened images of a whole-mounted uninjured heart stained with an anti-ZO1 antibody (red). Framed regions are enlarged below in the same scale as the enlarged panels of (C) to (E). ZO1 staining is outlined by dashed lines. Scale bar, 100 μm.

(C–E) Flattened images of whole-mounted adult hearts at 3 dpi (C), 5 dpi (D), and 14 dpi (E) stained with an anti-ZO1 antibody (red). Framed regions are enlarged below in the same scale, with epicardial ZO1 staining outlined by dashed lines. White arrows indicate the direction of regeneration. Scale bars, 50 μm.

(F–H) Quantifications of epicardial cell area and distance from the ventricular base at 3 dpi. Mononucleate cells (Mono) are represented by cyan dots (F) or bars (G and H), and multinucleate cells (Multi) by red dots (F) or bars (G and H). n = 94 for Mono and 161 for Multi. The blue lines in (F) show regression results for Mono and Multi, respectively. (F) p < 0.001, ANCOVA. (G and H) ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(I–K) Similar quantifications as (F) to (H), using samples at 5 dpi. n = 401 for Mono and 198 for Multi. (I) p < 0.001, ANCOVA. (J and K) ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(L) Quantification of multinucleation for uninjured and 3-, 5-, and 14-dpi hearts. n = 4 (uninjured), 5 (3 dpi), 4 (5 dpi), and 3 (14 dpi) hearts, respectively. ∗p < 0.05; ns, not significant; Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(M) Quantification of cell area distribution for uninjured and 3-, 5-, and 14-dpi hearts. n = 449 (uninjured), 255 (3 dpi), 599 (5 dpi) and 1,678 (14 dpi), respectively. Numbers on the plot indicate mean values. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate SD

See also Figures S1 and S2; Movie S1.

Emergence of Leader and Follower Cells Ex Vivo

(A) tcf21:nucEGFP epicardial cells migrating from an explant after 5 days of culture in a coated dish. The front of the leading edge is outlined with a magenta dashed line, and the mononucleate cell domain is outlined with a yellow dashed line. Scale bar, 100 μm.

(B) tcf21:nucEGFP explant culture stained for ZO1. White arrows indicate direction of migration. Scale bar, 100 μm.

(C) Quantification of cell area and distance to the explant for samples in (B). Mononucleate cells are represented by cyan dots and multinucleate cells by red dots. n = 224 (Mono) and n = 166 (Multi). The blue lines show regression results for Mono and Multi, respectively. p < 0.001, ANCOVA.

(D and E) Distribution of cell distances to explant (D) and cell areas (E) for mononucleate (Mono) and multinucleate cells (Multi). n = 224 (Mono) and n = 166 (Multi). ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(F–I) Polyploidy and hypertrophy of murine fetal epicardial cells. (F) Schematic of murine epicardial explant culture. The colors indicate location of regions quantified in (H). (G) A 72-hr culture stained for WT1 (red) and ZO1 (green) indicating epicardial cells at the leading edge (left) or the center (right). Nuclear staining is blue in merged images and white in bottom panels. Yellow arrows denote binucleate cells. (H) Quantification of cell areas at three different regions. n = 352 (center), 299 (200–400 μm from the front of leading edge), and 451 (0–200 μm), respectively. Numbers on the plot indicate mean values. (I) Quantification of cell areas at the leading edge (0–200 μm from the front) for mononucleate (Mono, n = 329) and multinucleate (Multi, n = 122) cells. Numbers on the plot indicate mean values. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD. Scale bar, 50 μm.

See also Figure S3.

Endomitosis and Endocycling Events Underlie Epicardial Cell Polyploidy

(A) (Top) Schematic of experimental design to detect epicardial cell fusion. (Middle) Image of the framed region represented in the cartoon acquired after 5 days of culture. (Bottom) The region framed in yellow, enlarged to show detail. Asterisk indicates actin meshwork (green) covering a red nucleus, not a fused cell. Scale bar, 100 μm.

(B) Video frames of FUCCI cell-cycle analysis of explanted epicardial cells. A 3-day culture of an explant carrying FUCCI reporters was subjected to live imaging for 13 hr. White dashed lines outline the explant and yellow dashed lines outline the leading edge. Arrows and lowercase letters denote nuclei shown in (C). Scale bar, 100 μm.

(C) Video frames of the nuclei highlighted in (B) showing cytokinesis (cell a), endomitosis (cell b), and endocycling (cell c). Timing, hr:min. Scale bar, 20 μm.

(D) Spatial distribution of cell-cycle behaviors. Cell positions were calculated at the point of nuclear membrane breakdown (for cytokinesis and endomitosis) or fluorescent change from green to red (for endocycling). n = 56 (cytokinesis), 59 (endomitosis), and 35 (endocycling), respectively. Numbers on the plot indicate mean values. Blue dashed line marks 200-μm position. ∗∗∗p < 0.001, Mann-Whitney rank-sum test (compared with cytokinesis). Error bars indicate mean ± SD.

(E) Video frames of explanted epicardial cells from a tcf21:LifeAct-EGFP; tcf21:H2A-mCherry line showing normal cytokinesis (top) and cytokinesis failure in an endomitotic cell (bottom). White arrows indicate start of cleavage furrow ingression, yellow arrows indicate formation of midbodies, and cyan arrows indicate furrow abscission or regression. Timing, hr:min. Scale bar, 20 μm.

(F) Spatial distribution of successful and failed cytokinesis. Cell positions were calculated at the point of nuclear membrane breakdown. n = 48 (Success) and n = 35 (Failure). Numbers on the plot indicate mean values. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(G–I) Quantifications of the durations for cleavage furrow ingression (G), midbody formation (H), and the total time of both (I), for successful and failed cytokinesis, respectively. Ingression time is defined as the time from the dense LifeAct-EGFP signal emergence between two daughter nuclei (white arrows in E) to the formation of the narrowest furrow (yellow arrows in E). Midbody time was defined as the time from the narrowest furrow formation to furrow abscission or furrow regression (cyan arrows in E). n = 48 (Success) and n = 35 (Failure). Numbers on the plot indicate mean values. ∗∗∗p < 0.001; ns, not significant; Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

See also Figure S4 and Movies S2–S5.

Leader Cells Display Higher Migration Velocity and Mechanical Tension than Followers

(A) Epicardial explant culture showing F-actin and nuclei by tcf21:LifeAct-EGFP;tcf21:H2A-mCherry reporters. The framed regions were enlarged for display in (D). LifeAct-EGFP is shown in green and H2A-mCherry in red. Scale bars, 100 μm.

(B) Same experiment as in (A) showing velocity vectors (white arrows) for each nucleus (H2A-mCherry, red) in a 5-hr window. Scale bars, 100 μm.

(C) Dot plot for each individual nucleus indicating the average speed in 15.5 hr over the average distance from the leading edge. The blue line indicates the regression result. n = 224.

(D) Magnified view of the framed regions in (A). LifeAct-EGFP is shown in inverted grayscale. Scale bars, 20 μm.

(E and F) A 5-day tcf21:H2A-EGFP (green) epicardial explant culture was stained with pMLC (Ser19, grayscale). The framed regions in (E) are enlarged to show details in (F). The yellow dashed lines approximately separate follower (top) and leader cell regions (bottom). Scale bars, 100 μm.

(G) Video frames of explanted epicardial cells from a tcf21:LifeAct-EGFP line showing laser incisions and recoil of the cells. LifeAct-EGFP is shown in grayscale. Red double arrows indicate position and length of incisions. F, follower cells; L, leader cells. Scale bar, 20 μm.

(H) Quantification of the initial recoil velocity of leader and follower cells after cutting in a direction parallel or perpendicular to the migration direction. n = 12 (parallel-follower), 16 (parallel-leader), 14 (perpendicular-follower), and 15 (perpendicular-leader), respectively. ∗∗∗p < 0.001; ns, not significant; Mann-Whitney rank-sum test. Error bars indicate SD.

See also Movies S4 and S6.

Mechanical Tissue Stretching Promotes Epicardial Endoreplication

(A) Schematic of experimental design in (C) to (F).

(B) An elastic chamber with culture surface areas of 7 × 23 mm2 (right) and 7 × 26 mm2 (left).

(C) Culture chambers before stretch (top) and after stretch (bottom). Enlarged views of a chamber with heart explants and PBS are shown on the right.

(D) Explant culture from tcf21:nucEGFP animals with (bottom) or without (top) 100% stretch. The double arrow indicates stretch direction. nucEGFP is shown in white. Scale bars, 100 μm.

(E) Explant culture from tcf21:LifeAct-EGFP;tcf21:H2A-mCherry animals after 100% stretch. (Top) LifeAct-EGFP is shown in green and H2A-mCherry in red. The framed region is enlarged to show LifeAct-EGFP (grayscale) below. The double arrow indicates stretch direction. Scale bars, 100 μm.

(F) Experiment as in (D) is shown, visualizing tcf21:nucEGFP tissue with cell shapes outlined using a wheat germ agglutinin stain (grayscale). Top, unstretched control; bottom, 100% stretch. The double arrow indicates stretch direction. White arrows denote multinucleate cells close to the explant. Scale bars, 100 μm.

(G) Quantification of epicardial cell multinucleation in a quadrant of the cell sheet shown in (F). n = 28 (Ctrl) and n = 26 (Stretched) explants. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(H) Distribution plot showing the percentages of multinucleate cells at different regions of the epicardial cell sheet for both unstretched (Ctrl) and stretched cultures. Distances of cells to explant were normalized to the migration distances of the cell sheets and expressed as a percentage. n = 27 (Ctrl) and n = 31 (Stretched) explants. ∗∗∗p < 0.001; ∗∗p < 0.01; ns, not significant; Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(I) Frequency plot showing the distances of cells to explants for mononucleate (Mono, green) and multinucleate (Multi, yellow) cells, respectively, for both unstretched (Ctrl) and stretched cultures. Distances of cells to explant were normalized to the migration distances of the cell sheets and expressed as a percentage. n = 7,183 (Ctrl, Mono), 3,390 (Ctrl, Multi), 3,959 (Stretched, Mono), and 3,994 (Stretched, Multi) cells, respectively. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate SD.

See also Figure S5.

Follower Cells Undergo Endoreplication after Leader Cell Ablation

(A) Schematic for experiments in (B) to (F).

(B and C) Ablation experiment using tcf21:nucEGFP explants (green). Twenty-four hours post laser ablation, the explant culture was stained to detect pMLC (Ser19, grayscale). The framed regions in (B) are enlarged to show details in (C). The yellow dashed lines approximately mark the region that was ablated. Scale bars, 200 μm (B) and 100 μm (C).

(D) Quantification of nucleation and cell area of de novo leader cells and pre-existing leader and follower cells in (B). Leader cell region is defined by the strong staining of pMLC. Mono, mononucleate; Multi, multinucleate. n = 111 (follower), 39 (new leader, Mono), 49 (new leader, Multi), 10 (existing leader, Mono), and 89 (existing leader, Multi), respectively. ∗∗∗p < 0.001; ∗∗p < 0.01; Mann-Whitney rank-sum test. Error bars indicate SD.

(E) Video frames of a tcf21:FUCCI epicardial explant culture subjected to live imaging and laser ablation. The top panel is an image before ablation; the middle panel, immediately after ablation; the lower panel, a reconstructed leader cell region at 42 hr 45 min after ablation. The white, cyan, and yellow arrows indicate nuclei that underwent endomitosis, endocycling, and cytokinesis, respectively. Timing, hr:min. Scale bar, 100 μm.

(F) Cropped video frames of the numbered cells indicated in the middle panel of (E). White arrows indicate the nuclei of cells that undergo endoreplication or cytokinesis. Timing, hr:min. Scale bar, 20 μm.

See also Movie S7.

High Regenerative Capacity in Endoreplicated Cells

(A) Schematic for experiments in (B) to (H). tcf21:NTR; tcf21:nucEGFP animals were used.

(B) Epicardial regeneration ex vivo in the presence of DMSO (vehicle), 10 μm SB431542, or 0.5 μm GSK1007102B. White dashed lines outline the explants. Framed regions are enlarged to show details below. Scale bar, 100 μm.

(C) Quantification of GFP fluorescence density from images captured at 12 dpi. n = 18 (vehicle), 18 (SB431542), and 13 (GSK1007102B), respectively. ∗∗∗p < 0.001, Student's t test (compared with vehicle). Error bars indicate mean ± SD.

(D) Quantification of timing to repopulate the ablated ventricular surface. n = 36 (vehicle), 26 (SB431542), and 21 (GSK1007102B), respectively. ∗∗p < 0.01; ns, not significant; Mann-Whitney rank-sum test (compared with vehicle). Error bars indicate mean ± SD.

(E) Flattened images of whole-mounted hearts with ZO1 staining (red). Hearts were treated with DMSO (vehicle) or SB431542 for 12 days (14 dpi). Framed regions are enlarged below, with nucEGFP shown in green, and ZO1 in grayscale and outlined in yellow. Scale bar, 100 μm.

(F) Quantification of epicardial cell multinucleation in hearts treated with DMSO (vehicle) or SB431542 for 12 days (14 dpi). n = 4 hearts for each. ∗p < 0.05, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(G) Quantification of epicardial cell area in hearts treated with DMSO (vehicle) or SB431542 for 12 days (14 dpi). n = 822 (vehicle) and n = 477 (SB431542). Numbers on the plot indicate mean values. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate SD.

(H) Regenerative capacity on a per cell basis, with or without SB431542 treatment, measured by area covered per day by a digitalized nucleus. Area was measured at day 0 (2 dpi) and day 10 (12 dpi) of treatment, and cell density was measured at day 10 (12 dpi). One digitalized nucleus is defined as 30 GFP pixels. n = 18 explants for each group. ∗∗∗p < 0.001, Mann-Whitney rank-sum test. Error bars indicate mean ± SD.

(I) Epicardial explant culture assays with vehicle, blebbistatin (10 μM), GSK1007102B (0.5 μM), or LY294002 (50 μM) treatment. tcf21:nucEGFP explants (shown in white) were plated for 5 days. Blebbistatin was added from day 3 to day 5, and GSK1007102B and LY294002 were added from day 0 to day 5. Framed regions are enlarged below to show details. Scale bar, 200 μm.

(J) Experiment as in (H) using tcf21:LifeAct-EGFP; tcf21:H2A-mCherry
(K) Quantification of epicardial growth. Skirt area is defined as the area covered by epicardial tissue growth from the explant. For migration distance and skirt area, n = 40 (vehicle), 30 (blebbistatin), 27 (GSK1007102B), and 19 (LY294002) explants, respectively. For nucleation, cell density, and average cell area, n = 10 explants for each treatment. ∗∗∗p < 0.001; ∗∗p < 0.01; Student's t test for migration distance and skirt area, Mann-Whitney rank-sum test for cell density, nucleation, and average cell area. All comparisons were versus vehicle-treated group. Error bars indicate mean ± SD.

See also Figures S6 and S7; Movie S8.

Analysis of Nucleation and Apoptosis in Polyploid Cells (Related to Figure 1)
(A) Orthogonal view of a z-stack image. A maximum projection image of the x-y plane is shown with nucEGFP in green and ZO1 staining in red. Yellow lines indicate positions for views of the y-z plane (right) and the x-z plane (bottom), respectively. Arrows and numbers denote nuclei. Scale bar, 20 μm.
(B) Two z-axis stacks of the image shown in (A). The numbers indicate the same nuclei shown in (A). The nuclei 1, 2, 3, and 5 are in the outermost layer, while nuclei 4, 6, and 7 are in the inner layer, embedded within the muscle (red autofluorescence).
(C) Video frames from Movie S1 with tcf21:H2A-EGFP shown in grayscale. N uclei of three cells are circled in low-magnification images (top), and two frames of each are displayed below. Nuclei within individual cells frequently overlap. Scale bar, 50 μm.
(D) Examples of mononucleate cells (top left, arrows) and multinucleate cells. Scale bar, 20 μm.
(E, F) Quantification of nuclear positions within each multinucleate cell at 3 and 5 dpi, respectively. The cell length was measured parallel to the direction of epicardial tissue regeneration. The nuclear position was defined as the center of an area encircling all of the nuclei in each cell and was normalized in percentiles to cell length. n = 50 for each timepoint. Bars indicate mean ± S.D.
(G) FACS analysis of purified epicardial cells. Epicardial cells were purified from uninjured (Vehicle, left) and regenerating samples (Mtz, right) and stained with PI for DNA content. tcf21:NTR; tcf21:nucEGFP ventricles were collected at 7 and 14 dpi. Percentages of 2N, 4N and >4N cells are indicated. Samples from regenerating hearts have 113% (7 dpi) or 56% (14 dpi) more >4N cells (red) than those from uninjured hearts.
(H) Schematic for experiments in (I-K).
(I) Video frames acquired at the ventricular apex at the final stages of epicardial regeneration, covering 61.5 h. tcf21:H2A-EGFP is shown in grayscale. Arrows and numbers denote nuclei shown in (J). Scale bar, 50 μm. See also Movie S1. (J) High-magnification view of nuclear doublets indicated in (I) undergoing nuclear fragmentation (red arrows). Scale bar, 20 μm.
(K) Video frames of an unconventional division of a nuclear doublet undergoing fragmentation. Yellow arrows denote multinucleate cell and daughter nuclei. Red arrows indicate nuclear fragmentation. One daughter nucleus divided again before fragmentation. Scale bar, 20 μm. Timing, hh:mm.

Cell Size and Nuclear Number during ex vivo Epicardial Regeneration (Related to Figure 1)
(A) A whole ventricular explant (with outflow tract at top) undergoing ex vivo epicardial regeneration. tcf21:NTR; tcf21:nucEGFP hearts were incubated in culture medium with 1 mM Mtz for 24 h. Explants were assessed daily for EGFP fluorescence. White dashed lines outline ventricles. Regions framed in red are enlarged to show details below each panel. White arrows denote nuclear doublets. Scale bar, 200 μm.
(B, C) Images of flattened explants stained with an anti-ZO1 antibody (red). Images of the framed region represented in the cartoons from 2 hearts are shown. Regions framed in cyan, green and blue were enlarged in the same scale at the right or the bottom. Optical sections are shown in enlarged images in (C) to more clearly indicate ZO1 staining. Scale bar, 100 μm.

Analysis of Cell Volume and the Relevance of ECM Components to Epicardial Regeneration (Related to Figure 2)
(A) A maximum projection view of an explant culture, showing tcf21:nucEGFP in green and b-catenin staining in grayscale. Three yellow lines (a, b and c) denote positions of views for the y-z planes in (B).
(B, C) y-z views for the planes denoted in (A). The framed regions in (B) are enlarged to show details in (C).
(D) Quantification of the cell depths by measuring junctions (b-catenin signals, By b-cat) or nuclei (nucEGFP signals, By GFP). n = 50 (By b-cat, follower), 64 (By b-cat, leader), 25 (By GFP, follower) and 10 (By GFP, leader), respectively. ** P < 0.01; ns, not significant; Mann-Whitney Rank Sum Test. Bars indicate mean ± S.D.
(E) Images of fibronectin (Fn) staining of regenerating hearts both in vivo and ex vivo. Fn is shown in green (merged images) or grayscale (single-channel images) and tcf21:nucEGFP is shown in red. The framed regions are enlarged to show details on the right. Scale bar, 100 μm.
(F-I) tcf21:nucEGFP; tcf21:DsRed2 epicardial explants were plated in differently coated dishes for 5 days. Dishes were coated with 0.01% type I collagen (F), 0.1% gelatin (G) or 0.01% poly-L-lysine (H) solutions overnight. Fibrin gel (I) was freshly made as described previously (Kim, et al., 2012). The framed regions of left panels are enlarged to show as middle (merged) and right panels (nucEGFP only). The yellow lines approximately separate follower (top) and leader cell regions (below). Scale bar, 100 μm.

New Reporter Lines for Live Imaging (Related to Figure 3)
(A, B) A tcf21:H2A-EGFP reporter line indicates chromatin in epicardial cells of a wholemounted heart (A) and in epicardial explant culture (B). Anti-phospho-Histone H3 staining (H3P) was performed to label condensed chromatin (red). Scale bars, 100 μm (A) or 10 μm (B).
(C) High-magnification view of tcf21:H2A-mCherry epicardial cells. Scale bar, 10 μm
(D, E) A tcf21:LifeAct-EGFP reporter labels F-actin in whole-mounted (D) or isolated epicardial cells (E). DAPI stains nuclei red in (E). Scale bars, 100 μm (D) or 10 μm (E).
(F, G) Epicardial explant culture from a tcf21:FUCCI heart, stained after a 1 h pulse of EdU to mark S phase. Arrows in (F) denotes different cell cycle phases, also highlighted in (G). Scale bars, 10 μm.

Mechanical Stretching of Epicardial Sheets (Related to Figure 5)
(A) The same experiment as shown in Figure 5, using tcf21:nucEGFP tissue with cell shapes outlined with a WGA staining (grayscale). (Top) Unstretched control. (Middle) Explants after 50% stretch. (Bottom) Explants after 200% stretch, which typically damaged the tissue. The dual arrow indicates stretch direction. Scale bars, 100 μm.
(B) Quantification of epicardial cell multinucleation within a quadrant of the epicardial sheet shown in (A) after 50% stretch. n = 8 (Ctrl) and 7 (Stretched) explants. * P < 0.05, Mann-Whitney Rank Sum Test. Bars indicate mean ± S.D.

Chemical Screening for Regulators of Epicardial Cell Proliferation (Related to Figure 7)
(A) Whole mounted tcf21:nucEGFP ventricles after 6 days of culture as explants, treated with DMSO (Vehicle) or GSK1007102B. Framed regions are enlarged and shown below.
(B) Quantification of EGFP fluorescence density. n = 8 (Vehicle) and 5 (GSK1007102B) explants, respectively. *** P < 0.001, Student’s t-test. Bars indicate mean ± S.D.
(C) Images of fixed and flattened tcf21:nucEGFP explants after 2 days of culture with DMSO (Vehicle) or SB431542. Samples were incubated with EdU for 1 h before fixation (shown in red and grayscale). DNA was stained with DAPI (blue). Scale bars, 100 μm.
(D) Quantification of EGFP+EdU+ nuclei per mm2 on the ventricular surface, from hearts in (C). n = 3 explants for each. * P < 0.05, Student’s t-test. All error bars indicate S.D.
(E, F) PI3K regulates epicardial regeneration. (E) Schematic for experiments in (F). tcf21:NTR; tcf21:nucEGFP animals were used. LY294002 (50 μM) was added for 8 days (3 - 11 dpi). Then, explants were rinsed twice with PBS and further cultured for 4 days. (F) Epicardial regeneration ex vivo in the presence of DMSO (Vehicle) or 50 μm LY294002. All vehicle-treated explants (16 of 16) displayed regeneration. All LY294002- treated explants displayed a block of regeneration (23 of 23), which resumed in each case after drug wash-out. Scale bar, 200 μm.

Chemical Treatment of Epicardial Explant Cultures (Related to Figure 7)
(A) Schematic for experimental design in (B-D). tcf21:nucEGFP explants were plated for 5 days. Blebbistatin was added from day 3 to day 5 (2 μM) or from day 2 to day 4 (10 μM). Images were acquired and analyzed at day 5.
(B) Epicardial explant culture assays with Vehicle or Blebbistatin (2 μM or 10 μM) treatment as shown in (A). Framed regions are enlarged below to show details. tcf21:nucEGFP is shown in green, WGA staining is shown in grayscale. Scale bar, 200 μm. (C) Quantification of epicardial tissue growth in (B). For migration distance and skirt area, n = 20 (Vehicle), 20 (2 μM), and 18 (10 μM) explants, respectively. For nucleation, cell density, and average cell area, n = 10 explants for each treatment. *** P < 0.001; ** P < 0.01; Mann-Whitney Rank Sum Test. All comparisons were versus Vehicle-treated group. Bars indicate mean ± S.D.
(D) Epicardial explant culture in (A) was stained with pMLC (Ser19, grayscale). Scale bar, 100 μm.
(E) Epicardial explant culture assays with Vehicle or SB431542 (10 μM) treatment. tcf21:nucEGFP explants (shown in green) were plated for 5 days. WGA staining is used to outline cells (grayscale). Drug was added from day 0 to day 5. Framed regions are enlarged below to show details. Scale bar, 200 μm.
(F) Quantification of epicardial tissue growth in (E). For migration distance and skirt area, n = 20 explants for each. For nucleation, cell density, and average cell area, n = 10 explants for each treatment. * P < 0.05; ns, not significant; Mann-Whitney Rank Sum Test. Bars indicate mean ± S.D.
(G) Epicardial explant culture assays with Vehicle, GSK1007102B (0.5 μM) or SB431542 (10 μM) treatment. tcf21:nucEGFP explants (shown in green) were plated for 5 days. WGA staining is used to outline cells (grayscale). Drugs were added from day 0 to day 5. Scale bar, 100 μm.
(H) Spatial distribution of nucleation after treatment with Vehicle, GSK1007102B (GSK), or SB431542 (SB) as shown in (G). n = 1,481 (Vehicle), 819 (GSK), and 439 (SB) cells, respectively. *** P < 0.001; ns, not significant; Mann-Whitney Rank Sum Test. Bars indicate S.D.