(A) The basement membrane of the epicardium was identified by staining for collagen IV (Col IV). The layer of mesothelial cells attached to the basement membrane on the outer surface of the heart is the epicardium. (B) BrdU labeling indicates epicardial cell proliferation. Arrowheads point to epicardial cells that are BrdU⁺. Scale bar = 20 µm. (C) Proliferation rate of epicardial cells at indicated time points. Six midcoronal sections per heart for each age were quantified. (D–E) WT1 and GFP from PDGFRα^GFP/+ mice were used to visualize epicardial cell location with ex vivo culture and aphidicolin treatment. (F) Quantification of WT1⁺ cells present within myocardium after treatment. (G) PDGFRα^GFP/+ hearts were used to visualize epicardial cell location with ex vivo culture and cyclin D1 over-expression at 48 hours. (H) Quantification of GFP⁺ within the myocardium after 48 hours of culture with control adenovirus or cyclin D1 adenovirus. Six midcoronal sections per heart were quantified. Scale bar = 10 µm. See also figure S1.

(A) Perpendicular and (B) parallel divisions can be observed in whole mount stained hearts. Podoplanin, p-Histone H3 and α-Tubulin stained: epicardial cells, mitotic cells and spindles respectively. Arrowheads point to the mitotic cells. Scale bars = 10 µm. See Table S1 for additional information.

(A,B) Spindle orientation of control and βcat^EKO epicardial cells at E12.5 and E13.5. For illustration purposes, spindle orientation events from only 9 hearts for each genotype and age are shown in the diagrams. Bar graphs at the right indicate the summary of spindle orientations. (C) In βcat^EKO hearts, adenoviruses overexpressg full length (Ad-βcat) or transactivation domain deleted β-caten (Ad-trβcat) rescue βcat^EKO EMT but Ad-GFP and Ad-dnTCF4 do not. (D) EMT results from wild type hearts transduced with Ad-dnTCF4 or Ad-TCF4. See also figure S3.

(A–C) NFP^EKO epicardial cells displayed abnormal apical basal polarity based on Par3, Par6 and aPKC localization. (D) Spindle orientation of control and NFP^EKO epicardial cells. Spindle orientation events from 9 hearts for each genotype, except control at E13.5 which used 7 hearts. (E) Indicates the ratio of spindle orientations at each embryonic stage. Scale bar = 10 µm. See also figure S5.

(A–B) E12.5 hearts stained with podoplanin, p-Histone H3 and γ-tubulin indicate epicardial mitotic spindles are either (A) perpendicular or (B) parallel to the basement membrane indicated by dotted line. Arrowheads point to epicardial cells positive for p-Histone H3 labeled centrosomes. (C–E) Quantification of epicardial cell spindle orientations at E12.5 and E13.5. Spindles were visualized by staining for γ-tubulin (as in A and B) or α-tubulin (as in Fig. S2). The basement membrane was considered 0° and is represented by the red line. Each line represents the spindle orientation of a dividing epicardial cell. Numbers in brackets quantify the divisions that occur between 0–30° and 60°–90°. (E) Ratio of spindle orientations at each embryonic stage. (F,G) Time lapse and Z stack images of E12.5 hearts cultured ex vivo. Tracing GFP positive cells, we observed divisions that were either (F) perpendicular or (G) parallel to the basement membrane. f1–f4 are consecutive images that were taken every 6 minutes. Arrowhead points to a cell that starts in (f1) pro-metaphase, then moves to (f2) metaphase, (f3) telophase, and (f4) produces two daughters. g1–g4 is similar to f1–f4 except this cell completed a parallel division. Images indicated by the primes are side views, and nuclei are indicated by red and white dots, respectively. Dotted lines indicate the basement membrane. Scale bar = 10 µm. See also figure S2.

Formation of adherens junctions (α-catenin and N-cadherin) and tight junctions (ZO-1) in (A) control and (B) βcat^EKO. Arrowheads point to the junctions. Podoplanin marked epicardial cells. (C–E). Wild type hearts were stained for the indicated cell polarity proteins. (F–G) Par3 localization in (F) control and (G) βcat^EKO mitotic epicardial cells. Arrow indicates accumulated Par3. Scale bar = 10 µm.

(A) Numb localized to adherens junctions in control but not in βcat^EKO epicardial cells. Arrowheads indicate adherens junctions. (B) Numb, Par3, N-cadherin (N-cad) and β-catenin (β-cat) were immunoprecipitated from primary epicardial cell lysates and blotted for the indicated proteins. (C) N-cadherin was immunoprecipitated from control and βcat^EKO epicardial cell lysates and blotted for interactions with the indicated proteins. Input was 5% of the lysate. (D) Fewer WT1⁺ cells were present in the myocardium of NFP^EKO than in control hearts. (E) Ad- GFP was used to trace epicardial cell EMT in an ex vivo culture system. (F) Adherens junctions (α-catenin and N-cadherin) and tight junctions (ZO-1) in NFP^EKO epicardial cells. Arrowheads point to junctions. Podoplanin was used to identify epicardial cells. Scale bar = 10 µm. See also figure S4.