RESEARCH ARTICLE

Nonmuscle myosin IIB regulates epicardial integrity andepicardium-derived mesenchymal cell maturationXuefei Ma1,*, Derek C. Sung1, Yanqin Yang2, Yoshi Wakabayashi2 and Robert S. Adelstein1

ABSTRACTNonmuscle myosin IIB (NMIIB; heavy chain encoded by MYH10)is essential for cardiac myocyte cytokinesis. The role of NMIIB inother cardiac cells is not known. Here, we show that NMIIB is requiredin epicardial formation and functions to support myocardialproliferation and coronary vessel development. Ablation of NMIIB inepicardial cells results in disruption of epicardial integrity with a loss ofE-cadherin at cell–cell junctions and a focal detachment of epicardialcells from the myocardium. NMIIB-knockout and blebbistatin-treatedepicardial explants demonstrate impaired mesenchymal cellmaturation during epicardial epithelial–mesenchymal transition.This is manifested by an impaired invasion of collagen gels by theepicardium-derived mesenchymal cells and the reorganization ofthe cytoskeletal structure. Although there is a marked decrease in theexpression of mesenchymal genes, there is no change in Snail (alsoknown as Snai1) or E-cadherin expression. Studies from epicardium-specific NMIIB-knockout mice confirm the importance of NMIIB forepicardial integrity and epicardial functions in promoting cardiacmyocyte proliferation and coronary vessel formation during heartdevelopment. Our findings provide a novel mechanism linkingepicardial formation and epicardial function to the activity of thecytoplasmic motor protein NMIIB.

KEYWORDS: Nonmuscle myosin IIB, Epicardial integrity, Epicardialepithelial–mesenchymal transition, Actin cytoskeleton

INTRODUCTIONThe development of a functional heart is orchestrated by variouslineages of cardiac cells including epicardial, endocardial,myocardial and cardiac non-myocyte interstitial cells (Tirziuet al., 2010; Brade et al., 2013), each of which contains thecytoplasmic contractile protein nonmuscle myosin IIB (NMIIB;heavy chain encoded by MYH10) (Ma and Adelstein, 2012). Theepicardium is a single cell layer of mesothelial cells that coversthe outer surface of the heart and protects the integrity of themyocardium (Von Gise and Pu, 2012). It is also the source of anumber of growth factors that promote myocardial growth duringembryonic heart development. Moreover the epicardium serves asthe source for many types of cardiac cells following the epicardialepithelial–mesenchymal transition (EMT). These include pericytes,vascular smooth muscle and endothelial cells, which are critical forcardiac coronary vessel formation, as well as interstitial fibroblasts,

which play important roles in cardiac architecture. They also may bea source for cardiac myocytes (Von Gise and Pu, 2012).

Decades of research have revealed the important extracellularsignals (TGFβ, retinoic acids, PDGF, FGFs etc.), and thedownstream Snail and E-cadherin canonical pathway regulatingepicardial EMT (Brade et al., 2013; Von Gise and Pu, 2012). Thesignificance of cytoskeletal dynamics in controlling EMT is lessfrequently addressed. However, there are several lines of evidencethat point to a role for NMII in EMT. First, epicardial EMT featuresan extensive actin cytoskeletal reorganization where epicardial cellslose their epithelial apical-basal polarity and strong cell–celladhesions, to acquire mesenchymal front-rear polarity and focaladhesions resulting in increased motility and invasiveness (Bradeet al., 2013; Von Gise and Pu, 2012). NMII plays important roles inregulating actin-cytoskeletal structures (Heissler and Manstein,2013; Vicente-Manzanares et al., 2009). Second, Rho activity isalso important in epicardial EMT and cardiac coronary vesselformation. Inhibiting Rho signaling blocks epicardial EMT both invivo and in vitro (Artamonov et al., 2015; Dokic and Dettman, 2006;Lu et al., 2001). Disruption of the Vangl2 and Rho kinase pathwayin the developing mouse heart impairs coronary vessel formation(Phillips et al., 2008). NMII is activated by phosphorylation of itsassociated regulatory light chain (MLC20; also known as MYL12Aand MYL12B) by myosin light chain kinase or Rho kinase (whichalso directly inhibits myosin phosphatase) (Vicente-Manzanareset al., 2009). Third, inhibition of NMII activity affects the behaviorof neural crest cells generated during neuroepithelial EMT in vivo inzebrafish brain development (Berndt et al., 2008). Moreover, fourth,NMII activity has been shown to affect stem cell lineagespecification (Buxboim et al., 2014; Engler et al., 2006; Kimet al., 2015; Wang et al., 2012). All of these findings suggest thatNMII may be involved in regulating epicardial EMT.

NMII is one of the major cellular motor proteins regulatingcytoskeletal structure and function by interacting with actin to eithergenerate tension on actin filaments or translocate actin filaments.Three isoforms of NMII have been identified in vertebratesincluding humans and mice, namely NMIIA, NMIIB and NMIICbased on three different heavy chain (NMHC) genes: Myh9encoding NMHCIIA, Myh10 encoding NMHCIIB and Myh14encoding NMHCIIC (Golomb et al., 2004; Berg et al., 2001). Eachisoform plays unique as well as overlapping roles during mouseembryonic development partially due to their differences indynamic motor activities and expression patterns in varioustissues (Ma and Adelstein, 2014b).

Compared to NMIIA and NMIIC, NMIIB is relatively enriched inthe brain and heart. Mice with a knockout for NMIIB die duringembryonic development by embryonic day (E)14.5 with severecongenital cardiac abnormalities. These include a hypoplasticmyocardium with reduced proliferative activity of the cardiacmyocytes and premature cardiac myocyte bi-nucleation, in additionto cardiac structural abnormalities such as a ventricular septalReceived 14 February 2017; Accepted 1 July 2017

1Laboratory of Molecular Cardiology, National Heart, Lung, and Blood Institute,National Institutes of Health, Bethesda, MD 20892-1762, USA. 2DNA SequencingandGenomics Core, National Heart, Lung, and Blood Institute, National Institutes ofHealth, Bethesda, MD 20892-1762, USA.

*Author for correspondence (max@nhlbi.nih.gov)

X.M., 0000-0002-9665-8282

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defect, double outlet of the right ventricle and pulmonary arterialstenosis (Tullio et al., 1997). Our previous studies on NMIIB in theheart primarily focused on cardiac myocytes. Knockout of NMIIBin cardiac myocytes resulted in a failure in cytokinesis (Takedaet al., 2003). Moreover, NMIIB exerts tension to drive contractilering constriction during cardiac myocyte cytokinesis (Ma et al.,2012). NMIIB is also required to disrupt the cardiac myocyte cell–cell adhesion complex during outflow tract myocardialization, theprocess necessary for normal alignment of the aorta to the leftventricle (Ma and Adelstein, 2014a), and to maintain the integrity ofcardiac intercalated discs in adult hearts (Ma et al., 2009). The rolesof NMIIB in other cardiac cells, such as the epicardium, have not yetbeen studied. The current study seeks to understand the role ofNMIIB in epicardial formation and function in vivo during mousecardiac development.

RESULTSAbnormal epicardium and coronary vessels in B−/B− heartsWe have previously shown that NMIIB is required for cardiacmyocyte cytokinesis duringmouse heart development (Takeda et al.,2003). In addition to its expression in cardiac myocytes, NMIIB isalso expressed in epicardial cells (Ma and Adelstein, 2012). Weexamined the localization of NMIIB in the developing epicardium offreshly isolated hearts from E14.5 mice expressing GFP-taggedNMHCIIB (denoted BGFP) (Bao et al., 2007). Confocal analysis ofE14.5 whole mouse hearts shows that NMIIB is concentrated at thecell–cell junctions of the epicardium (Fig. 1A, green). Super-resolution structured illumination microscopy (SIM) analysis furthershows paired NMIIB alignment between epicardial cells (Fig. 1B),reminiscent of NMII localization at epithelial cell–cell junctions(Ebrahim et al., 2013) and suggesting a role for NMIIB in regulatingepicardial cell–cell adhesion.Epicardial integrity is maintained by epicardial cell–cell

junctions, including adherens and tight junctions. We examinedthese junctions in mice with global knockout of NMHCIIB (i.e.Myh10–/–; hereafter denoted B−/B−) at E13.5 by whole-mountimmunofluorescence staining for localization of the adherensjunction molecule E-cadherin and the tight junction molecule ZO-1 (also known as TJP1). E-cadherin is enriched at the epicardialcell–cell junctions in the wild-type hearts (Fig. 1C, red). In contrast,enrichment for E-cadherin is markedly diminished at epicardialcell–cell junctions in B−/B− hearts (Fig. 1D, red). The localizationof ZO-1 at epicardial cell–cell junctions is observed both in B+/B+

(i.e. wild-type for NMIIB) and B−/B− hearts (Fig. S1, green).However, instead of forming a uniform continuous sheet ofZO-1-stained tight junctions, as seen in the wild-type epicardium(Fig. S1A, green), B−/B− epicardium shows irregular ZO-1staining with an uneven distribution apparent at different confocalz-sections (Fig. S1B–D, green). This uneven distribution of ZO-1may reflect its relocalization to the lateral sides of the cell due to thealtered epicardial apical-basal polarity. z-projections of ZO-1staining, however, show continuity of the tight junction in B−/B−

hearts. These results are consistent with the disorganization of theB−/B− epicardium. To further examine whether ablation of NMIIBaffects the epicardial barrier function, we carried out a biotinpermeability assay by pipetting biotin solution into the thoraciccavity of E13.5 mouse embryos. Detection of biotin (red) in heartsections through immunofluorescence shows that biotin is mostlyconfined to the epicardial layer in B+/B+ hearts (Fig. 1E, red,arrowheads) but, in contrast, is detected throughout the variouslayers of the heart in B−/B− mice (Fig. 1F, red) indicating acompromised barrier function. Thus, NMIIB is important for

epicardial integrity because it functions in the maintenance ofepicardial cell–cell junctions.

In addition to protecting the heart, the epicardium also playsimportant roles in coronary vessel formation during heartdevelopment. We next examined the coronary vessels in E13.5B−/B− hearts through whole-mount staining with antibodies againstPECAM1 (CD31, green), a marker for endothelial cells. Whereasthe developing coronary vasculature of B+/B+ hearts shows normal‘chicken wire’ coronary plexuses covering the entire dorsal surfaceof the ventricles (Fig. 2A, enlarged in 2C), the B−/B− heartsshow abnormal coronary vascular morphogenesis, with markedlyreduced coverage by vascular plexuses (Fig. 2B, enlarged in 2D).

Fig. 1. Localization of NMIIB in epicardium and abnormalities of B−/B−

epicardium. (A,B) Confocal images of freshly isolated E14.5 heartsexpressing EGFP–NMHCIIB (BGFP) show localization of NMIIB at cell–celljunctions of the epicardium (A, green). Scale bar: 20 µm. Super-resolution SIMshows paired alignments of NMIIB at the cell–cell junctions (B). (C,D) Whole-mount immunofluorescence confocal images of E13.5 mouse epicardiumshowing E-cadherin (red) at the epicardial cell–cell junctions in B+/B+ mousehearts (C). In B−/B−mouse hearts, E-cadherin is greatly diminished at the cell–cell junctions (D). Nuclei were stained blue with DAPI. Scale bar: 20 µm.(E,F) Biotin permeability assay of E13.5 mouse epicardium showing impairedepicardial integrity in B−/B− hearts. Biotin was detected with Rhodamine-conjugated streptavidin (red) and shows deep penetrance throughout theentire ventricle in B−/B− hearts (F, red). Biotin is limited near the epicardiallayer in B+/B+ hearts (E, red). Vimentin (green) stains cardiac nonmyocytes.Nuclei were stained blue with DAPI. Arrowheads point to the epicardium.Scale bars: 50 µm.

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These phenotypes in B−/B− hearts are consistent with abnormalitiesin the epicardium.

NMIIB is important for epicardial function to supportmyocardial growthThere is extensive cross-talk between the epicardium andmyocardium during embryonic heart development. Defects inmyocardial development such as those seen in our B−/B− heartscould conversely also affect epicardial formation. To confirmthe importance of epicardial NMIIB in heart development, wegenerated epicardial-specific NMIIB-knockout mice by crossingNMIIB floxed mice (Bflox/Bflox) with a mouse line expressing Crerecombinase driven by the WT-1 promoter and examined heartdevelopment and coronary vessel formation in these mice(BWT-1/BWT-1). Although WT-1-mediated Cre recombinase hasbeen reported to be transiently activated in other cell types (Rudatand Kispert, 2012), the BWT-1/BWT-1 mouse hearts studied here showspecific knockout of NMIIB in epicardial cells but not in endocardialand myocardial cells (Fig. S2). Knockout of NMIIB in theepicardium was confirmed by immunofluorescence confocalmicroscopy using antibodies to NMHCIIB (Fig. S2C,D comparedto Fig. S2A,B, red, arrows). The expression of NMIIB in themyocardium (desmin, green, a marker for myocytes) andendocardium (arrowheads) in BWT-1/BWT-1 hearts (Fig. S2C,D) wascomparable to that in Bflox/Bflox hearts (Fig. S2A,B).Knockout of NMIIB does not affect NMIIA expression in

BWT-1/BWT-1 epicardial cells (Fig. S3E, red, arrows) compared towhat is seen in Bflox/Bflox cells (Fig. S3B, red, arrows). There is also nochange in the phosphorylation of serine 19 of the regulatory myosinlight chain (pMLC20) in BWT-1/BWT-1 epicardial cells (Fig. S3D,green, arrowheads) compared to what is seen in Bflox/Bflox cells(Fig. S3A, green, arrowheads). Note that the number of NMIIA-positive non-myocytes in compact myocardium is markedlyreduced in BWT-1/BWT-1 hearts (Fig. S3F, arrows) compared tothat in Bflox/Bflox hearts (Fig. S3C, arrows) indicating a defect inepicardium-derived cell development (see below for details). Theseresults indicate that WT-1 Cre-mediated knockout of NMIIBin mice primarily affects epicardial expression of NMIIB inBWT-1/BWT-1 hearts and not myocardial or endocardial expression.

Histological analysis shows that the compact myocardiumis markedly thinner in BWT-1/BWT-1 hearts compared to that inBflox/Bflox hearts at E15.5 (Fig. 3A,B, brackets). BWT-1/BWT-1 heartsat E12.5 show focal detachment of the epicardium from themyocardium (Fig. 3D, arrows). This detachment is not seen incontrol hearts (Fig. 3C, Bflox/Bflox). It has been reported thatepicardial N-cadherin is required to maintain heterotypicepicardial–myocardial cell–cell interactions (Luo et al., 2006).Immunoconfocal microscopy analysis shows that N-cadherin isexpressed in both epicardial and myocardial cells in Bflox/Bflox

hearts (Fig. 3E, red). Knockout of NMIIB in epicardial cells doesnot affect N-cadherin expression in myocardial cells in BWT-1/BWT-1

hearts. However, the expression of N-cadherin in epicardial cellsis markedly diminished in BWT-1/BWT-1 hearts (Fig. 3F, red,arrowheads) compared to that seen in the Bflox/Bflox epicardial

Fig. 2. Abnormal coronary vessel formation in B−/B− hearts. Confocalz-projections of the dorsal surface of whole-mount E14.5 hearts stained forCD31 to reveal coronal vessels showing that the coronary plexuses cover thewhole B+/B+ ventricular surface (A, enlarged in C). The normal ‘chicken wire’coronary plexus is not obvious in the B−/B− heart (B, enlarged in D).

Fig. 3. Epicardial knockout of NMIIB results in a hypoplastic myocardiumand focal detachment of epicardium from myocardium. (A–D) H&E-stained heart sections of Bflox/Bflox (A,C) and BWT-1/BWT-1 (B,D) hearts showingreduced thickness of the compact myocardium in BWT-1/BWT-1

hearts (B, bracket) compared to Bflox/Bflox hearts (A, bracket) at E15.5.Detachment of epicardial cells from myocardium is more often seen in BWT-1/BWT-1 hearts at E12.5 (D, arrows) compared to in Bflox/Bflox hearts (C). Scalebars: 200 µm. (E,F) Immunofluorescence confocal microscope images ofE13.5mouse hearts stainedwith an antibody for N-cadherin (red) showing lossof expression in the epicardium in BWT-1/BWT-1 hearts (F, arrowheads)compared to in Bflox/Bflox hearts (E, arrowheads). Nuclei were stained blue withDAPI. Scale bar: 100 µm.

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cells (Fig. 3E, red, arrowheads). The expression of β1-integrin is notaffected in BWT-1/BWT-1 epicardium (Fig. S4). These results indicatethat epicardial NMIIB is important for epicardial–myocardialattachment by, at least in part, maintaining N-cadherin-mediatedepicardial–myocardial cell–cell adhesion. Unlike the B−/B− heart,no evidence of bi-nucleated cardiac myocytes was found inBWT-1/BWT-1 hearts, suggesting that NMIIB functions normally inBWT-1/BWT-1 cardiac myocyte cytokinesis. BWT-1/BWT-1 embryonichearts, however, showed a reduced proliferation of cardiac myocytes,as evaluated by the BrdU-labeling assay (Fig. 4A,B, green). BrdU-positive cardiac myocytes in BWT-1/BWT-1 hearts (Fig. 4B, 21±4%;mean±s.d., n=3 mice) are fewer in number than in Bflox/Bflox hearts(Fig. 4A, 28±3%; mean±s.d., n=3 mice, P

Bflox/Bflox compact myocardium (Fig. 5E, arrows), whereas only16.9±3.9% of EPDCs are labeled by BrdU in BWT-1/BWT-1 compactmyocardium (Fig. 5H, arrow). During embryonic heart development,EPDCs differentiate into pericytes and smooth muscle cells (SMCs)to support coronary vessels.We next examinedwhether pericytes andSMCs are properly positioned surrounding the coronary vessels inBWT-1/BWT-1 hearts. Both SMCs (Fig. S5A, red, BASM) andpericytes (Fig. S5C, red, NG2) are found surrounding coronaryvessels (Fig. S5A,C, green, CD34, arrows) in Bflox/Bflox hearts. Veryfew pericytes and SMCs are detected surrounding BWT-1/BWT-1

coronary vessels (Fig. S5B,D). Quantification of SMCs in compactmyocardium shows that BWT-1/BWT-1 hearts have significantly fewer

SMCs (0.132±0.026/µm2) than Bflox/Bflox hearts (0.219±0.019/µm2,P

E11.5 mouse hearts. Following 3 days of stimulation with 10%fetal bovine serum (FBS) to induce EMT, the explants werestained with phalloidin to show F-actin (Fig. 6A,B, red). In wild-type explants, EPDCs generated following epicardial EMTmigrate into the collagen gel (similar to mesenchymal cells) andform vessel-like projections (Fig. 6A). In contrast, B−/B− explantsshow a marked reduction of the number of cells migrating into thecollagen gel (Fig. 6B), consistent with impaired epicardial EMT.Note that the total number of cells present in the explants iscomparable between B+/B+ and B−/B− explants. The lower part ofthe panels of Fig. 6A,B are x-z plots of the explants showing cellnuclei stained with DAPI. Again, B−/B− explants showsignificantly fewer cells migrating downward into the gelcompared to for B+/B+ explants. The average percentages ofcells migrating into the gel are 34±1% and 14±3% for B+/B+ andB−/B− explants, respectively (mean±s.d., n=3 each genotype,P

defect, we examined the expression of genes known to be importantfor EMT as well as the expression of genes encoding epithelial andmesenchymal markers in wild-type and B−/B− epicardial explantsby performing quantitative real-time PCR (qRT-PCR). Comparedto the wild-type explants, B−/B− epicardial explants showsignificantly reduced expression of genes for mesenchymalmarkers including Cdh6 (K-cadherin), Acta2 (smooth muscle α-actin, SMA), Tagln (SM22α) and Postn (Table 1, P

number of total SMA-positive cells migrating from explants for theentire panel are: 598±151 (DMSO, control), 249±71 (10 nMlatrunculin-A, P

mesenchymal cells associated with extensive reorganization of theactomyosin cytoskeleton is of major interest and is important forcardiac development. One of the novel findings from this study isthe role of NMIIB in regulating EPDC proliferation and maturationfollowing epicardial EMT. Following EMT, EPDCs migrate,proliferate and differentiate into pericytes, smooth muscle cellsand cardiac fibroblasts to support coronary vessel formation. In thisstudy, we provide evidence that knockout of NMIIB specifically inmouse epicardial cells results in a marked reduction of EPDCproliferation, resulting in fewer EPDCs being found in the compactmyocardium. It is likely that EPDCs also receive proliferativesignals from the epicardium, similar to for the cardiac myocytes asdiscussed above. Defects in epicardial function and attachment ofthe epicardium to the myocardium may block the epicardialsignaling for EDPC proliferation in BWT-1/BWT-1 hearts.Although NMIIB knockout affects neither activation of canonical

Snail/E-cadherin signaling nor the decreased expression ofepithelial genes during epicardial EMT, the increased expressionof mesenchymal genes such as Acta2 (smooth muscle actin) andTagln (SM22α) is markedly impaired during epicardial EMT.Whilethe defects in NMIIB-knockout epicardium during EMT are notsecondary to the insufficiency of canonical Snail/E-cadherinsignaling, NMIIB is required for full acquisition of themesenchymal phenotype by EPDCs following epicardial EMT.Our finding of decreased periostin expression during B−/B−

epicardial EMT is also consistent with a lack of EPDCmaturation, since mice with a knockout for periostin show adefect in mesenchymal differentiation of the cardiac cushions intofibroblast tissue during cardiac valve formation (Snider et al., 2008).This is also consistent with a previous report showing a NMIIC toNMIIB isoform switch, and a reduced mesenchymal cell invasionfollowing siRNA knockdown of NMIIB during EMT in mousemammary epithelial cells (NMuMG) in response to TGFβ (Beachet al., 2011). Recently, NMIIB was shown to generate tension fornuclear translocation during migration in 3D collagen gels (Thomaset al., 2015).EMT features an extensive reorganization of cytoskeletal

structures, including formation of intracellular actin stress-fibersand formation of focal adhesion complexes in mesenchymal cells,which are required for cell morphological changes and cellmigration during EMT (Lamouille et al., 2014). Increasedexpression of the ERM (ezrin/radixin/moesin) protein moesin alsocontributes to actin cytoskeleton remodeling and morphologicalchanges during EMT (Haynes et al., 2011). It was previouslyunclear whether actin cytoskeleton remodeling is required for EMT.Shankar et al. were among the first to propose that actin dynamicscontrols EMT in metastatic cancer cells. This hypothesis was basedon their studies with cultured metastatic cancer cell lines showingthat knockdown of pseudopodia-enriched proteins (Shankar et al.,2010) or cytochalasin D-induced disruption of actin filaments(Shankar and Nabi, 2015) decreased pseudopod formation andtumor cell invasion. This was associated with reduced actin filamentstability, the loss of N-cadherin and vimentin expression, and theregaining of E-cadherin expression. These findings suggest thatactin stability plays a role in maintaining the mesenchymalproperties of metastatic cancer cells in culture. The resultspresented here provide direct evidence showing that NMIIB-mediated actin filament formation is actively involved in maturationof the mesenchymal phenotype during epicardial EMT in explantsand in vivo during mouse heart development. NMII regulates bothactin stress fiber formation and focal adhesion formation (le Ducet al., 2010; Pasapera et al., 2010). Activation of NMII promotes

actin stress fiber formation, while NMII tension on actin stress fiberspromotes maturation of nascent focal adhesion complexes.Moreover, we demonstrate that ablation of NMIIB or inhibition ofNMIIB activity impairs mesenchymal gene expression duringepicardial EMT associated with defects in actin stress fiberformation and focal adhesion maturation. Since interference withactin dynamics directly by destabilizing actin stress fibers blocksmesenchymal phenotype maturation during epicardial EMT, wesuggest that NMIIB regulates epicardial EMT in vivo and in vitro byaltering actin dynamics.

Taken together, our results support the idea that epicardialEMT may be divided into two steps: one that does not rely onNMIIB, including the initial induction of EMT Snail signaling andloss of the epithelial phenotype, and a second step that requiresNMIIB activity to acquire the mesenchymal phenotype, such aschanges in cell polarity and cytoskeletal reorganization that isneeded for cell migration, and an increase in mesenchymal geneexpression. While these two programs may not be completelyindependent of each other, both are ultimately necessary forcompletion of epicardial EMT.

MATERIALS AND METHODSAnimalsB−/B−, Bflox/Bflox, and BGFP/BGFP mice were generated as previouslydescribed (Bao et al., 2007; Ma et al., 2009; Tullio et al., 1997) and areavailable through the Mutant Mouse Regional Resource Centers (MMRRC,#16991and #37053). WT-1 Cre mice were generously provided byDr William Pu (Boston Children’s Hospital, Boston, MA). All procedureswere conducted using an approved animal protocol (H0053R3) inaccordance with National Heart, Lung, and Blood Institute Animal Careand Use Committee guidelines.

Histology and immunofluorescence stainingThe mouse embryos or hearts were collected in PBS and directly immersedin 4% paraformaldehyde (PFA) in PBS (pH 7.4) overnight. Paraffin sectionsat a thickness of 5 µm were prepared by Histoserv, Inc. (Germantown, MD).Primary antibodies for immunostaining were incubated at 4°C overnightfollowing antigen retrieval in 10 mM citrate buffer (pH 6). The followingprimary antibodies were used in this study: polyclonal antibodies againstpMLC20 (1:400, Cell Signaling Technology), NG2 (1:400, MilliporeSigma), NMHCIIB (1:3000, Covance), vimentin Alexa Fluor 488 conjugate(1:200, Cell Signaling Technology), bovine aorta smooth musclemyosin heavy chain (BASM, 1:200; Kelley and Adelstein, 1990) andWT-1 (1:100, Neomarker); and monoclonal antibodies against CD34(1:500, GeneTex), E-cadherin (1:500, BD Biosciences, San Jose, CA),desmin (1:100, DakoCytomation, Denmark), FGF-9 (1:100, Santa CruzBiotechnology, Santa Cruz Biotechnology, CA), β1 integrin (1:300, BDTransduction), N-cadherin (1:200, Invitrogen), NMHCIIA (1:300, Abcam),PECAM1 (CD31, 1:200, BD Pharmingen), smooth muscle α-actin(1:2000, Sigma), vinculin (1:1000, Sigma) and ZO-1 (1:200, Invitrogen).Fluorescence secondary antibodies used were: Alexa Fluor 488-conjugatedgoat anti-rabbit-IgG or Alexa Fluor 594-conjugated goat anti-mouse-IgG(1:250, Invitrogen, Carlsbad, CA). The slides were mounted with ProlongGold antifade mounting medium (Invitrogen) and observed under aconfocal microscope. The confocal images were collected using a ZeissLSM 510-META. In all cases, when possible, comparison was made amonglittermates. For each genotype, we analyzed at least three to five mice.

Biotin epicardial permeability assayEZ-Link Sulfo-NHS-LC-Biotin (Thermo Scientific) solution was freshlyprepared at 10 mg/ml in 1× PBS. Mouse embryos were dissected and placedin PBS. An opening in the chest wall, which broke the pericardium, wasmadeusing forceps, and 10 µl of biotin was pipetted into the opening and allowedto perfuse for 15 min. Embryos were then fixed in 4% PFA overnight,paraffin embedded and sectioned. Following deparaffinization, antigenretrieval, and blocking in 10% goat serum, sections were incubated with

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Alexa Fluor 488-conjugated vimentin (1:200, Cell Signaling Technology), tovisualize cardiac non-myocytes, and Rhodamine-conjugated streptavidin, todetect biotin, for 30 min prior to confocal imaging.

Epicardial explants3D epicardial explants were prepared from E11.5 mouse hearts with the atriaand outflow tract removed. The hearts were placed on a 1% collagen gel(prepared from rat tail collagen I in MEM) with the dorsal surface againstthe gel. The epicardial cells were allowed to grow out over the gel for 2 dayswith only one drop of Dulbecco’s modified Eagle’s medium (DMEM) overthe hearts. The hearts were then removed, and the explants were cultured foranother 3 days in 10% FBS in DMEMwith pencillin-streptomycin to induceEMT. To study the effect of various chemicals, as indicated in the text, onepicardial explants, the explants were prepared from E11.5 wild-type(C57BL6, Jackson Lab) mouse hearts and then the explants were randomlydivided into two groups. Following removal of hearts from explants at day 2,each group of explants was further cultured in medium with variouschemicals or the solvent (DMSO) as control. 2D epicardial explants wereprepared by placing E11.5 hearts on gelatin-coated coverslips with 10%FBS in DMEM with pencillin-streptomycin for 2 days and then removingthe hearts. The explants were grown for 2 additional days. For confocalanalysis, the explants were fixed in 4% PFA, permeabilized with TritonX-100 and then incubated with various antibodies of interest.

RNAseq and qRT-PCR AnalysisRNA was extracted from 3D epicardial explants after 72 h incubation with10% FBS in DMEM using the RNeasy Mini plus Kit (Qiagen) andquantified by Qubit Fluorometer. RNA-seq libraries were constructed usingOvation RNA-Seq System V2 kit (NuGen, Inc., San Carlos, CA) andNextera XT DNA library preparation kit (Illumina, Inc., San Diego, CA) byfollowing the manufacturer’s instructions. After the final amplification step,the PCR products were separated on 2% agarose gel to excise 250–450 bpfragments. The resulting barcoded RNA-seq libraries were then pooled andsubjected to 2×50 bp paired-end sequencing using the Illumina HiSeq3000platform (Illumina, San Diego, CA, USA). Raw sequencing data weredemultiplexed and converted into a FASTQ format. Real-time PCR wasperformed using an ABI 7500 real-time PCR instrument following themanufacturer’s instruction. Glyceraldehyde-3-phosphate dehydrogenase(GAPDH) expression level was used as a control for normalization.

Quantification and statistical analysesThe BrdU labeling index of cardiac myocytes was calculated as a percentageof the number of BrdU- and desmin-positive cells over the total number ofdesmin-positive cells. Embryonic heart sections were stained withantibodies against BrdU and desmin. Nuclei were stained by DAPI.Confocal images were captured with a 40× objective. The images wereanalyzed with IDL Software (programmed by Christian A. Combs,NHLBI). The index was scored from three different embryos per eachgenotype. For each mouse, more than 1000 total cells of the compactmyocardium were counted.

The percentage of epicardial cells that dissociated from myocardium overthe total epicardial cells was calculated from H&E images with ImageJsoftware. Hearts from three mice per genotype were quantified and morethan 1000 total epicardial cells were counted per mouse.

Epicardial explants were quantified from three experiments per genotype(or treatment) with ImageJ software. For the 3D collagen gel migrationassay, ∼200 total cells were counted for each explant. The focal adhesionsize and actin filament thickness were calculated as an average from threedifferent explants per genotype; 10 cells were measured for each explant.On average 15 to 20 focal adhesions and actin-filaments were measured foreach cell.

Data are expressed as mean±s.d. The Student’s t-test was performed tocompare two means.

AcknowledgementsWe thank Dr Mary Anne Conti for her significant contributions to this manuscript.Dr Sachiyo Kawamoto and members of the Laboratory of Molecular Cardiology alsoprovided critical comments on the manuscript. Special thanks for Dr Keekwang Kim

for his help with qRT-PCR analysis. We also thank Drs Chengyu Liu and Yubin Du[National Heart, Lung, and Blood Institute (NHLBI) Transgenic Core], Drs ChristianA. Combs and Daniela Malide (NHLBI Light Microscopy Core). Antoine Smith andDalton Saunders provided technical assistance.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsConceptualization: X.M., R.S.A.; Methodology: X.M.; Formal analysis: X.M., Y.Y.;Investigation: R.S.A.; Data curation: X.M., D.C.S., Y.W.; Writing - original draft: X.M.,D.C.S., R.S.A.; Writing - review & editing: X.M., D.C.S., R.S.A.; Supervision: R.S.A.;Funding acquisition: R.S.A.

FundingThis research was supported by the National Institutes of Health, National Heart,Lung, and Blood Institute (HL-004228). Deposited in PMC for release after12 months.

Data availabilityRNAseq data reported in this paper has been deposited in the Gene ExpressionOmnibus under (GEO) accession number GSE101701 (https:////www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE101701).

Supplementary informationSupplementary information available online athttp://jcs.biologists.org/lookup/doi/10.1242/jcs.202564.supplemental

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