DEVELOPMENT AND STEM CELLS RESEARCH ARTICLE 3531

Development 139, 3531-3542 (2012) doi:10.1242/dev.082222© 2012. Published by The Company of Biologists Ltd

INTRODUCTIONAngiogenesis and vascular patterning are dependent upon thecapacity of vascular smooth muscle cells (SMCs) to transducebiomechanical and humoral signals that influence celldifferentiation, migration, proliferation and survival (Owens, 1998;Owens et al., 2004). Under homeostatic conditions, vascular SMCsassume a spindle-like morphology with a rich, well-organizedcytoskeleton expressing abundant SMC-restricted contractileproteins that together define the contractile properties of thismuscle cell lineage. Within the arterial wall, signals transduceddirectly from endothelial cells and the extracellular matrix (ECM)regulate the physiological properties of SMCs (Astrof and Hynes,2009; Hayward et al., 1995; Thyberg and Hultgårdh-Nilsson,1994). The signals transduced between ECM components andvascular SMCs play crucial roles in regulating embryonicangiogenesis and contribute to pathogenesis of atherosclerosis andthe formation of arterial aneurysms (Raines, 2000).

Our group and others have shown that the MADS boxtranscription factor, serum response factor (SRF), acting in concertwith transcriptional co-activators in the myocardin-relatedtranscription factor (MRTF) family, including myocardin,myocardin-like protein 1 (MKL1) and myocardin-like protein 2(MKL2), promote the contractile SMC phenotype (Parmacek,2007). Forced expression of any member of the MRTF family inembryonic stem (ES) cells activates transcription of endogenous

genes encoding SMC-restricted contractile proteins (Du et al.,2004; Du et al., 2003). In response to Rho/actin signaling, MKL1and MKL2 localize to the nucleus where they physically associatewith SRF and activate transcription of a subset of genes associatedwith cytoskeletal organization (Sotiropoulos et al., 1999).

Two lines of genetically engineered mice that harbor loss-of-function mutations in the Mkl2 gene have been described (Li etal., 2005; Oh et al., 2005; Wei et al., 2007). Mice containing anMkl2 insertional gene trap mutation located between exons 10and 11 exhibit a hypomorphic phenotype. Gene trap mutant micesurvive to birth, but die within 48 hours, exhibiting a spectrumof cardiac outflow tract and great artery patterning defects thatare attributable to a cell-autonomous block in differentiation ofneural crest-derived vascular SMCs (Li et al., 2005; Wei et al.,2007). Defects in development of the vitelline system thatproduces the liver sinusoids are also observed in Mkl2 gene trapmutant embryos (Wei et al., 2007). By contrast, Mkl2–/– embryossurvive until embryonic day (E) 13.5-14.5 and they alsodemonstrate defective remodeling of the pharyngeal arch arteriesand cardiac outflow tract, recapitulating common forms ofcongenital heart disease (Oh et al., 2005). In addition, Mkl2–/–

embryos develop pericardial edema and hemorrhage (Oh et al.,2005). However, the observed defects in vascular patterning inMkl2–/– null embryos fails to explain lethality at mid-gestation,raising questions of what other functions are mediated by MKL2in differentiating cells and the embryo.

Multiple studies have shown that TGF signaling plays a crucialrole in development of the great arteries (Pardali et al., 2010).TGF signaling influences vascular SMC shape, migration,homing and location through processes that are controlled by cell-surface transmembrane receptors and components of theextracellular matrix (ECM) (Massagué, 1990). TGF2 and, to alesser extent, TGF3 are expressed by vascular SMCs (Molin etal., 2003). Genetic studies in mice and humans have shown that

1University of Pennsylvania Cardiovascular Institute, University of Pennsylvania,Philadelphia, PA 19104-4283, USA. 2Department of Medicine, University ofPennsylvania, Philadelphia, PA 19104-4283, USA. 3Department of Surgery, Universityof Pennsylvania, Philadelphia, PA 19104-4283, USA. 4University of Maryland Centerfor Bioinformatics and Computational Biology, College Park, MD 20742, USA.

*Author for correspondence (michael.parmacek@uphs.upenn.edu)

Accepted 6 July 2012

SUMMARYThe molecular mechanisms that regulate and coordinate signaling between the extracellular matrix (ECM) and cells contributing tothe developing vasculature are complex and poorly understood. Myocardin-like protein 2 (MKL2) is a transcriptional co-activatorthat in response to RhoA and cytoskeletal actin signals physically associates with serum response factor (SRF), activating a subset ofSRF-regulated genes. We now report the discovery of a previously undescribed MKL2/TGF signaling pathway in embryonic stem(ES) cells that is required for maturation and stabilization of the embryonic vasculature. Mkl2–/– null embryos exhibit profoundderangements in the tunica media of select arteries and arterial beds, which leads to aneurysmal dilation, dissection andhemorrhage. Remarkably, TGF expression, TGF signaling and TGF-regulated genes encoding ECM are downregulated in Mkl2–/–

ES cells and the vasculature of Mkl2–/– embryos. The gene encoding TGF2, the predominant TGF isoform expressed in vascularsmooth muscle cells and embryonic vasculature, is activated directly via binding of an MKL2/SRF protein complex to a conservedCArG box in the TGF2 promoter. Moreover, Mkl2–/– ES cells exhibit derangements in cytoskeletal organization, cell adhesion andexpression of ECM that are rescued by forced expression of TGF2. Taken together, these data demonstrate that MKL2 regulates aconserved TGF- signaling pathway that is required for angiogenesis and ultimately embryonic survival.

KEY WORDS: Angiogenesis, Embryonic stem cell, Myocardin-related transcription factor B/MKL2, Mouse, Transforming growth factor

Myocardin-like protein 2 regulates TGF signaling inembryonic stem cells and the developing vasculatureJian Li1,2, Nina Bowens1,3, Lan Cheng1,2, Xiaohong Zhu1,2, Mary Chen1,2, Sridhar Hannenhalli4, Thomas P. Cappola1,2 and Michael S. Parmacek1,2,*

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disruption of TGF signaling pathways results in defects invasculogenesis and angiogenesis that are attributable, at least inpart, to vascular SMCs (Pardali et al., 2010). Mice in which theTgbr2 gene was conditionally ablated in vascular SMC precursorsdisplay arterial dilation and aneurysm formation, which leads tolate embryonic lethality (Choudhary et al., 2009).

To examine the function of MKL2 in the developing embryo, wegenerated and characterized Mkl2–/– ES cells and mouse embryos.By mid-gestation, Mkl2–/– embryos develop aneurysmal dilationand dissection of the aorta, carotid arteries and select arterial bedsthroughout the embryo. Mkl2–/– mutant arteries display disruptionof the tunica media with alterations in SMC morphology, alignmentand investment of ECM. Surprisingly, analysis of Mkl2–/– ES cellsrevealed defects in cell adhesion that are attributable to a block inTGF signaling and TGF-regulated genes that encode ECM.Consistent with these data, TGF expression, TGF signaling andthe expression of TGF-regulated genes encoding ECM aredisrupted in the vasculature of Mkl2–/– embryos. Moreover, Tgfb2is activated directly via binding of an MKL2/SRF complex to theTgfb2 promoter. These data reveal an MKL2/TGF-dependentsignaling pathway in ES cells that is also required for developmentand structural integrity of the embryonic vasculature.

MATERIALS AND METHODSGeneration and characterization of Mkl2 null miceAn Mkl2 gene-targeting vector was constructed via recombineering with aBAC (BAC/PAC Resources, clone number BAC RP23-402A16) asdescribed previously (Lee et al., 2001) (supplementary material Fig. S1).Conditionally targeted Mkl2+/F ES cells were transiently transfected with thepCMV-Cre expression plasmid to generate heterozygous Mkl2+/– ES cells.Mkl2+/– ES cells were re-targeted via electroporation with the linearized Mkl2conditional targeting vector (supplementary material Fig. S1A). Mkl2–/F EScells were transfected with the pTurbo-Cre plasmid (Washington University,St Louis, MO) generating homozygous Mkl2–/– ES cells. To generate Mkl2–/–

ES cells that stably express TGF2, Mkl2–/– and wild-type ES cells werestably transfected with the pIRES2-TGF2-EGFP vector that expressesTGF2 and EGFP. Conditionally targeted Mkl2+/F ES cells weremicroinjected into C57BL/6 donor blastocysts as described previously(Morrisey et al., 1998). Southern blot analysis was performed to confirm thatthe conditionally targeted Mkl2+/F allele was passed through the germline(supplementary material Fig. S1). To create a germline mutation in the Mkl2gene, Mkl2F/F mice were interbred with CMV-Cre transgenic mice. Genotypeanalyses were performed by Southern blot or by PCR as described previously(Li et al., 2005). PCR genotyping primers can be found in supplementarymaterial Table S1. All animal experimentation was performed underprotocols approved by the University of Pennsylvania IACUC and inaccordance with NIH guidelines.

Histology and immunohistochemistryStandard histology and immunohistochemistry protocols are available athttp://www.med.upenn.edu/mcrc/histology_core (IHC in paraffin sectionand H&E staining) and http://www.med.upenn.edu/mcrc/histology_core/culturedcells/shtml. F-actin expression was assessed by staining withrhodamine-tagged phalloidin (Invitrogen, catalog code R415) and G-actinwas identified with FITC-stained DNase I (Invitrogen, catalog codeD12371). A list of the antibodies used is provided in supplementarymaterial Table S2.

Immunoblot and ELISA analysesImmunoblot analyses were performed as described previously (Li et al.,2005). The concentration of secreted fibronectin in ES cells wasdetermined using the mouse fibronectin ELISA (Cell Application,catalog code CL0351). ES cells (1.0�106/well) were seeded in six-wellplates with 2 ml medium for 1 hour and the medium was collected formeasurement of secreted fibronectin. Data are expressed as ngfibronectin/ml±s.e.m.

Microarray analysisTotal RNA was extracted from wild-type and Mkl2–/– ES cells using theRNeasy protocol (Invitrogen), yielding RNA for two wild-type and threeMkl2–/– samples. Each sample was hybridized with Affymetrix MouseGene ST 1.0 microarrays according to standard protocols. Raw.cel fileswere normalized using the RMA algorithm, and exploratory analysescompared fold change in expression in Mkl2–/– versus control. These datawere deposited in the Gene Expression Omnibus (GEO) public database(Accession Number GSE38316). To screen for signaling pathways affectedby Mkl2 deletion, Gene Set Enrichment Analysis (GSEA) was performedusing all signaling pathways described in the Kyoto Encyclopedia of Genesand Genomes (KEGG) in the Molecular Signature Database (Subramanianet al., 2005).

qRT-PCRThe sequences of the PCR primers are listed in supplementary materialTable S1. Target genes were amplified by PCR with Power SYBR green(Applied Biosystems, catalog number 4309155) as described previously(Du et al., 2004). All reactions were run in triplicate, and relative geneexpression was calculated against a GAPDH standard as describedpreviously (Du et al., 2004). Data are expressed as mean gene expression(arbitrary units)±s.e.m.

Transient transfection analysesThe pTGF2.luc luciferase reporter plasmid contains the mouse 1.7 kbmouse TGF2 promoter subcloned into pGL3-Basic plasmid (Promega).The pTGF2 CArG1.luc plasmid is identical to pTGF2.luc, except theTGF2 promoter contains a 2 bp mutation in CArG1 that abolishes bindingof SRF to the TGF2 promoter. Cos-7 cells (2�105/well) were transientlyco-transfected with 400 ng luciferase reporter plasmid, 100-400 ng ofpcDNA3 control or pcDNA3-based expression plasmid and 5ng Renillareference plasmid (pRL-TK) using Lipofectamine 2000 (Invitrogen).Luciferase and Renilla assays were performed with the Dual-LuciferaseReporter Assay System (Promega, catalog code TM040). Smad signalingactivity was measured in Mkl2–/– ES cells using Cignal SMAD ReporterKit (Qiagen, catalog code CCS-017L). All experiments were performedwith duplicate plates of cells run in triplicate for each time point. Data areexpressed as mean relative luciferase activity±s.e.m.

Chromatin immunoprecipitation analysesChromatin immunoprecipitation (ChIP) assays were performed with DNAharvested from wild-type and Mkl2–/– ES cells using a ChIP assay kitaccording to the manufacturer’s directions (Millipore, catalog code 17-295). Polyclonal anti-SRF IgG (Santa Cruz, catalog code Sc335) or controlrabbit IgG was used to immunoprecipitate cross-linked chromatin.Quantitative PCR was performed with Power SYBR Green PCRMastermix and the PRISM 7500 (ABI). The nucleotide sequence of PCRprimers used to amplify Tgf2 CArG boxes 1-5 are shown insupplementary material Table S1.

Cell adhesion assaysCell adhesion assays were performed using the Vybrant Cell AdhesionAssay kit (Invitrogen, cat. V-13181). ES cells (5�106/ml) were grownin 0.5% fetal bovine serum for 2 hours and then stained with calcein-AM. Following 1 hour of incubation on plastic or fibronectin-coatedtissue culture wells, the fluorescence intensity (O.D.) of each well wasmeasured. The percentage of adherent cells was determined by dividingthe fluorescence intensity (O.D.) by the total fluorescence intensity of adherent cells in each well. Each experiment was repeated at leastthree times. Data are reported as the mean percentage of adherentcells±s.e.m.

Statistical analysesStatistical significance was determined using the Student’s t-test:*P

RESULTSMkl2–/– null mutant mice survive only until E13.5Heterozygous Mkl2 (Mkl2+/–) mice were generated in which exon8 was deleted, rendering the protein functionally null(supplementary material Fig. S1) (Wang et al., 2002). Mkl2–/–

embryos survive until E13.5, but die between E13.5 and E16.5(Table 1). At E15.5, fewer than 10% of the anticipated Mkl2–/–

mutants survived and no viable Mkl2–/– embryos were observedbeyond E16.5. Of note, the timing of embryonic demise is earlierthan we observed in Mkl2 gene trap mutants that exhibit ahypomorphic phenotype and survive until postnatal day (P) 1-2 (Liet al., 2005). Consistent with previous reports (Li et al., 2005; Ohet al., 2005), cardiac outflow tract patterning defects were observedin E14.5-16.5 Mkl2–/– embryos (data not shown). However, thesedefects failed to explain lethality at E13.5-16.5, which is prior tothe transition from the fetal to the adult circulation that occurs inthe immediate postnatal period.

Mkl2 null embryos develop arterial aneurysmsand hemorrhageAnalyses of 859 embryos revealed that through E10.5, wild-type(Mkl2+/+), heterozygous (Mkl2+/–) and null (Mkl2–/–) embryos weregrossly indistinguishable. However, as early as E11.5, prior toaorticopulmonary septation, some Mkl2–/– embryos exhibited areasof focal hemorrhage. By E12.5, all Mkl2–/– mutants displayedregions of hemorrhage and all surviving E15.5 mutant embryosdisplayed large areas of hemorrhage covering their bodies (Fig.1A,B). Histological analyses revealed dilation of the midlinederivative of the ventral aorta (Ao) in all E11.0 Mkl2–/– embryos(Fig. 1C,D, white arrowheads). By E12.5, aneurysmal dilation(white arrows) of the aortic arch (Ao) (Fig. 1G,H) and 3rdpharyngeal arch artery (3PA) (Fig. 1E,F) was commonly observedin Mkl2–/– embryos. Aneurysmal dilation and arterial rupture (blackarrows) was also observed in other arteries supplying the head andneck of Mkl2–/– embryos (Fig. 1F, arrows). In mutant embryos thatsurvived to E15.5, massive aneurysmal dilation of the commoncarotid artery (CCA) was observed displacing the trachea (Tr) andesophagus (Es) (Fig. 1I,J). In addition, intraparenchymalhemorrhage of the embryonic liver and lung (white arrows) wasobserved in E12.5-15.5 Mkl2–/– mutant embryos (Fig. 1K,L). Takentogether, these data suggest strongly that hemorrhage attributableto defects in select arteries contributed to the demise of Mkl2–/–

embryos.

Disruption of the tunica media and structuralintegrity of Mkl2–/– arteriesStriking defects in the structural organization of the tunica mediawas observed in E12.5-15.5 Mkl2–/– arteries. In E12.5 Mkl2–/–

embryos, the aortic sac (Ao) had enlarged and expanded rostrally

into the neck beyond the level normally occupied by the 6thpharyngeal arch artery (Fig. 2A,B). The tunica media of the mutantaorta was thickened in some sections and reduced to a single layerof cells (arrow) in other sections (Fig. 2C,D). At E15.5, SMCspopulating the aortic arch of control embryos are distinguished bytheir spindle-like morphology (arrows) and layered structure (Fig.

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Fig. 1. Mkl2–/– embryos exhibit hemorrhage and aneurysmaldilation of the great arteries. (A,B)E15.5 wild-type (+/+) and Mkl2–/–(–/–) embryos demonstrating diffuse hemorrhage in the E15.5 mutantembryo. (C,D)Hematoxylin and Eosin-stained transverse sections ofE11.0 wild-type and Mkl2–/– embryo, demonstrating dilated aortic sac(Ao) (arrowheads) in the mutant embryo. Original magnification was�20. ICA, internal carotid artery. (E,F)Hematoxylin and Eosin-stainedtransverse section of E12.5 wild-type and Mkl2–/– embryo,demonstrating dilation of the 3rd PA artery and dilation andhemorrhage (black arrowheads) of the lingual vessels in the Mkl2–/–

mutant embryo. Original magnification was �20. White arrowheadsindicate aneurysm. (G,H)Hematoxylin and Eosin-stained section ofE12.5 wild-type and Mkl2–/– embryo, showing dilation of the mutantaorta (Ao, white arrowheads) extending to the level of the commoncarotid artery (CCA). Original magnification was �20. ES, esophagus;Tr, trachea. (I,J)Hematoxylin and Eosin-stained transverse section ofE14.5 wild-type and Mkl2–/– embryo, demonstrating aneurysmaldilation of the mutant common carotid artery (CCA). Originalmagnification was �20. Arrowheads indicate aneurysm.(K,L)Hematoxylin and Eosin-stained frontal sections of E15.5 wild-typeand Mkl2–/– embryos showing intra-parenchymal hemorrhages (whitearrowheads) in the liver (Li) and lung (Lu) of the mutant embryo.Original magnification was �20.

Table 1. Genotype analysis of embryos harvested from Mkl2+/– � Mkl2+/– staged matingsAge Mkl2+/+ (wild type) Mkl2+/– (heterozygous) Mkl2–/– (null)

E9.5 6 22 13 (20%)E10.5 14 32 11 (17%)E11.5 38 90 40 (22%)E12.5 45 82 48 (29%)E13.5 28 57 12* (10%)E14.5 33 60 12* (10%)E15.5 36 73 3* (2%)E16.5 32 69 3* (2%)P0 33 71 0

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2E,G). By contrast, the tunica media of Mkl2–/– embryos waspopulated with rounded heterogeneous cells lacking a predominantorientation with obvious gaps appearing between cells (Fig. 2F,H).Disruption of the tunica media was even more striking in thecommon carotid arteries (CCA) of E13.5-15.5 Mkl2–/– embryos(Fig. 2I-P). At E13.5, aneurysmal dilation, dissection and rupture(arrow) of the CCA was commonly observed in Mkl2–/– embryos(Fig. 2J,L). By contrast, three or four layers of circumferentiallyoriented vascular SMCs comprise the tunica media of wild-typelittermates (Fig. 2I,K). Immunostaining with anti-tropoelastinantibody demonstrates disruption of cell-cell and cell-matrixrelationships in Mkl2–/– carotid arteries compared with controls(supplementary material Fig. S2). Complex aneurysm formationand dissection with formation of a neointima that recapitulatedpathological changes observed in humans was commonly observedin E15.5 Mkl2–/– carotid arteries (Fig. 2M-P). At E15.5, the tunicamedia of the carotid artery in wild-type embryos consists of fouror five layers of well-organized, spindle-shaped SMCs (Fig.2M,O). By contrast, gaps in the tunica media with penetratinghemorrhage (arrow) were observed in Mkl2–/– embryos and bothmedial and neointimal vascular SMCs were characterized bymarked heterogeneity in size and orientation (Fig. 2N,P).

Inspection of the cerebral vasculature revealed dilation of thebasilar artery in Mkl2–/– embryos compared with controls (Fig. 2Q-T). At E13.5, the lumen of the basilar (Bas) artery is typicallysurrounded by a single layer of circumferentially oriented SMA-positive vascular SMCs (Fig. 2Q,R). However, the basilar artery ofMkl2–/– mutants was markedly dilated and the lumen was surroundedby multiple layers of heterogeneous appearing cells (arrowheads)expressing SMC markers (Fig. 2S,T; supplementary material Fig.S5). Of note, SMCs contributing to the basilar artery are not derivedfrom the cardiac neural crest. By contrast, only subtle differenceswere also observed in the descending thoracic aorta of E15.5 Mkl2–/–

mutant compared with control littermates (Fig. 2U-X). In mutantembryos, the lumen of the thoracic aorta appeared eccentric withsome thinning of the tunica media (arrows) compared with control

littermates (Fig. 2V,X). Moreover, no obvious changes wereobserved in the iliac or femoral arteries of Mkl2–/– and controllittermates. However, intra-parenchymal hemorrhage (white arrows)of the liver (Li) and lung (Lu) was commonly observed in E12.5-15.5 Mkl2–/– embryos (Fig. 1L). Taken together, these datademonstrate that the hemorrhage observed in Mkl2–/– embryos isattributable to defects in the structural integrity of select arteriesowing to alterations in medial SMC morphology, orientation, andcell-cell and cell-ECM relationships.

MKL2 regulates TGF signaling in embryonic stemcellsMicroarray analyses were performed with mRNA harvested fromundifferentiated Mkl2–/– embryonic stem (ES) cells and from theparental wild-type SV129 ES cell line. We chose to compare

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Fig. 2. Disruption of the tunica media and aneurysm formation inMkl2–/– embryos. (A-D)Hematoxylin and Eosin-stained transversesection of E12.5 wild-type (+/+) and Mkl2–/– (–/–) embryos,demonstrating dilation of the mutant aortic sac in the Mkl2–/– mutantaorta (Ao). Tr, trachea. (C,D)High-power magnification of the wall ofthe aortic sac reveals thinning of the tunica media to a single layer ofcells (arrowheads) in some segments of the Mkl2–/– mutant aortic sac.Original magnifications were �100 (A,B) and �400 (C,D). Tr, trachea. (E-H)Hematoxylin and Eosin-stained transverse section of the aorticarch (Ao) of E15.5 wild-type and Mkl2–/– embryo, demonstratingdilation of mutant aorta arch (Ao). The red rectangles in E and Findicate the locations of G and H. (G,H)High-power magnification ofthe tunica media (arrowhead) of the control artery reveals spindle-shaped SMCs oriented circumferentially (G); by contrast, medial SMCspopulating the mutant artery appear polygonal lacking predominantorientation with obvious gaps between cells (H). Original magnificationswere �40 (E,F) and �1000 (G,H). (I-L)Hematoxylin and Eosin-stainedtransverse section demonstrating aneurysmal dilation of the commoncarotid arteries of E13.5 wild-type and Mkl2–/– embryos. The location ofthe jugular vein (JV) is indicated. The red rectangles indicate thelocations of K and L. (K,L)High-power magnification reveals thinning ofthe tunica media and rupture through the arterial wall (arrowheads) ofthe in the Mkl2–/– embryo (L). By contrast, spindle-like SMCs composethe media of the control artery (K). Original magnifications were �20(I,J) and �200 (K,L). (M-P)Hematoxylin and Eosin-stained sectionshowing the common carotid artery of E15.5 wild-type and Mkl2–/–

embryo, demonstrating aneurysmal dilation, dissection and rupture(arrowhead) in the Mkl2–/– embryo (N). (O,P)High-power magnificationdemonstrates alignment of spindle-shaped SMCs surrounding thelumen of the control carotid artery (O). By contrast, SMCs of theMkl2–/– artery are polygonal, lacking predominant orientation withintramural hemorrhage observed between cells (P). Originalmagnifications were �100 (M,N) and �1000 (O,P). (Q-T)Hematoxylinand Eosin-stained section of the basilar artery (Bas) of E13.5 wild-typeand Mkl2–/– embryos demonstrating dilation of the mutant basilarartery (Bas) (R,T). The red rectangles in Q and R indicate the locations ofS and T. (S,T)High-power magnification reveals disorganization of thetunica media (arrowheads) in the Mkl2–/– artery compared with thecontrol basilar artery, which comprises a single layer of SMCs. Originalmagnifications were �40 (Q,R) and �400 (S,T). ICA, internal carotidartery. (U-X)Hematoxylin and Eosin-stained section showing thedescending thoracic aorta of E15.5 wild-type (U,W) and Mkl2–/– (V,X)embryos. The red rectangles in U and V indicate the locations of W andX. Slight thinning of the tunica media (arrowheads) is observed in theMkl2–/– mutant artery (X) compared with the control aorta (W). Originalmagnifications were �40 (U,V) and �200 (W,X). IVC, inferior venacava. See also supplementary material Fig. S2.

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undifferentiated Mkl2–/– ES cells with control ES cells to identifyMKL 2-regulated pathways that underlie basic developmentalprograms and to maximize signal-to-noise in the first stage of theseanalyses. These data were deposited in the Gene ExpressionOmnibus (GEO) public database (Accession Number GSE38316).In exploratory analyses, more genes were downregulated thanupregulated in response to Mkl2 deletion, consistent with thefunction of MKL2 as a transcriptional co-activator (supplementarymaterial Table S3). To screen for canonical molecular pathways

regulated directly or indirectly by Mkl2 deletion, Gene SetEnrichment Analysis (GSEA) was applied to the microarray data.Remarkably, no Kyoto Encyclopedia of Genes and Genomes(KEGG)-defined molecular pathway was upregulated in Mkl2–/– EScells compared with wild-type cells, whereas 55 KEGG-definedpathways showed evidence of repression (FDR q value

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Fig. 4. TGF signaling is downregulated in Mkl2–/– ES cells and the great arteries of Mkl2–/– embryos. (A)qRT-PCR analyses performed withmRNA harvested from wild-type and Mkl2–/– ES cells following 6 days of differentiation in vitro. The experiment was performed three times withthree biological replicates used in each experiment. Data are expressed as the percentage of wild-type gene expression observed in Mkl2–/– EScells±s.e.m. (*P

Mkl2–/– ES cells compared with control cells (Fig. 4A) (P

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wild-type ES cells located on the margins of embryoid bodiesspread and begin to migrate, demonstrating numerous lamellopodiaand filopodia (Fig. 6A,C,E). By contrast, Mkl2–/– ES cells assumeda polygonal shape that rarely demonstrated lamellopodia orfilopodia (Fig. 6B,D,F). Paxillin (pPax) (green stain), a componentof focal adhesions, was observed in discrete complexes across thesurface of wild-type ES cells (Fig. 6E, arrows). By contrast,relatively low levels of dot-like phosphorylated paxillin complexeswere observed on the margins of Mkl2–/– ES cells (Fig. 6F). Wild-type ES cells exhibited dense F-actin networks (red stain)throughout their cytoplasm and at the leading edge oflamellopodium (Fig. 6G, arrows). By contrast, centrally locateddot-like focal adhesions that are indicative of a less-motile cellwere visualized in Mkl2–/– ES cells (Fig. 6H). DNase I stainingrevealed comparable expression of G-actin (green stain) in wild-type and Mkl2–/– ES cells, strongly suggesting that the F:G actinratio is decreased in Mkl2–/– ES cells (Fig. 6I,J). Finally, discretecomplexes containing fibronectin (Fn) were observed in wild-typeES cells, often localizing to lamellpodia and filopodia (Fig. 6K,arrows). By contrast, very low levels of fibronectin were observedin the perinuclear region of Mkl2–/– ES cells (Fig. 6L).

A quantitative fluorescence-based cell adhesion assaydemonstrated that Mkl2–/– ES cells exhibit profound defects in celladhesion to bovine serum albumin (BSA)-coated tissue cultureplates. After 1 hour of incubation, 21.2±3.1% of wild-type (WT)cells adhere to the uncoated plates, whereas only 8.0±1.8% ofMkl2–/– ES cells adhere (P

embryonic development and in response to environmental cues andsignals. Our group and others have reported that Mkl2 loss-of-function mutant embryos exhibit defects in vascular patterningattributable to a cell-autonomous block in differentiation of neuralcrest-derived vascular SMCs (Li et al., 2005; Oh et al., 2005; Weiet al., 2007). However, this defect fails to explain the lethality inMkl2–/– embryos that occurs between E13.5 and E15.5. The studiesdescribed in this report demonstrate that Mkl2–/– embryos exhibithemorrhage caused by aneurysmal dilation and dissection of selectarterial beds throughout the embryo. Surprisingly, microarraystudies and qRT-PCR experiments identified a conserved MKL2-regulated TGF signaling pathway in ES cells. Moreover, Mkl2–/–ES cells exhibit profound defects in cell adhesion that are rescuedby forced expression of TGF2. Consistent with this observation,

TGF2 expression, TGF signaling and TGF-regulated genesencoding key components of the ECM are downregulated in thedeveloping vasculature of Mkl2–/– embryos.

The identification of an MKL2/TGF signaling pathwayprovides important new insights into the molecular mechanismsunderlying MKL2/SRF function in the embryonic vasculature. Asreported previously, an SRF/MKL2 complex plays a crucial role inactivating a subset of SRF-dependent genes that encode SMCcontractile proteins in neural crest-derived vascular SMCs (Li etal., 2005; Oh et al., 2005; Wei et al., 2007). Indeed, expression ofSM--actin and SM-MyHC is severely attenuated in vascularSMCs populating the aorta and pharyngeal arch arteries in E12.5Mkl2–/– embryos compared with wild-type (WT) littermates(supplementary material Fig. S5A-H). Surprisingly, in Mkl2–/–

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Fig. 6. Mkl2–/– ES cells exhibit derangements in cytoskeletal organization and cell adhesion that are rescued by TGF2. (A-D)Phase-contrast micrographs showing wild-type (A,C) and Mkl2–/– (B,D) ES cells 24 hours after plating on fibronectin-coated chamber slides. Wild-type EScells spread and form numerous protrusive lamellpodia and filopodia (arrows) (C). Original magnifications were �20 (A,B) and �200 (C,D). (E-L)Immunostaining of wild-type and Mkl2–/– ES cells for expression of proteins associated with cell adhesion and cytoskeletal organization.(E,F)Wild-type and Mkl2–/– ES cells immunostained with anti-phospho-paxillin (green) and counterstained with DAPI (blue), demonstratingabundant expression in complexes across wild-type ES cells and low level expression at margins of Mkl2–/– ES cell. Arrows indicate focal adhesions.(G,H)Staining with rhodamine-phalloidin, which localizes to F-actin (red), demonstrates a rich array of actin filaments in control ES cells andabsence of stress fibers in Mkl2–/– ES cells. Arrows indicate actin filaments. (I,J)ES cells were exposed to DNase I to identify monomeric G-actin(green). (K,L)ES cells immunostained with anti-fibronectin antibody demonstrate abundant expression in wild-type ES cells with localization tolamellopodia and filopodia (K, white arrows). By contrast, relatively low levels of fibronectin are observed in the perinuclear regions of Mkl2–/– EScells (L). Original magnification �400. (M)A fluorometric cell adhesion assay was performed comparing the capacity of wild-type (WT) ES cells(black bars), Mkl2–/– null ES cells (light-gray bars) and Mkl2–/– null ES cells stably transfected with an expression plasmid encoding TGF2 (gray bars)to adhere to BSA- or fibronectin-coated tissue culture plates. Data are expressed as % adherent cells±s.e.m. (**P

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embryos expression of SM-MyHC and SM--actin is alsodramatically downregulated in a subset of non-neural crest-derivedvascular SMCs, including those populating the mutant basilarartery (supplementary material Fig. S5I,J) and hepatic vasculature(J.L., unpublished observations). These findings demonstrate thatthe role of MKL2 in the vasculature is not restricted to cardiacneural crest-derived SMCs. This raises the crucial issue of whetherdefects in vascular patterning and stabilization in the embryoresulted primarily from an MKL2-mediated block in expression ofgenes encoding contractile SMC proteins versus a block in TGFsignaling. Indeed, mutations of the ACTA2 gene are associated withfamilial thoracic aneurysms in humans (Guo et al., 2007; Morisakiet al., 2009; Regalado et al., 2011). However, it is noteworthy thatconditional ablation of the myocardin (Myocd) gene in cardiacneural crest-derived SMCs results in dramatic suppression of theSMC contractile proteins that was not accompanied by vascularaneurysm, dissection or hemorrhage (Huang et al., 2008).Ultimately, as discussed below and shown in Fig. 7, we believe thatMKL2/SRF-mediated loss of SMC contractile proteins in Mkl2–/–

embryos directly contributes to the vascular phenotype observed inMkl2–/– embryos and the loss of SMC contractile proteins indirectlyinfluences TGF signaling in the vasculature. Conversely, TGFsignaling, in turn, directly and indirectly influences the contractileSMC gene program. This model highlights the crucial role that thetranscriptional co-activator MKL2 plays in regulating angiogenesisand vascular patterning in the embryo.

The data provided in this report expands our understanding ofthe function of MKL2 in the embryo by revealing that MKL2 liesupstream in a transcriptional program that regulates TGFexpression, TGF signaling and TGF-regulated genes encodingECM. Surprisingly, we observed that MKL2 profoundly influencesthe morphology and adhesive properties of ES cells that act at leastin part via TGF/BMP signaling. As shown in Fig. 7, in theembryonic vasculature, developmental cues and extracellularsignals transduced via RhoA and cytoskeletal actin promote the

translocation of G-actin/MKL2 complexes from the cytoplasm tothe SMC nucleus (Mouilleron et al., 2011). In the nucleus, MKL2physically associates with SRF, which promotes the binding of theMKL2/SRF complex to the TGF2 promoter (and related TGFfamily members), activating TGF signaling in the arterial wall.TGF signaling, in turn, activates expression of a subset of genesencoding ECM, including fibronectin (Fbn), fibrillin 1 (Fbn1) andCol4a1 (Col4a1). The ECM feeds back upon the vascular SMC,modulating its structure, morphology, adhesive and migratoryproperties (Astrof and Hynes, 2009; Pardali et al., 2010). Thesenew data support a model wherein the loss of MKL2 results in ablock in TGF signaling in select arterial beds, including the greatarteries, causing alterations in cell adhesion with disruption of thetunica media, which ultimately leads to aneurysmal dilation andhemorrhage. Consistent with this model, in E10.5 EIIIA/EIIIBfibronectin-double null embryos defective association of vascularSMCs with the endothelium of the aorta is observed. Interestingly,in fibronectin mutant embryos, vascular SMCs assume a roundedmorphology resembling that observed in Mkl2–/– mutant embryos(Astrof et al., 2007). As such, these new data serve to identify asecond arm (shown in red) of the MKL2 signaling pathway thatplays a crucial role in regulating angiogenesis and vascularstabilization required for embryonic survival (Fig. 7).

Other data supporting this model include the overlappingpatterns of MKL2 and TGF2 expression in the embryonicvasculature. Fate-mapping studies have shown that, in the E8.0mouse embryo, MKL2 mRNA is observed in cardiac neural crestcells located in the hindbrain prior to their migration to the cardiacoutflow tract and great arteries and MKL2 is expressed abundantlyin these arteries throughout development (Li et al., 2005).Similarly, TGF2 is the predominant TGF isoform expressed inthe great arteries and the gene is expressed abundantly in SMCspopulating the aortic sac and pharyngeal arch arteries in a temporalwindow consistent with MKL2-induced activation of the Tgfb2gene (Molin et al., 2003). Therefore, it is not surprising that Mkl2–/–

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Fig. 7. An MKL2/TGF signaling pathway is required for vascular patterning and stabilization of the great arteries during embryonicdevelopment. The transcriptional co-activator MKL2 transduces multiple signals in vascular SMCs that promote translocation of MKL2 (bound toG-actin) to the nucleus, where it physically associates with SRF. This, in turn, promotes SRF binding to CArG boxes, which activates transcription ofthe Tgfb2 gene and other genes encoding TGF signaling molecules. Activation of TGF signaling activates expression genes that encode keycomponents of the ECM, including fibronectin, fibrillin and Col4a1. The ECM, in turn, feeds back on vascular SMCs, reinforcing the contractileSMC phenotype and leading to organization of the tunica media that is required for stabilization of the great arteries. In addition, MKL2 promotesSMC differentiation of the great arteries via binding to SRF, which in turn binds to CArG boxes that control expression of genes encoding SMCcontractile proteins. Expression of SMC contractile proteins influences expression of TGF signaling and TGF-regulated genes encoding ECM, andvisa versa, serving to define an MKL2/SRF molecular program required for vascular development and patterning in the embryo. Pathways identifiedin the experiments described in this work are shown in red. D

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and Tgfb2–/– embryos exhibit vascular defects that are attributable,at least in part, to a block of TGF signaling in aortic sac and the4th pharyngeal arch artery, which gives rise to the aortic arch.Consistent with this observation, abundant nuclear Smad2expression is observed in the 4th pharyngeal arch segment of theaortic arch, but not in other pharyngeal arch segments or theascending or descending aorta (Molin et al., 2004). Similarly,fibronectin, a TGF target gene that is dramatically downregulatedin Mkl2–/– ES cells and arteries, is expressed abundantly in theaortic sac and pharyngeal arch arteries. Therefore, vulnerability ofthe aorta and fourth pharyngeal arch arteries may be linked to alocalized reduced vascular Smad2/3 signaling and relatedexpression of fibronectin in Mkl2–/– embryos. The findings thatTGF2, the predominant TGF- isoform expressed in the cardiacoutflow tract and great arteries in the embryo, as well as SMADsignaling and fibronectin are dramatically downregulated in thegreat arteries of Mkl2–/– embryos is consistent with this model.Taken together, these data support the conclusion that the vascularpathology observed in Mkl2–/– embryos results from a block inMKL2-induced expression of TGF2 (and probably TGF3) andTGF signaling in the developing arterial wall, reflecting thecrucial role that SRF and MKL2 play in development of the aortaand great arteries in the embryo.

This raises the important question of how does the block inTGF signaling observed in ES cells relate to the vascularphenotype observed in Mkl2–/– mutant embryos? Remarkably,multiple genes encoding TGF/BMP signaling molecules weredownregulated in undifferentiated Mkl2–/– ES cells (Fig. 3; Fig. 4Aand supplementary material Fig. S3). Some, but not all, of thesegenes are expressed in the developing vasculature. In this regard,it is noteworthy that MKL1 and MKL2 are expressed inundifferentiated ES cells, whereas myocardin is expressed severaldays after the initiation of differentiation in vitro. Nevertheless, thedemonstrated block in multiple genes encoding TGF/BMP familymembers in Mkl2–/– ES cells reveals a non-redundant function forMKL2 in ES cells in this context. Similarly, even thoughmyocardin, MKL1 and MKL2 are co-expressed in vascular SMCs,only Mkl2–/– embryos demonstrate a block in TGF signaling inthe embryonic vasculature, revealing a unique and non-redundantfunction of MKL2 in select arteries and vascular beds. However, itremains possible, indeed likely, that other TGF-/BMP signalingmolecules contributed to the vascular phenotype observed inMkl2–/– mutant embryos. In support of this hypothesis, Tgfb2–/–

embryos exhibit defects in pharyngeal arch development, but failto phenocopy Mkl2–/– embryos (Gittenberger-de Groot et al., 2006;Sanford et al., 1997). By contrast, Tgfb2/Tgfb3 compound mutantembryos recapitulate multiple aspects of the vascular changesobserved in Mkl2–/– embryos (Dünker and Krieglstein, 2002).

Previous studies have shown that TGF2 and its cognatereceptors, TGFBR2 and TGFBR1/ALK5, play a crucial role inembryonic angiogenesis and cardiovascular development (for areview, see Pardali et al., 2010). Both Mkl2 and Tgfb2 knockoutmice exhibit aortic arch malformations involving the 4thpharyngeal arch artery (Gittenberger-de Groot et al., 2006; Sanfordet al., 1997). However, Mkl2–/– embryos exhibit aneurysmaldilation of the aortic arch and carotid arteries, whereasTgfb2–/–embryos exhibit defects in vascular patterning attributableto the 4th pharyngeal arch artery. What explains these differences?As a transcriptional co-activator, MKL2 regulates and coordinatesexpression of multiple SRF-dependent genes involved inangiogenesis. Indeed, TGF2 and TGF3 are expressed in the greatarteries during embryonic development (supplementary material

Fig. S4A-L) and both isoforms were markedly repressed in Mkl2–/–

ES cells (Fig. 3A; supplementary material Fig. S3). Moreover,expression of genes encoding TGF receptors and multiple Smadswere downregulated in Mkl2–/– ES cells (Fig. 3; supplementarymaterial Fig. S3). As such, the observed differences in phenotypebetween Mkl2–/– and Tgfb2–/– embryos most probably result fromthe differential expression of SRF/MKL2-regulated genes beyondTGF2. As discussed above, these include both genes encodingSMC contractile proteins, as well as related TGF/BMP signalingmolecules.

The observation that Mkl2–/– mutant embryos develop aorticaneurysms accompanied by downregulation of TGF isoforms,TGF signaling and TGF-regulated genes encoding ECMstrongly supports a model wherein loss of function of TGFsignaling in the vasculature leads to aneurysm formation duringembryonic angiogenesis. Consistent with this observation,conditional ablation of the Tgfbr2 gene in vascular SMCsrecapitulated many of the features observed in Mkl2–/– embryos,including aneurysmal dilation and dissection of the aorta duringlate embryonic development (Choudhary et al., 2009). However,these conclusions conflict with the observation that individualswith Marfan syndrome and Loeys-Dietz syndrome exhibitactivation of TGF signaling and gain of TGF function in thearterial wall (Pearson et al., 2008). Interestingly, however, theNHLBI Go Exome Sequencing Project recently described a loss-of-function mutation in TGFB2 that is associated with familialthoracic aortic aneurysms and acute aortic dissections associatedwith mild system features of Marfan syndrome (Boileau et al.,2012). These data highlight gaps in current understanding of therole that TGF signaling plays in the embryonic and adultvasculature, and suggests that TGF signals are differentiallytransduced during embryonic and postnatal development. In anycase, further studies examining the role of MKL2 and/or MKL1 inmaintenance and adaptation of the adult vasculature tohemodynamic stress promise to provide important new insightsinto the pathogenesis of heritable and acquired forms of vasculardisease involving aneurysm formation and dissection.

AcknowledgementsWe thank Jon Epstein, Mark Kahn and Ed Morrisey for their advice and helpfulcomments.

FundingThis work was supported in part by the National Institutes of Health (NIH)[R01-HL102968 and R01-HL094520 to M.S.P., T32-HL0783 to N.B.] and by theCommonwealth of Pennsylvania [to the University of PennsylvaniaCardiovascular Institute]. Deposited in PMC for release after 12 months.

Competing interests statementThe authors declare no competing financial interests.

Supplementary materialSupplementary material available online athttp://dev.biologists.org/lookup/suppl/doi:10.1242/dev.082222/-/DC1

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SUMMARYKEY WORDS: Angiogenesis, Embryonic stem cell, Myocardin-related transcription factor B/MKL2,INTRODUCTIONMATERIALS AND METHODSGeneration and characterization of Mkl2 null miceHistology and immunohistochemistryImmunoblot and ELISA analysesMicroarray analysisqRT-PCRTransient transfection analysesChromatin immunoprecipitation analysesCell adhesion assaysStatistical analyses

RESULTSMkl2-/- null mutant mice survive only until E13.5Mkl2 null embryos develop arterial aneurysms and hemorrhageDisruption of the tunica media and structural integrity of Mkl2-/-MKL2 regulates TGFb signaling in embryonic stem cellsMkl2-/- mutants exhibit a block in TGFb signaling in theThe TGFb2 gene is a direct transcriptional target of MKL2Mkl2-/- ES cells exhibit defects in cytoskeletal organization and cell

Fig. 1.Table 1.Fig. 2.Fig. 3.Fig. 4.DISCUSSIONFig. 5.Fig. 6.Fig. 7.Supplementary materialReferences