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1.  Whole animal knockout of smooth muscle alpha-actin does not alter excisional wound healing or the fibroblast-to-myofibroblast transition 
The contractile phenotype and function of myofibroblasts have been proposed to play a critical role in wound closure. It has been hypothesized smooth muscle alpha-actin expressed in myofibroblasts is critical for their formation and function. We have used smooth muscle α-actin-null mice to test this hypothesis. Full-thickness excisional wounds closed at a similar rate in smooth muscle α-actin -null and wild type mice. In addition, fibroblasts in smooth muscle α-actin-null granulation tissue when immunostained with a monoclonal antibody that recognizes all muscle actin isoforms exhibited a myofibroblast-like distribution and a stress fiber-like pattern, demonstrating that these cells acquired the myofibroblast phenotype. Dermal fibroblasts from smooth muscle α-actin-null and wild type mice formed stress fibers and supermature focal adhesions, and generated similar amounts of contractile force in response to transforming growth factor-β1. Smooth muscle γ-actin and skeletal muscle alpha-actin were expressed in smooth muscle α-actin-null myofibroblasts, as demonstrated by immunostaining, real-time PCR, and mass spectrometry. These results demonstrate that smooth muscle α-actin is not necessary for myofibroblast formation and function and for wound closure, and that smooth muscle γ-actin and skeletal muscle α-actin may be able to functionally compensate for the lack of smooth muscle α-actin in myofibroblasts.
doi:10.1111/wrr.12001
PMCID: PMC3540133  PMID: 23253249
wound healing; smooth muscle α-actin; myofibroblast; cytoskeleton; stress fiber; focal adhesion
2.  Correction: MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(12):10.1371/annotation/90602bc3-5052-49ac-a7fb-33210d7c8b4d.
doi:10.1371/annotation/90602bc3-5052-49ac-a7fb-33210d7c8b4d
PMCID: PMC3867558
3.  MicroRNA-17-92, a Direct Ap-2α Transcriptional Target, Modulates T-Box Factor Activity in Orofacial Clefting 
PLoS Genetics  2013;9(9):e1003785.
Among the most common human congenital anomalies, cleft lip and palate (CL/P) affects up to 1 in 700 live births. MicroRNA (miR)s are small, non-coding RNAs that repress gene expression post-transcriptionally. The miR-17-92 cluster encodes six miRs that have been implicated in human cancers and heart development. We discovered that miR-17-92 mutant embryos had severe craniofacial phenotypes, including incompletely penetrant CL/P and mandibular hypoplasia. Embryos that were compound mutant for miR-17-92 and the related miR-106b-25 cluster had completely penetrant CL/P. Expression of Tbx1 and Tbx3, the DiGeorge/velo-cardio-facial (DGS) and Ulnar-mammary syndrome (UMS) disease genes, was expanded in miR-17-92 mutant craniofacial structures. Both Tbx1 and Tbx3 had functional miR seed sequences that mediated gene repression. Analysis of miR-17-92 regulatory regions uncovered conserved and functional AP-2α recognition elements that directed miR-17-92 expression. Together, our data indicate that miR-17-92 modulates expression of critical T-box transcriptional regulators during midface development and is itself a target of Bmp-signaling and the craniofacial pioneer factor AP-2α. Our data are the first genetic evidence that an individual miR or miR cluster is functionally important in mammalian CL/P.
Author Summary
CL/P are very common birth defects in humans. The genetic mechanism underlying CL/P pathogenesis is poorly understood. MiRs, small non-coding RNAs that function to post-transcriptionally regulate gene expression, have been identified as pivotal modulators of various developmental events and diseases. To date, there is no individual miR or miR cluster that has been identified as functionally essential in mammalian CL/P. Here, we have discovered that deletion of miR-17-92 cluster in mice results in craniofacial malformations including CL/P. Importantly, MIR-17-92 is located on a critical human chromosome region associated with 13q deletion syndrome, a chromosomal disorder that presents with defects including CL/P, suggesting the advantages of our animal model to study human disease. Moreover, our work demonstrated that miR-17-92 cluster directly repressed T-box factors, which have critical functions during craniofacial development. We further showed that miR-17-92 was directly activated by Bmp-signaling and transcription factor AP-2α. Together, our work identified a novel miR-mediated transcriptional network underlying CL/P, providing new insights into craniofacial developmental biology.
doi:10.1371/journal.pgen.1003785
PMCID: PMC3777996  PMID: 24068957
4.  Hemogenic endocardium contributes to transient definitive hematopoiesis 
Nature communications  2013;4:1564.
Hematopoietic cells arise from spatiotemporally restricted domains in the developing embryo. Although studies of non-mammalian animal and in vitro embryonic stem cell models suggest a close relationship among cardiac, endocardial, and hematopoietic lineages, it remains unknown whether the mammalian heart tube serves as a hemogenic organ akin to the dorsal aorta. Here we examine the hemogenic activity of the developing endocardium. Mouse heart explants generate myeloid and erythroid colonies in the absence of circulation. Hemogenic activity arises from a subset of endocardial cells in the outflow cushion and atria earlier than in the aorta-gonad-mesonephros region, and is transient and definitive in nature. Interestingly, key cardiac transcription factors, Nkx2-5 and Isl1, are expressed in and required for the hemogenic population of the endocardium. Together, these data suggest that a subset of endocardial/endothelial cells expressing cardiac markers serve as a de novo source for transient definitive hematopoietic progenitors.
doi:10.1038/ncomms2569
PMCID: PMC3612528  PMID: 23463007
5.  Subepicardial endothelial cells invade the embryonic ventricle wall to form coronary arteries 
Cell Research  2013;23(9):1075-1090.
Coronary arteries bring blood flow to the heart muscle. Understanding the developmental program of the coronary arteries provides insights into the treatment of coronary artery diseases. Multiple sources have been described as contributing to coronary arteries including the proepicardium, sinus venosus (SV), and endocardium. However, the developmental origins of coronary vessels are still under intense study. We have produced a new genetic tool for studying coronary development, an AplnCreER mouse line, which expresses an inducible Cre recombinase specifically in developing coronary vessels. Quantitative analysis of coronary development and timed induction of AplnCreER fate tracing showed that the progenies of subepicardial endothelial cells (ECs) both invade the compact myocardium to form coronary arteries and remain on the surface to produce veins. We found that these subepicardial ECs are the major sources of intramyocardial coronary vessels in the developing heart. In vitro explant assays indicate that the majority of these subepicardial ECs arise from endocardium of the SV and atrium, but not from ventricular endocardium. Clonal analysis of Apln-positive cells indicates that a single subepicardial EC contributes equally to both coronary arteries and veins. Collectively, these data suggested that subepicardial ECs are the major source of intramyocardial coronary arteries in the ventricle wall, and that coronary arteries and veins have a common origin in the developing heart.
doi:10.1038/cr.2013.83
PMCID: PMC3760626  PMID: 23797856
coronary artery; origin; development; subepicardial endothelial cell; angiogenesis
6.  Sumoylation and regulation of cardiac gene expression 
Circulation research  2010;107(1):19-29.
Sumoylation is a post-translational modification process in which SUMO proteins are covalently and reversibly conjugated to their targets via enzymatic cascade reactions. Since the discovery of SUMO-1 in 1996, the SUMO pathway has garnered increased attention due to its role in a number of important biological activities such as cell cycle progression, epigenetic modulation, signal transduction, and DNA replication/repair, as well as its potential implication in human pathogenesis such as in cancer development and metastasis, neurodegenerative disorders and craniofacial defects. The role of the SUMO pathway in regulating cardiogenic gene activity, development and/or disorders is just emerging. Our review is based on recent advances that highlight the regulation of cardiac gene activity in cardiac development and disease by the SUMO conjugation pathway.
doi:10.1161/CIRCRESAHA.110.220491
PMCID: PMC3744328  PMID: 20616338
sumoylation; cardiac gene expression; cardiac development; transcription factor; review
7.  Regulation of Insulin-Like Growth Factor Signaling by Yap Governs Cardiomyocyte Proliferation and Embryonic Heart Size 
Science signaling  2011;4(196):ra70.
The Hippo signaling pathway regulates growth of the heart and other tissues. Hippo pathway kinases influence the activity of various targets, including the transcriptional coactivator Yap, but the specific role of Yap in heart growth has not been investigated. We show that Yap is necessary and sufficient for embryonic cardiac growth in mice. Deletion of Yap in the embryonic mouse heart impeded cardiomyocyte proliferation, causing myocardial hypoplasia and lethality at embryonic stage 10.5. Conversely, forced expression of a constitutively active form of Yap in the embryonic heart increased cardiomyocyte number and heart size. Yap activated the insulin-like growth factor (IGF) signaling pathway in cardiomyocytes, resulting in inactivation of glycogen synthase kinase 3β, which led to increased abundance of β-catenin, a positive regulator of cardiac growth. Our results point to Yap as a critical downstream effector of the Hippo pathway in the control of cardiomyocyte proliferation and a nexus for coupling the IGF, Wnt, and Hippo signaling pathways with the developmental program for heart growth.
doi:10.1126/scisignal.2002278
PMCID: PMC3440872  PMID: 22028467
8.  Pax3 is essential for normal cardiac neural crest morphogenesis but is not required during migration nor outflow tract septation 
Developmental biology  2011;356(2):308-322.
Systemic loss-of-function studies have demonstrated that Pax3 transcription factor expression is essential for dorsal neural tube, early neural crest and muscle cell lineage morphogenesis. Cardiac neural crest cells participate in both remodeling of the pharyngeal arch arteries and outflow tract septation during heart development, but the lineage specific role of Pax3 in neural crest function has not yet been determined. To gain insight into the requirement of Pax3 within the neural crest, we conditionally deleted Pax3 in both the premigratory and migratory neural crest populations via Wnt1-Cre and Ap2α-Cre and via P0-Cre in only the migratory neural crest, and compared these phenotypes to the pulmonary atresia phenotype observed following the systemic loss of Pax3. Surprisingly, using Wnt1-Cre deletion there are no resultant heart defects despite the loss of Pax3 from the premigratory and migratory neural crest. In contrast, earlier premigratory and migratory Ap2α-Cre mediated deletion resulted in double outlet right ventricle alignment heart defects. In order to assess the tissue-specific contribution of neural crest to heart development, genetic ablation of neural crest lineage using a Wnt1-Cre-activated diphtheria toxin fragment-A cell-killing system was employed. Significantly, ablation of Wnt1-Cre-expressing neural crest cells resulted in fully penetrant persistent truncus arteriosus malformations. Combined, the data show that Pax3 is essential for early neural crest progenitor formation, but is not required for subsequent cardiac neural crest progeny morphogenesis involving their migration to the heart or septation of the outflow tract.
doi:10.1016/j.ydbio.2011.05.583
PMCID: PMC3143301  PMID: 21600894
mouse embryo; Pax3; cardiac neural crest; congenital heart defects; lineage mapping; conditional knockout; genetic cell abaltion
9.  Myoepithelial Cell Contraction and Milk Ejection Are Impaired in Mammary Glands of Mice Lacking Smooth Muscle Alpha-Actin1 
Biology of Reproduction  2011;85(1):13-21.
Mammary myoepithelial cells are specialized smooth musclelike epithelial cells that express the smooth muscle actin isoform: smooth muscle alpha-actin (ACTA2). These cells contract in response to oxytocin to generate the contractile force required for milk ejection during lactation. It is believed that ACTA2 contributes to myoepithelial contractile force generation; however, this hypothesis has not been directly tested. To evaluate the contribution of ACTA2 to mammary myoepithelial cell contraction, Acta2 null mice were utilized and milk ejection and myoepithelial cell contractile force generation were evaluated. Pups suckling on Acta2 null dams had a significant reduction in weight gain starting immediately postbirth. Cross-fostering demonstrated the lactation defect is with the Acta2 null dams. Carmine alum whole mounts and conventional histology revealed no underlying structural defects in Acta2 null mammary glands that could account for the lactation defect. In addition, myoepithelial cell formation and organization appeared normal in Acta2 null lactating mammary glands as evaluated using an Acta2 promoter-GFP transgene or phalloidin staining to visualize myoepithelial cells. However, mammary myoepithelial cell contraction in response to oxytocin was significantly reduced in isolated Acta2 null lactating mammary glands and in in vivo studies using Acta2 null lactating dams. These results demonstrate that lack of ACTA2 expression impairs mammary myoepithelial cell contraction and milk ejection and suggests that ACTA2 expression in mammary myoepithelial cells has the functional consequence of enhancing contractile force generation required for milk ejection.
Female mice lacking smooth muscle alpha-actin have a lactation defect that results from the inability of myoepithelial cells to generate sufficient contractile force in response to oxytocin to promote milk ejection.
doi:10.1095/biolreprod.110.090639
PMCID: PMC3123380  PMID: 21368298
cytoskeleton; lactation; mammary glands; milk ejection; myoepithelial cell; pregnancy; smooth muscle alpha-actin; transgenic/knockout model
10.  Protein Tyrosine Phosphatase-Like A Regulates Myoblast Proliferation and Differentiation through MyoG and the Cell Cycling Signaling Pathway 
Molecular and Cellular Biology  2012;32(2):297-308.
Protein tyrosine phosphatase-like A (PTPLa) has been implicated in skeletal myogenesis and cardiogenesis. Mutations in PTPLa correlated with arrhythmogenic right ventricular dysplasia in humans and congenital centronuclear myopathy with severe hypotonia in dogs. The molecular mechanisms of PTPLa in myogenesis are unknown. In this report, we demonstrate that PTPLa is required for myoblast growth and differentiation. The cells lacking PTPLa remained immature and failed to differentiate into mature myotubes. The repressed MyoG expression was responsible for the impaired myoblast differentiation. Meanwhile, impeded cell growth, with an obvious S-phase arrest and compromised G2/M transition, was observed in PTPLa-deficient myoblasts. Further study demonstrated that the upregulation of cyclin D1 and cyclin E2 complexes, along with a compromised G2/M transition due to the decreased CDK1 (cyclin-dependent kinase 1) activity and upregulated p21, contributed to the mutant cell S-phase arrest and eventually led to the retarded cell growth. Finally, the transcriptional regulation of the PTPLa gene was explored. We identified PTPLa as a new target gene of the serum response factor (SRF). Skeletal- and cardiac-muscle-specific SRF knockouts resulted in significant decreases in PTPLa expression, suggesting a conserved transcriptional regulation of the PTPLa gene in mice.
doi:10.1128/MCB.05484-11
PMCID: PMC3255775  PMID: 22106411
11.  A cell-autonomous role of Cited2 in controlling myocardial and coronary vascular development 
European Heart Journal  2012;34(32):2557-2565.
Aims
Myocardial development is dependent on concomitant growth of cardiomyocytes and a supporting vascular network. The coupling of myocardial and coronary vascular development is partly mediated by vascular endothelial growth factor (VEGFA) signalling and additional unknown mechanisms. We examined the cardiomyocyte specific role of the transcriptional co-activator Cited2 on myocardial microstructure and vessel growth, in relation to Vegfa expression.
Methods and results
A cardiomyocyte-specific knockout of mouse Cited2 (Cited2Nkx) was analysed using magnetic resonance imaging and histology. Ventricular septal defects and significant compact layer thinning (P< 0.02 at right ventricular apex, P< 0.009 at the left ventricular apex in Cited2Nkx vs. controls, n= 11 vs. n= 7, respectively) were found. This was associated with a significant decrease in the number of capillaries to larger vessels (ratio 1.56 ± 0.56 vs. 3.25 ± 1.63, P= 2.7 × 10−6 Cited2Nkx vs. controls, n= 11 vs. n= 7, respectively) concomitant with a 1.5-fold reduction in Vegfa expression (P< 0.02, Cited2Nkx vs. controls, n= 12 vs. n= 12, respectively). CITED2 was subsequently found at the Vegfa promoter in mouse embryonic hearts using chromatin immunoprecipitation, and moreover found to stimulate human VEGFA promoter activity in cooperation with TFAP2 transcription factors in transient transfection assays. There was no change in the myocardial expression of the left-right patterning gene Pitx2c, a previously known target of CITED2.
Conclusions
This study delineates a novel cell-autonomous role of Cited2 in regulating VEGFA transcription and the development of myocardium and coronary vasculature in the mouse. We suggest that coupling of myocardial and coronary growth in the developing heart may occur in part through a Cited2→Vegfa pathway.
doi:10.1093/eurheartj/ehs056
PMCID: PMC3748368  PMID: 22504313
CITED2; VEGFA; Myocardial development; Capillary growth
12.  Serum Response Factor Micro-Managing Cardiogenesis 
Current opinion in cell biology  2007;19(6):618-627.
Serum response factor (SRF), a cardiac enriched transcription factor, is required for the appearance of beating sarcomeres in the heart. SRF may also direct the expression of microRNAs (miRs) that inhibit the expression of cardiac regulatory factors. The recent knockout of miR-1-2, an SRF gene target, showed defective heart development, caused in part by the induction of GATA6, Irx4/5, and Hand2, that may alter cardiac morphogenesis, channel activity and cell cycling. SRF and co-factors play an obligatory role in cardiogenesis, as major determinants of myocyte differentiation not only by regulating the biogenesis of muscle contractile proteins but also by driving the expression of silencer miRNA.
doi:10.1016/j.ceb.2007.09.013
PMCID: PMC2735128  PMID: 18023168
13.  Targeted Expression of Cre Recombinase Provokes Placental-Specific DNA Recombination in Transgenic Mice 
PLoS ONE  2012;7(2):e29236.
Background
Inadequate placental development is associated with a high incidence of early embryonic lethality and serious pregnancy disorders in both humans and mice. However, the lack of well-defined trophoblast-specific gene regulatory elements has hampered investigations regarding the role of specific genes in placental development and fetal growth.
Principal Findings
By random assembly of placental enhancers from two previously characterized genes, trophoblast specific protein α (Tpbpa) and adenosine deaminase (Ada), we identified a chimeric Tpbpa/Ada enhancer that when combined with the basal Ada promoter provided the highest luciferase activity in cultured human trophoblast cells, in comparison with non-trophoblast cell lines. We used this chimeric enhancer arrangement to drive the expression of a Cre recombinase transgene in the placentas of transgenic mice. Cre transgene expression occurred throughout the placenta but not in maternal organs examined or in the fetus.
Significance
In conclusion, we have provided both in vitro and in vivo evidence for a novel genetic system to achieve placental transgene expression by the use of a chimeric Tpbpa/Ada enhancer driven transgene. The availability of this expression vector provides transgenic opportunities to direct the production of desired proteins to the placenta.
doi:10.1371/journal.pone.0029236
PMCID: PMC3281813  PMID: 22363401
14.  Conditional Ablation of Ezh2 in Murine Hearts Reveals Its Essential Roles in Endocardial Cushion Formation, Cardiomyocyte Proliferation and Survival 
PLoS ONE  2012;7(2):e31005.
Ezh2 is a histone trimethyltransferase that silences genes mainly via catalyzing trimethylation of histone 3 lysine 27 (H3K27Me3). The role of Ezh2 as a regulator of gene silencing and cell proliferation in cancer development has been extensively investigated; however, its function in heart development during embryonic cardiogenesis has not been well studied. In the present study, we used a genetically modified mouse system in which Ezh2 was specifically ablated in the mouse heart. We identified a wide spectrum of cardiovascular malformations in the Ezh2 mutant mice, which collectively led to perinatal death. In the Ezh2 mutant heart, the endocardial cushions (ECs) were hypoplastic and the endothelial-to-mesenchymal transition (EMT) process was impaired. The hearts of Ezh2 mutant mice also exhibited decreased cardiomyocyte proliferation and increased apoptosis. We further identified that the Hey2 gene, which is important for cardiomyocyte proliferation and cardiac morphogenesis, is a downstream target of Ezh2. The regulation of Hey2 expression by Ezh2 may be independent of Notch signaling activity. Our work defines an indispensible role of the chromatin remodeling factor Ezh2 in normal cardiovascular development.
doi:10.1371/journal.pone.0031005
PMCID: PMC3270034  PMID: 22312437
15.  Frs2α-deficiency in cardiac progenitors disrupts a subset of FGF signals required for outflow tract morphogenesis 
Development (Cambridge, England)  2008;135(21):3611-3622.
Summary
The cardiac outflow tract (OFT) is a developmentally complex structure derived from multiple lineages and is often defective in human congenital anomalies. While emerging evidence shows that the fibroblast growth factor (FGF) is essential for OFT development, the downstream pathways mediating FGF-signaling in cardiac progenitors remain poorly understood. Here, we report that FRS2α, an adaptor protein that links FGF receptor kinases to multiple signaling pathways, mediates critical aspects of FGF-dependent OFT development. Ablation of Frs2α in mesodermal OFT progenitor cells that originate in the second heart field (SHF) affects their expansion into the OFT myocardium, resulting in OFT misalignment and hypoplasia. Moreover, Frs2α mutants had defective endothelial-mesenchymal-transition and neural crest cell recruitment into the OFT cushions, resulting in OFT septation defects. The results provide new insight into the signaling molecules downstream of FGF receptor tyrosine kinases in cardiac progenitors.
doi:10.1242/dev.025361
PMCID: PMC3262991  PMID: 18832393
receptor tyrosine kinase; cell signaling; heart development; second heart field; mouse model
16.  The FGF-BMP signaling axis regulates outflow tract valve primordium formation by promoting cushion neural crest cell differentiation 
Circulation research  2010;107(10):1209-1219.
Rationale
Heart valves develop from precursor structures called cardiac cushions, an endothelial-lined cardiac jelly that resides in the inner side of the heart tube. The cushions are then invaded by cells from different sources, undergo a series of complicated and poorly understood remodeling processes, and give rise to valves. Disruption of the fibroblast growth factor (FGF) signaling axis impairs morphogenesis of the outflow tract (OFT). Yet, whether FGF signaling regulates OFT valve formation is unknown.
Objective
To study how OFT valve formation is regulated and how aberrant cell signaling causes valve defects.
Methods and results
By employing mouse genetic manipulation, cell lineage tracing, ex vivo heart culture, and molecular biology approaches, we demonstrated that FGF signaling in the OFT myocardium upregulated Bmp4 expression, which then enhanced smooth muscle differentiation of neural crest cells (NCCs) in the cushion. FGF signaling also promoted OFT myocardial cell invasion to the cushion. Disrupting FGF signaling interrupted cushion remodeling with reduced NCCs differentiation into smooth muscle and less cardiomyocyte invasion, and resulted in malformed OFT valves.
Conclusions
The results demonstrate a novel mechanism by which the FGF-BMP signaling axis regulates formation of OFT valve primordia by controlling smooth muscle differentiation of cushion NCCs.
doi:10.1161/CIRCRESAHA.110.225318
PMCID: PMC3052773  PMID: 20847311
FGF; BMP; heart development; NCC differentiation; cardiac valve defect
17.  SUMO-Specific Protease 2 Is Essential for Suppression of Polycomb Group Proteins Mediated Gene Silencing During Embryonic Development 
Molecular cell  2010;38(2):191-201.
SUMMARY
SUMO-specific protease 2 (SENP2) has a broad de-SUMOylation activity in vitro. However, the biological function of SENP2 is largely unknown. Here, we show that deletion of SENP2 gene in mouse causes defects in the embryonic heart and reduces the expression of Gata4 and Gata6, which are essential for cardiac development. SENP2 regulates transcription of Gata4 and Gata6 mainly through alteration of occupancy of Pc2/CBX4, a Polycomb Repressive Complex 1 (PRC1) subunit, on its promoters. We demonstrate that Pc2/CBX4 is a target of SENP2 in vivo and that SUMOylation is essential for Pc2/CBX4-mediated PRC1 recruitment to methylated histone 3 at K27 (H3K27me3). In SENP2 null embryo, SUMOylated Pc2/CBX4 accumulates and Pc2/CBX4 occupancy on the promoters of PcG target genes is markedly increased, leading to repression of Gata4 and Gata6 transcription. Our results reveal a critical role for de-SUMOylation in the regulation of PcG target gene expression through a novel mechanism.
doi:10.1016/j.molcel.2010.03.005
PMCID: PMC2879644  PMID: 20417598
18.  Characterization and in vivo pharmacological rescue of a Wnt2-Gata6 pathway required for cardiac inflow tract development 
Developmental cell  2010;18(2):275-287.
Although the molecular pathways governing the development of the anterior pole of the heart have been the subject of intense investigation, little is understood about the molecular mechanisms underlying the morphogenesis of the posterior pole of the heart which generates the atria, pulmonary veins and portions of the atrio-ventricular canal. Here we show that Wnt2 is expressed specifically in the developing inflow tract mesoderm in a domain encompassing the dorsal mesocardium and dorsal mesenchymal protrusion which generates portions of the atria and atrio-ventricular cushions. Loss of Wnt2 results in defective development of the atrial myocardium, atrio-ventricular canal, and pulmonary veins resulting in a phenotype resembling the human congenital heart syndrome complete common atrio-ventricular canal. The dorsal mesocardium and dorsal mesenchymal protrusion overlaps spatially with posterior second heart field progenitors and we show that the number and proliferation of these progenitors is reduced in Wnt2-/- mutants. Remarkably, these defects can be rescued in vivo in a temporally restricted manner through pharmacological inhibition of Gsk-3β, indicating that Wnt2 regulates canonical Wnt signaling in the posterior cardiac mesoderm. Molecular and genetic analysis shows that Wnt2 works in a feed-forward transcriptional loop with Gata6 to regulate posterior cardiac development. These data reveal an important new molecular pathway regulating cardiac inflow tract development and demonstrates that such defects in the second heart field can be rescued pharmacologically in vivo.
doi:10.1016/j.devcel.2010.01.008
PMCID: PMC2846539  PMID: 20159597
19.  Role of the homeodomain transcription factor Bapx1 in mouse distal stomach development 
Gastroenterology  2009;136(5):1701-1710.
Background & Aims
Expansion and patterning of the endoderm generate a highly ordered, multi-organ digestive system in vertebrate animals. Among distal foregut derivatives, the gastric corpus, antrum, pylorus and duodenum are distinct structures with sharp boundaries. Some homeodomain transcription factors expressed in gut mesenchyme convey positional information required for anterior-posterior patterning of the digestive tract. Barx1, in particular, controls stomach differentiation and morphogenesis. The NK homeobox gene Bapx1 (Nkx3-2) has an established role in skeletal development but its function in the mammalian gut is less clear.
Methods
We generated a Bapx1Cre knock-in allele to fate map Bapx1-expressing cells and evaluate its function in gastrointestinal development.
Results
Bapx1-expressing cells populate the gut mesenchyme with a rostral boundary in the hindstomach, near the junction of the gastric corpus and antrum. Smooth muscle differentiation and distribution of early regional markers are ostensibly normal in Bapx1Cre/Cre gut, but there are distinctive morphologic abnormalities near this rostral Bapx1 domain: the antral segment of the stomach is markedly shortened and the pyloric constriction is lost. Comparison of expression domains and examination of stomach phenotypes in single and compound Barx1 and Bapx1 mutant mice suggest a hierarchy between these two factors; Bapx1 expression is lost in the absence of Barx1.
Conclusions
This study reveals the non-redundant requirement for Bapx1 in distal stomach development, places it within a Barx1-dependent pathway, and illustrates the pervasive influence of gut mesenchyme homeobox genes on endoderm differentiation and digestive organogenesis.
doi:10.1053/j.gastro.2009.01.009
PMCID: PMC2955323  PMID: 19208343
Gastrointestinal development; stomach; mesoderm; endoderm; Nkx3-2; Bapx1; pyloric sphincter; Barx1; antrum
20.  Rho kinase-1 mediates cardiac fibrosis by regulating fibroblast precursor cell differentiation 
Cardiovascular Research  2009;83(3):511-518.
Aims
Highly proliferative, CD34+/CD45+ fibroblasts derived from monocytic, blood-borne precursor cells play a critical role in the development of fibrosis in a murine ischaemic/reperfusion cardiomyopathy (I/RC) model. The differentiation of human monocytes into fibroblasts in vitro occurs after transendothelial migration (TEM) induced by monocyte chemoattractant protein 1 (MCP-1). Because Rho-associated kinase-1 (ROCK-1) has been implicated in fibrosis and leukocyte TEM, we investigated its involvement in I/RC.
Methods and results
We subjected mice with genetic deletion of ROCK-1 to I/RC. We found that ROCK-1−/− mice did not develop the fibrosis and cardiac dysfunction characteristic for I/RC: compared with wild-type, ROCK-1−/− hearts showed markedly lower numbers of I/RC-induced α-smooth muscle actin+ fibroblasts and CD34+/CD45+ fibroblast precursors. Isolated cardiac fibroblasts from ROCK-1−/− mice undergoing I/RC were large and slowly proliferating, similar to fibroblasts isolated from sham-treated hearts. We also performed in vitro assays in which human peripheral blood mononuclear cells (PBMC) migrated through endothelial cells in response to MCP-1. Prior to migration, PBMC were incubated with ROCK-1-targeting small interfering RNA to silence ROCK-1 expression. We found that an 80% reduction of ROCK-1 protein did not inhibit TEM, but significantly reduced the amount of mononuclear cells that differentiated into fibroblasts by >20-fold.
Conclusion
Our data implicate an important role for ROCK-1 in the differentiation, but not in the TEM of monocytes that mature into cardiac fibroblasts. These cells mediate non-adaptive fibrosis.
doi:10.1093/cvr/cvp135
PMCID: PMC2709463  PMID: 19406912
Cardiac fibroblasts; Monocytes; Rho-associated kinase-1; Endothelial transmigration; Fibrosis
21.  Genetic Fate Mapping Identifies Second Heart Field Progenitor Cells As a Source of Adipocytes in Arrhythmogenic Right Ventricular Cardiomyopathy 
Circulation research  2009;104(9):1076-1084.
The phenotypic hallmark of arrhythmogenic right ventricular cardiomyopathy, a genetic disease of desmosomal proteins, is fibroadipocytic replacement of the right ventricle. Cellular origin of excess adipocytes, the responsible mechanism(s) and the basis for predominant involvement of the right ventricle are unknown. We generated 3 sets of lineage tracer mice regulated by cardiac lineage promoters α-myosin heavy chain (αMyHC), Nkx2.5, or Mef2C. We conditionally expressed the reporter enhanced yellow fluorescent protein while concomitantly deleting the desmosomal protein desmoplakin in cardiac myocyte lineages using the Cre-LoxP technique. Lineage tracer mice showed excess fibroadiposis and increased numbers of adipocytes in the hearts. Few adipocytes in the hearts of αMyHC-regulated lineage tracer mice, but the majority of adipocytes in the hearts of Nkx2.5- and Mef2C-regulated lineage tracer mice, expressed enhanced yellow fluorescent protein. In addition, rare cells coexpressed adipogenic transcription factors and the second heart field markers Isl1 and Mef2C in the lineage tracer mouse hearts and in human myocardium from patients with arrhythmogenic right ventricular cardiomyopathy. To delineate the responsible mechanism, we generated transgenic mice expressing desmosomal protein plakoglobin in myocyte lineages. Transgene plakoglobin translocated to nucleus, detected by immunoblotting and immunofluorescence staining and coimmunoprecipitated with Tcf7l2, a canonical Wnt signaling transcription factor. Expression levels of canonical Wnt/Tcf7l2 targets bone morphogenetic protein 7 and Wnt5b, which promote adipogenesis, were increased and expression level of connective tissue growth factor, an inhibitor of adipogenesis, was decreased. We conclude adipocytes in arrhythmogenic right ventricular cardiomyopathy originate from the second heart field cardiac progenitors, which switch to an adipogenic fate because of suppressed canonical Wnt signaling by nuclear plakoglobin.
doi:10.1161/CIRCRESAHA.109.196899
PMCID: PMC2767296  PMID: 19359597
adipocytes; progenitor cells; Wnt signaling; desmosomes; heart failure
22.  SMYD1, the myogenic activator, is a direct target of serum response factor and myogenin 
Nucleic Acids Research  2009;37(21):7059-7071.
SMYD1 is a heart and muscle specific SET-MYND domain containing protein, which functions as a histone methyltransferase and regulates downstream gene transcription. We demonstrated that the expression of SMYD1 is restricted in the heart and skeletal muscle tissues in human. To reveal the regulatory mechanisms of SMYD1 expression during myogenesis and cardiogenesis, we cloned and characterized the human SMYD1 promoter, which contains highly conserved serum response factor (SRF) and myogenin binding sites. Overexpression of SRF and myogenin significantly increased the endogenous expression level of Smyd1 in C2C12 cells, respectively. Deletion of Srf in the heart of mouse embryos dramatically decreased the expression level of Smyd1 mRNA and the expression of Smyd1 can be rescued by exogenous SRF introduction in SRF null ES cells during differentiation. Furthermore, we demonstrated that SRF binds to the CArG site and myogenin binds to the E-box element on Smyd1 promoter region using EMSA and ChIP assays. Moreover, forced expression of SMYD1 accelerates myoblast differentiation and myotube formation in C2C12 cells. Taken together, these studies demonstrated that SMYD1 is a key regulator of myogenic differentiation and acts as a downstream target of muscle regulatory factors, SRF and myogenin.
doi:10.1093/nar/gkp773
PMCID: PMC2790895  PMID: 19783823
23.  Prox1 maintains muscle structure and growth in the developing heart 
Development (Cambridge, England)  2008;136(3):495-505.
Impaired cardiac muscle growth and aberrant myocyte arrangement underlie congenital heart disease and cardiomyopathy. We show that cardiac-specific inactivation of the homeobox transcription factor Prox1 results in disruption of the expression and localisation of sarcomeric proteins, gross myofibril disarray and growth retarded hearts. Furthermore, we demonstrate that Prox1 is required for direct transcriptional regulation of structural proteins α-actinin, N-RAP and Zyxin which collectively function to maintain an actin-α-actinin interaction as the fundamental association of the sarcomere. Aspects of abnormal heart development and manifestation of a subset of muscular-based disease have previously been attributed to mutations in key structural proteins. Our study demonstrates an essential requirement for direct transcriptional regulation of sarcomere integrity, in the context of enabling fetal cardiomyocyte hypertrophy, maintenance of contractile function and progression towards inherited or acquired myopathic disease.
doi:10.1242/dev.030007
PMCID: PMC2655234  PMID: 19091769
Prox1; heart development; myocardium; sarcomere; hypertrophy; myopathy
24.  MicroRNA Regulation of Cell Lineages in Mouse and Human Embryonic Stem Cells 
Cell stem cell  2008;2(3):219-229.
SUMMARY
Cell fate decisions of pluripotent embryonic stem (ES) cells are dictated by activation and repression of lineage-specific genes. Numerous signaling and transcriptional networks progressively narrow and specify the potential of ES cells. Whether specific microRNAs help refine and limit gene expression, and thereby could be used to manipulate ES cell differentiation, has largely been unexplored. Here, we show that two serum response factor (SRF)-dependent muscle-specific microRNAs, miR-1 and miR-133 promote mesoderm formation from ES cells but have opposing functions during further differentiation into cardiac muscle progenitors. Furthermore, miR-1 and miR-133 were potent repressors of nonmuscle gene expression and cell fate during mouse and human ES cell differentiation. miR-1’s effects were in part mediated by translational repression of the Notch ligand Delta-like 1 (Dll-1). Our findings indicate that muscle-specific miRNAs reinforce the silencing of nonmuscle genes during cell lineage commitment and suggest that miRNAs may have general utility in regulating cell fate decisions from pluripotent ES cells.
doi:10.1016/j.stem.2008.01.016
PMCID: PMC2293325  PMID: 18371447
25.  Double-Inducible Gene Activation System for Caspase 3 and 9 in Epidermis 
Genesis (New York, N.Y. : 2000)  2007;45(4):194-199.
Summary
Expression of genes with tight and precise temporal and spatial control is desired in a wide variety of applications ranging from cultured cells and transgenic animals to gene therapy. While current inducible systems, such as RU486 and chemical inducers of dimerization (CID), have improved earlier inducible models (Gossen et al., 1995) (Wang et al., 1994), no single system is perfect at present. One potential drawback of these systems is leakage of transgene expression, causing limitations of each system. We have developed an inducible model containing both RU486 and CID systems, which in addition to inducing caspase activation, has potential applicability specifically to other genes encoding proteins that require a dimerization event for activation. This Double-Inducible Gene Activation System generates two barriers for the target gene expression and protein activation thereby minimizing leakage.
doi:10.1002/dvg.20288
PMCID: PMC2566959  PMID: 17417788
RU486; CID; keratin 14; inducible gene expression; double-inducible system

Results 1-25 (32)