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1.  Lack of Genetic Interaction between Tbx20 and Tbx3 in Early Mouse Heart Development 
PLoS ONE  2013;8(7):e70149.
Members of the T-box family of transcription factors are important regulators orchestrating the complex regionalization of the developing mammalian heart. Individual mutations in Tbx20 and Tbx3 cause distinct congenital heart abnormalities in the mouse: Tbx20 mutations result in failure of heart looping, developmental arrest and lack of chamber differentiation, while hearts of Tbx3 mutants progress further, loop normally but show atrioventricular convergence and outflow tract defects. The two genes have overlapping areas of expression in the atrioventricular canal and outflow tract of the heart but their potential genetic interaction has not been previously investigated. In this study we produced compound mutants to investigate potential genetic interactions at the earliest stages of heart development. We find that Tbx20; Tbx3 double heterozygous mice are viable and fertile with no apparent abnormalities, while double homozygous mutants are embryonic lethal by midgestation. Double homozygous mutant embryos display abnormal cardiac morphogenesis, lack of heart looping, expression patterns of cardiac genes and time of death that are indistinguishable from Tbx20 homozygous mutants. Prior to death, the double homozygotes show an overall developmental delay similar to Tbx3 homozygous mutants. Thus the effects of Tbx20 are epistatic to Tbx3 in the heart but Tbx3 is epistatic to Tbx20 with respect to developmental delay.
PMCID: PMC3723716  PMID: 23936153
2.  Functional Characterization of a Novel Mutation in NKX2-5 Associated with Congenital Heart Disease and Adult-Onset Cardiomyopathy 
Circulation. Cardiovascular genetics  2013;6(3):10.1161/CIRCGENETICS.113.000057.
The transcription factor NKX2-5 is crucial for heart development and mutations in this gene have been implicated in diverse congenital heart diseases (CHD) and conduction defects (CD) in mouse models and humans. Whether NKX2-5 mutations have a role in adult-onset heart disease is unknown.
Methods and Results
Mutation screening was performed in 220 probands with adult-onset dilated cardiomypathy (DCM). Six NKX2-5 coding sequence variants were identified, including 3 non-synonymous variants. A novel heterozygous mutation, I184M, located within the NKX2-5 homeodomain (HD), was identified in one family. A subset of family members had CHD, but there was an unexpectedly high prevalence of DCM. Functional analysis of I184M in vitro demonstrated a striking increase in protein expression when transfected into COS-7 cells or HL-1 cardiomyocytes, due to reduced degradation by the ubiquitin-proteasome system (UPS). In functional assays, DNA binding activity of I184M was reduced, resulting in impaired activation of target genes, despite increased expression levels of mutant protein.
Certain NKX2-5 HD mutations show abnormal protein degradation via the UPS and partially impaired transcriptional activity. We propose that this class of mutation can impair heart development and mature heart function, and contribute to NKX2-5-related cardiomyopathies with graded severity.
PMCID: PMC3816146  PMID: 23661673
dilated cardiomyopathy; transcription factors; gene mutations; ubiquitin-proteasome system; NKX2-5
3.  Antisense-mediated exon skipping: a therapeutic strategy for titin-based dilated cardiomyopathy 
EMBO Molecular Medicine  2015;7(5):562-576.
Frameshift mutations in the TTN gene encoding titin are a major cause for inherited forms of dilated cardiomyopathy (DCM), a heart disease characterized by ventricular dilatation, systolic dysfunction, and progressive heart failure. To date, there are no specific treatment options for DCM patients but heart transplantation. Here, we show the beneficial potential of reframing titin transcripts by antisense oligonucleotide (AON)-mediated exon skipping in human and murine models of DCM carrying a previously identified autosomal-dominant frameshift mutation in titin exon 326. Correction of TTN reading frame in patient-specific cardiomyocytes derived from induced pluripotent stem cells rescued defective myofibril assembly and stability and normalized the sarcomeric protein expression. AON treatment in Ttn knock-in mice improved sarcomere formation and contractile performance in homozygous embryos and prevented the development of the DCM phenotype in heterozygous animals. These results demonstrate that disruption of the titin reading frame due to a truncating DCM mutation can be restored by exon skipping in both patient cardiomyocytes in vitro and mouse heart in vivo, indicating RNA-based strategies as a potential treatment option for DCM.
PMCID: PMC4492817  PMID: 25759365
dilated cardiomyopathy; exon skipping; induced pluripotent stem cells; titin
4.  Cardiogenic Genes Expressed in Cardiac Fibroblasts Contribute to Heart Development and Repair 
Circulation research  2014;114(9):1422-1434.
Cardiac fibroblasts are critical to proper heart function through multiple interactions with the myocardial compartment but appreciation of their contribution has suffered from incomplete characterization and lack of cell-specific markers.
To generate an unbiased comparative gene expression profile of the cardiac fibroblast pool, identify and characterize the role of key genes in cardiac fibroblast function, and determine their contribution to myocardial development and regeneration.
Methods and Results
High-throughput cell surface and intracellular profiling of cardiac and tail fibroblasts identified canonical MSC and a surprising number of cardiogenic genes, some expressed at higher levels than in whole heart. Whilst genetically marked fibroblasts contributed heterogeneously to interstitial but not cardiomyocyte compartments in infarcted hearts, fibroblast-restricted depletion of one highly expressed cardiogenic marker, Tbx20, caused marked myocardial dysmorphology and perturbations in scar formation upon myocardial infarction.
The surprising transcriptional identity of cardiac fibroblasts, the adoption of cardiogenic gene programs and direct contribution to cardiac development and repair provokes alternative interpretations for studies on more specialized cardiac progenitors, offering a novel perspective for reinterpreting cardiac regenerative therapies.
PMCID: PMC4083003  PMID: 24650916
Heart; fibroblast; transcription factors; transcriptional network; cardiac fibroblasts
5.  Bioengineering and Stem Cell Technology in the Treatment of Congenital Heart Disease 
Journal of Clinical Medicine  2015;4(4):768-781.
Congenital heart disease places a significant burden on the individual, family and community despite significant advances in our understanding of aetiology and treatment. Early research in ischaemic heart disease has paved the way for stem cell technology and bioengineering, which promises to improve both structural and functional aspects of disease. Stem cell therapy has demonstrated significant improvements in cardiac function in adults with ischaemic heart disease. This finding, together with promising case studies in the paediatric setting, demonstrates the potential for this treatment in congenital heart disease. Furthermore, induced pluripotent stems cell technology, provides a unique opportunity to address aetiological, as well as therapeutic, aspects of disease.
PMCID: PMC4470166
congenital heart disease; hypoplastic left heart; inducible pluripotential stem cells; bioengineered myocardium
6.  Nkx2-5 Mediates Differential Cardiac Differentiation Through Interaction with Hoxa10 
Stem Cells and Development  2013;22(15):2211-2220.
The regulation of cardiac differentiation is complex and incompletely understood. Recent studies have documented that Nkx2-5-positive cells are not limited to the cardiac lineage, but can give rise to endothelial and smooth muscle lineages. Other work has elucidated that, in addition to promoting cardiac development, Nkx2-5 plays a larger role in mesodermal patterning although the transcriptional networks that govern this developmental patterning are undefined. By profiling early Nkx2-5-positive progenitor cells, we discovered that the progenitor pools of the bisected cardiac crescent are differentiating asynchronously. This asymmetry requires Nkx2-5 as it is lost in the Nkx2-5 mutant. Surprisingly, the posterior Hox genes Hoxa9 and Hoxa10 were expressed on the right side of the cardiac crescent, independently of Nkx2-5. We describe a novel, transient, and asymmetric cardiac-specific expression pattern of the posterior Hox genes, Hoxa9 and Hoxa10, and utilize the embryonic stem cell/embryoid body (ES/EB) model system to illustrate that Hoxa10 impairs cardiac differentiation. We suggest a model whereby Hoxa10 cooperates with Nkx2-5 to regulate the timing of cardiac mesoderm differentiation.
PMCID: PMC3715789  PMID: 23477547
7.  Heart field origin of great vessel precursors relies on nkx2.5-mediated vasculogenesis 
Nature cell biology  2013;15(11):10.1038/ncb2862.
The pharyngeal arch arteries (PAAs) are transient embryonic blood vessels that make indispensable contributions to the carotid arteries and great vessels of the heart, including the aorta and pulmonary artery1, 2. During embryogenesis, the PAAs appear in a craniocaudal sequence to connect pre-existing segments of the primitive circulation after de novo vasculogenic assembly from angioblast precursors3, 4. Despite the unique spatiotemporal characteristics of PAA development, the embryonic origins of PAA angioblasts and the genetic factors regulating their emergence remain unknown. Here, we identify the embryonic source of PAA endothelium as nkx2.5+ progenitors in lateral plate mesoderm long considered to adopt cell fates within the heart exclusively5, 6. Further, we report that PAA endothelial differentiation relies on Nkx2.5, a canonical cardiac transcription factor not previously implicated in blood vessel formation. Together, these studies reveal the heart field origin of PAA endothelium and attribute a novel vasculogenic function to the cardiac transcription factor nkx2.5 during great vessel precursor development.
PMCID: PMC3864813  PMID: 24161929
8.  Precardiac deletion of Numb and Numblike reveals renewal of cardiac progenitors 
eLife  2014;3:e02164.
Cardiac progenitor cells (CPCs) must control their number and fate to sustain the rapid heart growth during development, yet the intrinsic factors and environment governing these processes remain unclear. Here, we show that deletion of the ancient cell-fate regulator Numb (Nb) and its homologue Numblike (Nbl) depletes CPCs in second pharyngeal arches (PA2s) and is associated with an atrophic heart. With histological, flow cytometric and functional analyses, we find that CPCs remain undifferentiated and expansive in the PA2, but differentiate into cardiac cells as they exit the arch. Tracing of Nb- and Nbl-deficient CPCs by lineage-specific mosaicism reveals that the CPCs normally populate in the PA2, but lose their expansion potential in the PA2. These findings demonstrate that Nb and Nbl are intrinsic factors crucial for the renewal of CPCs in the PA2 and that the PA2 serves as a microenvironment for their expansion.
eLife digest
Human embryos contain cells called ‘cardiac progenitor cells’ that serve as the building blocks to make the heart. Cardiac progenitor cells, or CPCs for short, initially move into areas of the embryo called the first and second heart fields, and then undergo a change to become specific types of heart cells: such as cardiac muscle cells. However, it is not known if CPCs are maintained during the development of the heart.
Now, Shenje, Andersen et al. have shown that Numb and Numblike—two proteins that are needed for the development of nerve cells—are also involved in the development of the heart. Mouse embryos without the genes for Numb and Numblike failed to develop hearts normally; and these mutants also had fewer CPCs in the ‘second pharyngeal arch’: a part of the embryo that becomes the sides and front of the neck. Experiments on wild-type mice showed that the CPCs multiplied within this arch, and then changed into specific heart cells as they left this structure. Furthermore, mixing CPCs in a petri dish with cells taken from this arch encouraged the CPCs to multiply without changing into specific cell types.
To investigate the importance of these two proteins further, Shenje, Andersen et al. engineered ‘chimeric’ mice in which some CPCs contained the Numb and Numblike genes and other CPCs did not. In most of these chimeric mice, the hearts developed normally, but the CPCs without the Numb or Numblike genes failed to multiply in the second pharyngeal arch. This shows that these genes must be present within an individual CPC to regulate the multiplication of that cell within this arch.
By uncovering how problems with the maintenance of CPCs can lead to heart defects—a very common birth defect in humans—this work may lead to new ways to prevent or treat congenital heart disease. Furthermore, identifying the other factors or mechanisms that can allow the long-term maintenance of CPCs in the laboratory will be crucial for research into heart regeneration, and for CPC-based treatments to repair the heart.
PMCID: PMC4007206  PMID: 24843018
cardiac progenitor; self-renewal; niche; numb; microenvironment; heart; mouse
9.  Nkx2-5+Islet1+ mesenchymal precursors generate distinct spleen stromal cell subsets and participate in restoring stromal network integrity 
Immunity  2013;38(4):782-791.
Lymphoid organ stromal cells comprise different subsets whose origin remains unknown. Herein, we exploit a genetic lineage-tracing approach to show that splenic fibroblastic reticular cells (FRCs), follicular dendritic cells (FDCs), marginal reticular cells (MRCs), and mural cells, but not endothelial cells, originated from embryonic mesenchymal progenitors of the Nkx2-5+Islet1+ lineage. This lineage included embryonic mesenchymal cells with lymphoid tissue organizer (LTo) activity capable of supporting ectopic lymphoid-like structures, and a subset of resident spleen stromal cells that proliferated and regenerated the splenic stromal microenvironment following resolution of a viral infection. These findings identify progenitor cells that generate stromal diversity in spleen development and repair, and suggest the existence of multipotent stromal progenitors in the adult spleen with regenerative capacity.
PMCID: PMC3652017  PMID: 23601687
10.  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.
PMCID: PMC3612528  PMID: 23463007
11.  Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module 
Developmental Cell  2012;22(5):913-926.
The molecular determinants of spleen organogenesis and the etiology of isolated congenital asplenia (ICA), a life-threatening human condition, are unknown. We previously reported that Pbx1 deficiency causes organ growth defects including asplenia. Here, we show that mice with splenic mesenchyme-specific Pbx1 inactivation exhibit hyposplenia. Moreover, the loss of Pbx causes down-regulation of Nkx2-5 and derepression of p15Ink4b in spleen mesenchymal progenitors, perturbing the cell cycle. Removal of p15Ink4b in Pbx1 spleen-specific mutants partially rescues spleen growth. By whole-exome sequencing of a multiplex kindred with ICA, we identify a heterozygous missense mutation (P236H) in NKX2-5 showing reduced transactivation in vitro. This study establishes that a Pbx/Nkx2-5/p15 regulatory module is essential for spleen development.
PMCID: PMC3356505  PMID: 22560297
Spleen; Organ growth; Human Isolated Congenital Asplenia; Cell cycle; Pbx; p15Ink4b; Nkx2-5
12.  Adult Cardiac-Resident MSC-like Stem Cells with a Proepicardial Origin 
Cell stem cell  2011;9(6):527-540.
Colony-forming units – fibroblast (CFU-Fs), analogous to those giving rise to bone marrow (BM) mesenchymal stem cells (MSCs), are present in many organs, although the relationship between BM and organ-specific CFU-Fs in homeostasis and tissue repair is unknown. Here we describe a population of adult cardiac-resident CFU-Fs (cCFU-Fs) that occupy a perivascular, adventitial niche and show broad trans-germ layer potency in vitro and in vivo. CRE lineage tracing and embryo analysis demonstrated a proepicardial origin for cCFU-Fs. Furthermore, in BM transplantation chimeras, we found no interchange between BM and cCFU-Fs after aging, myocardial infarction, or BM stem cell mobilization. BM and cardiac and aortic CFU-Fs had distinct CRE lineage signatures, indicating that they arise from different progenitor beds during development. These diverse origins for CFU-Fs suggest an underlying basis for differentiation biases seen in different CFU-F populations, and could also influence their capacity for participating in tissue repair.
PMCID: PMC3652240  PMID: 22136928
13.  Rotary ATPases 
Bioarchitecture  2013;3(1):2-12.
Rotary ATPases are molecular rotary motors involved in biological energy conversion. They either synthesize or hydrolyze the universal biological energy carrier adenosine triphosphate. Recent work has elucidated the general architecture and subunit compositions of all three sub-types of rotary ATPases. Composite models of the intact F-, V- and A-type ATPases have been constructed by fitting high-resolution X-ray structures of individual subunits or sub-complexes into low-resolution electron densities of the intact enzymes derived from electron cryo-microscopy. Electron cryo-tomography has provided new insights into the supra-molecular arrangement of eukaryotic ATP synthases within mitochondria and mass-spectrometry has started to identify specifically bound lipids presumed to be essential for function. Taken together these molecular snapshots show that nano-scale rotary engines have much in common with basic design principles of man made machines from the function of individual “machine elements” to the requirement of the right “fuel” and “oil” for different types of motors.
PMCID: PMC3639240  PMID: 23369889
biological motors; rotary motors; energy conversion; ATP synthase; vacuolar ATPase; A-type ATPase; structural biology; X-ray crystallography; electron microscopy
14.  Combined Mutation Screening of NKX2-5, GATA4, and TBX5 in Congenital Heart Disease: Multiple Heterozygosity and Novel Mutations 
Congenital heart disease  2011;7(2):151-159.
Variants of several genes encoding transcription modulators, signal transduction, and structural proteins are known to cause Mendelian congenital heart disease (CHD). NKX2-5 and GATA4 were the first CHD-causing genes identified by linkage analysis in large affected families. Mutations of TBX5 cause Holt–Oram syndrome, which includes CHD as a clinical feature. All three genes have a well-established role in cardiac development.
In order to investigate the possible role of multiple mutations in CHD, a combined mutation screening was performed in NKX2-5, GATA4, and TBX5 in the same patient cohort. Samples from a cohort of 331 CHD patients were analyzed by polymerase chain reaction, double high-performance liquid chromatography and sequencing in order to identify changes in the NKX2-5, GATA4, and TBX5 genes.
Two cases of multiple heterozygosity of putative disease-causing mutations were identified. One patient was found with a novel L122P NKX2-5 mutation in combination with the private A1443D mutation of MYH6. A patient heterozygote for a D425N GATA4 mutation carries also a private mutation of the MYH6 gene (V700M).
In addition to reporting two novel mutations of NKX2-5 in CHD, we describe families where multiple individual mutations seem to have an additive effect over the pathogenesis of CHD. Our findings highlight the usefulness of multiple gene mutational analysis of large CHD cohorts.
PMCID: PMC3370385  PMID: 22011241
Congenital Heart Disease; Mutations; Multiple Heterozygosity
15.  Nkx2-5 Represses Gata1 Gene Expression and Modulates the Cellular Fate of Cardiac Progenitors during Embryogenesis 
Circulation  2011;123(15):1633-1641.
Recent studies suggest that the hematopoietic and cardiac lineages have close ontogenic origins, and that an early mesodermal cell population has the potential to differentiate into both lineages. Studies also suggest that specification of these lineages is inversely regulated. However, the transcriptional networks that govern the cell fate specification of these progenitors are incompletely defined.
Methods and Results
Here, we show that Nkx2-5 regulates the hematopoietic/erythroid fate of the mesoderm precursors early during cardiac morphogenesis. Utilizing transgenic technologies to isolate Nkx2-5 expressing cells, we observed an induction of the erythroid molecular program, including Gata1, in the Nkx2-5 null embryos. We further observed that overexpression of Nkx2-5 using an Nkx2-5-inducible embryonic stem (ES) cell system significantly repressed Gata1 gene expression and suppressed the hematopoietic/erythroid potential but not the endothelial potential of the ES cells. This suppression was cell-autonomous and was partially rescued by overexpressing Gata1. In addition, we demonstrated that Nkx2-5 binds to the Gata1 gene enhancer and represses the transcriptional activity of the Gata1 gene.
Our results demonstrate that the hematopoietic/erythroid cell fate is suppressed via Nkx2-5 during mesodermal fate determination and that the Gata1 gene is one of the targets that are suppressed by Nkx2-5.
PMCID: PMC3110259  PMID: 21464046
Nkx2-5; Gata1; cardiac progenitors; gene regulation
16.  Tinman/Nkx2-5 acts via miR-1 and upstream of Cdc42 to regulate heart function across species 
The Journal of Cell Biology  2011;193(7):1181-1196.
Cdc42 regulates cardiac function in mice and flies downstream of a conserved Tinman/Nkx2-5–miR-1 signaling network.
Unraveling the gene regulatory networks that govern development and function of the mammalian heart is critical for the rational design of therapeutic interventions in human heart disease. Using the Drosophila heart as a platform for identifying novel gene interactions leading to heart disease, we found that the Rho-GTPase Cdc42 cooperates with the cardiac transcription factor Tinman/Nkx2-5. Compound Cdc42, tinman heterozygous mutant flies exhibited impaired cardiac output and altered myofibrillar architecture, and adult heart–specific interference with Cdc42 function is sufficient to cause these same defects. We also identified K+ channels, encoded by dSUR and slowpoke, as potential effectors of the Cdc42–Tinman interaction. To determine whether a Cdc42–Nkx2-5 interaction is conserved in the mammalian heart, we examined compound heterozygous mutant mice and found conduction system and cardiac output defects. In exploring the mechanism of Nkx2-5 interaction with Cdc42, we demonstrated that mouse Cdc42 was a target of, and negatively regulated by miR-1, which itself was negatively regulated by Nkx2-5 in the mouse heart and by Tinman in the fly heart. We conclude that Cdc42 plays a conserved role in regulating heart function and is an indirect target of Tinman/Nkx2-5 via miR-1.
PMCID: PMC3216339  PMID: 21690310
17.  Expression of Slit and Robo Genes in the Developing Mouse Heart 
Development of the mammalian heart is mediated by complex interactions between myocardial, endocardial, and neural crest-derived cells. Studies in Drosophila have shown that the Slit-Robo signaling pathway controls cardiac cell shape changes and lumen formation of the heart tube. Here, we demonstrate by in situ hybridization that multiple Slit ligands and Robo receptors are expressed in the developing mouse heart. Slit3 is the predominant ligand transcribed in the early mouse heart and is expressed in the ventral wall of the linear heart tube and subsequently in chamber but not in atrioventricular canal myocardium. Furthermore, we identify that the homeobox gene Nkx2-5 is required for early ventral restriction of Slit3 and that the T-box transcription factor Tbx2 mediates repression of Slit3 in nonchamber myocardium. Our results suggest that patterned Slit-Robo signaling may contribute to the control of oriented cell growth during chamber morphogenesis of the mammalian heart.
PMCID: PMC2996720  PMID: 20941780
Slit/Robo pathway; cardiac development; mouse; Tbx; atrioventricular canal
18.  Complex SUMO-1 Regulation of Cardiac Transcription Factor Nkx2-5 
PLoS ONE  2011;6(9):e24812.
Reversible post-translational protein modifications such as SUMOylation add complexity to cardiac transcriptional regulation. The homeodomain transcription factor Nkx2-5/Csx is essential for heart specification and morphogenesis. It has been previously suggested that SUMOylation of lysine 51 (K51) of Nkx2-5 is essential for its DNA binding and transcriptional activation. Here, we confirm that SUMOylation strongly enhances Nkx2-5 transcriptional activity and that residue K51 of Nkx2-5 is a SUMOylation target. However, in a range of cultured cell lines we find that a point mutation of K51 to arginine (K51R) does not affect Nkx2-5 activity or DNA binding, suggesting the existence of additional Nkx2-5 SUMOylated residues. Using biochemical assays, we demonstrate that Nkx2-5 is SUMOylated on at least one additional site, and this is the predominant site in cardiac cells. The second site is either non-canonical or a “shifting” site, as mutation of predicted consensus sites and indeed every individual lysine in the context of the K51R mutation failed to impair Nkx2-5 transcriptional synergism with SUMO, or its nuclear localization and DNA binding. We also observe SUMOylation of Nkx2-5 cofactors, which may be critical to Nkx2-5 regulation. Our data reveal highly complex regulatory mechanisms driven by SUMOylation to modulate Nkx2-5 activity.
PMCID: PMC3171482  PMID: 21931855
19.  Investigation of Association between PFO Complicated by Cryptogenic Stroke and a Common Variant of the Cardiac Transcription Factor GATA4 
PLoS ONE  2011;6(6):e20711.
Patent foramen ovale (PFO) is associated with clinical conditions including cryptogenic stroke, migraine and varicose veins. Data from studies in humans and mouse suggest that PFO and the secundum form of atrial septal defect (ASDII) exist in an anatomical continuum of septal dysmorphogenesis with a common genetic basis. Mutations in multiple members of the evolutionarily conserved cardiac transcription factor network, including GATA4, cause or predispose to ASDII and PFO. Here, we assessed whether the most prevalent variant of the GATA4 gene, S377G, was significantly associated with PFO or ASD. Our analysis of world indigenous populations showed that GATA4 S377G was largely Caucasian-specific, and so subjects were restricted to those of Caucasian descent. To select for patients with larger PFO, we limited our analysis to those with cryptogenic stroke in which PFO was a subsequent finding. In an initial study of Australian subjects, we observed a weak association between GATA4 S377G and PFO/Stroke relative to Caucasian controls in whom ASD and PFO had been excluded (OR = 2.16; p = 0.02). However, in a follow up study of German Caucasians no association was found with either PFO or ASD. Analysis of combined Australian and German data confirmed the lack of a significant association. Thus, the common GATA4 variant S377G is likely to be relatively benign in terms of its participation in CHD and PFO/Stroke.
PMCID: PMC3108965  PMID: 21673957
20.  Chromatin remodelling complex dosage modulates transcription factor function in heart development 
Nature communications  2011;2:187.
Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.
PMCID: PMC3096875  PMID: 21304516
21.  RNA toxicity in myotonic muscular dystrophy induces NKX2-5 expression 
Nature genetics  2007;40(1):61-68.
Myotonic muscular dystrophy (DM1) is the most common inherited neuromuscular disorder in adults and is considered the first example of a disease caused by RNA toxicity. Using a reversible transgenic mouse model of RNA toxicity in DM1, we provide evidence that DM1 is associated with induced NKX2-5 expression. Transgene expression resulted in cardiac conduction defects, increased expression of the cardiac-specific transcription factor NKX2-5 and profound disturbances in connexin 40 and connexin 43. Notably, overexpression of the DMPK 3′ UTR mRNA in mouse skeletal muscle also induced transcriptional activation of Nkx2-5 and its targets. In human muscles, these changes were specific to DM1 and were not present in other muscular dystrophies. The effects on NKX2-5 and its downstream targets were reversed by silencing toxic RNA expression. Furthermore, using Nkx2-5+/− mice, we show that NKX2-5 is the first genetic modifier of DM1-associated RNA toxicity in the heart.
PMCID: PMC2909759  PMID: 18084293
22.  A gain-of-function TBX20 mutation causes congenital atrial septal defects, patent foramen ovale and cardiac valve defects 
Journal of Medical Genetics  2010;47(4):230-235.
Ostium secundum atrial septal defects (ASDII) account for approximately 10% of all congenital heart defects (CHD), and mutations in cardiac transcription factors, including TBX20, were identified as an underlying cause for ASDII. However, very little is known about disease penetrance in families and functional consequences of inherited TBX20 mutations.
The coding region of TBX20 was directly sequenced in 170 ASDII patients. Functional consequences of one novel mutation were investigated by surface plasmon resonance, CD spectropolarymetry, fluorescence spectrophotometry, luciferase assay and chromatin immunoprecipitation.
We found a novel mutation in a highly conserved residue in the T-box DNA binding domain (I121M) segregating with CHD in a three generation kindred. Four mutation carriers revealed cardiac phenotypes in terms of cribriform ASDII, large patent foramen ovale or cardiac valve defects. Interestingly, tertiary hydrophobic interactions within the mutant TBX20 T-box were significantly altered leading to a more dynamic structure of the protein. Moreover, Tbx20-I121M resulted in a significantly enhanced transcriptional activity, which was further increased in the presence of co-transcription factors GATA4/5 and NKX2-5. Occupancy of DNA binding sites on target genes was also increased.
We suggest that TBX20-I121M adopts a more fluid tertiary structure leading to enhanced interactions with cofactors and more stable transcriptional complexes on target DNA sequences. Our data, combined with that of others, suggest that human ASDII may be related to loss-of-function as well as gain-of-function TBX20 mutations.
PMCID: PMC2981023  PMID: 19762328
Congenital heart defect; atrial septal defect; patent foramen ovale; TBX20; cardiovascular medicine; clinical genetics; molecular genetics
23.  Cardiac Deletion of Smyd2 Is Dispensable for Mouse Heart Development 
PLoS ONE  2010;5(3):e9748.
Chromatin modifying enzymes play a critical role in cardiac differentiation. Previously, it has been shown that the targeted deletion of the histone methyltransferase, Smyd1, the founding member of the SET and MYND domain containing (Smyd) family, interferes with cardiomyocyte maturation and proper formation of the right heart ventricle. The highly related paralogue, Smyd2 is a histone 3 lysine 4- and lysine 36-specific methyltransferase expressed in heart and brain. Here, we report that Smyd2 is differentially expressed during cardiac development with highest expression in the neonatal heart. To elucidate the functional role of Smyd2 in the heart, we generated conditional knockout (cKO) mice harboring a cardiomyocyte-specific deletion of Smyd2 and performed histological, functional and molecular analyses. Unexpectedly, cardiac deletion of Smyd2 was dispensable for proper morphological and functional development of the murine heart and had no effect on global histone 3 lysine 4 or 36 methylation. However, we provide evidence for a potential role of Smyd2 in the transcriptional regulation of genes associated with translation and reveal that Smyd2, similar to Smyd3, interacts with RNA Polymerase II as well as to the RNA helicase, HELZ.
PMCID: PMC2840034  PMID: 20305823
24.  An Nkx2-5/Bmp2/Smad1 negative feedback loop controls second heart field progenitor specification and proliferation 
Cell  2007;128(5):947-959.
During heart development the second heart field (SHF) provides progenitor cells for most cardiomyocytes and expresses the homeodomain factor Nkx2-5. We now show that feedback repression of Bmp2/Smad1 signaling by Nkx2-5 critically regulates SHF proliferation and outflow tract (OFT) morphology. In the cardiac fields of Nkx2-5 mutants, genes controlling cardiac specification (including Bmp2) and maintenance of the progenitor state were up-regulated, leading initially to progenitor over-specification, but subsequently to failed SHF proliferation and OFT truncation. In Smad1 mutants, SHF proliferation and deployment to the OFT were increased, while Smad1 deletion in Nkx2-5 mutants rescued SHF proliferation and OFT development. In Nkx2-5 hypomorphic mice, which recapitulate human congenital heart disease (CHD), OFT anomalies were also rescued by Smad1 deletion. Our findings demonstrate that Nkx2-5 orchestrates the transition between periods of cardiac induction, progenitor proliferation and OFT morphogenesis via a Smad1-dependent negative feedback loop, which may be a frequent molecular target in CHD.
PMCID: PMC2092439  PMID: 17350578
25.  The Hlx homeobox transcription factor is required early in enteric nervous system development 
Development of the enteric nervous system (ENS) requires interactions between migrating neural crest cells and the nascent gastrointestinal tract that are dependent upon genes expressed by both cell compartments. Hlx, a homeobox transcription factor gene that is expressed in mouse intestinal and hepatic mesenchyme, is required for normal embryonic growth of intestine and liver, and the Hlx-/- genotype is embryonic lethal. We hypothesized that Hlx is required for ENS development.
Enteric neurons were identified in Hlx+/+ and Hlx-/- mouse embryos by immunostaining of embryo sections for the neural markers PGP9.5 and Phox2b, or by staining for β-galactosidase in whole-mount embryos containing the dopamine β-hydroxylase-nLacZ transgene. In Hlx+/+ embryos, neural crest cells/enteric neurons have moved from the stomach into the intestine by E10.5. By contrast, neural crest cells/enteric neurons remain largely restricted to the lateral stomach mesenchyme of Hlx-/- embryos, with only a few scattered neural crest cells/enteric neurons in the intestine between E10.5–16.5.
The Hlx homeobox transcription factor is required for early aspects of ENS development.
PMCID: PMC1564389  PMID: 16854219

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