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With the emergence of high throughput single cell techniques, the understanding of the molecular and cellular diversity of mammalian organs have rapidly increased. In order to understand the spatial organization of this diversity, single cell data is often integrated with spatial data to create probabilistic cell maps. However, targeted cell typing approaches relying on existing single cell data achieve incomplete and biased maps that could mask the true diversity present in a tissue slide. Here we applied a de novo technique to spatially resolve and characterize cellular diversity of in situ sequencing data during human heart development. We obtained and made accessible well defined spatial cell-type maps of fetal hearts from 4.5 to 9 post conception weeks, not biased by probabilistic cell typing approaches. With our analysis, we could characterize previously unreported molecular diversity within cardiomyocytes and epicardial cells and identified their characteristic expression signatures, comparing them with specific subpopulations found in single cell RNA sequencing datasets. We further characterized the differentiation trajectories of epicardial cells, identifying a clear spatial component on it. All in all, our study provides a novel technique for conducting de novo spatial-temporal analyses in developmental tissue samples and a useful resource for online exploration of cell-type differentiation during heart development at sub-cellular image resolution.

Congenital heart disease (CHD) affects about 1 in 100 newborns and its causes are multifactorial. In the embryo, blood flow within the heart and vasculature is essential for proper heart development, with abnormal blood flow leading to CHD. Here, we discuss how blood flow (hemodynamics) affects heart development from embryonic to fetal stages, and how abnormal blood flow solely can lead to CHD. We emphasize studies performed using avian models of heart development, because those models allow for hemodynamic interventions, in vivo imaging, and follow up, while they closely recapitulate heart defects observed in humans. We conclude with recommendations on investigations that must be performed to bridge the gaps in understanding how blood flow alone, or together with other factors, contributes to CHD.

Parental exposure to bisphenol A (BPA) has been linked to a greater incidence of congenital diseases. We have demonstrated that BPA induces in zebrafish males an increase in the acetylation of sperm histones that is transmitted to the blastomeres of the unexposed progeny. This work is aimed to determine whether histone hyperacetylation promoted by paternal exposure to BPA is the molecular mechanism underlying the cardiogenesis impairment in the descendants. Zebrafish males were exposed to 100 and 2000 µg/L BPA during early spermatogenesis and mated with non-exposed females. We analyzed in the progeny the expression of genes involved in cardiogenesis and the epigenetic profile. Once the histone hyperacetylation was confirmed, treatment with epigallocatechin gallate (EGCG), an inhibitor of histone acetyltransferases, was assayed on F1 embryos. Embryos from males exposed to 2000 µg/L BPA overexpressed the transcription factor hand2 and the receptor esr2b, showing their own promoters-as well as that of kat6a-an enrichment in H3K9ac. In embryos treated with EGCG, both gene expression and histone acetylation (global and specific) returned to basal levels, and the phenotype was recovered. As shown by the results, the histone hyperacetylated landscape promoted by BPA in the sperm alters the chromatin structure of the progeny, leading to the overexpression of the histone acetyltransferase and genes involved in cardiogenesis.

Birth defects are the leading cause of infant death and the most common are congenital heart defects (CHD). In the majority of cases, the underlying genetic cause is unknown. There is increasing evidence that variants in non-coding DNA within the functional cis-regulatory elements (CREs) controlling gene expression can cause CHDs. However, little is known about the human CREs that are crucial for cardiovascular development. To address this knowledge gap, we bioinformatically identified cardiovascular CREs based on the occupancy of the CRE by the homeodomain protein NKX2-5 and cardiac chromatin histone modifications. This search defined several putative CREs within the loci of genes specifically implicated in heart development and disease. We show that human regulatory elements are functionally conserved and capable of driving reporter transgene expression in vivo throughout the developing cardiovascular system of medaka (Oryzias latipes). Using heart muscle cells and endothelial cells derived from human embryonic stem cells in which these CREs were deleted, we identified molecular perturbations in the gene regulatory network controlling vascular development. Among the CREs identified, we show that the deletion of the human enFLT1 enhancer in vitro, a highly conserved enhancer within the FLT1 locus, using endothelial tube formation assays did not disrupt vessel formation. However, deletion of the endogenous enFLT1 enhancer in medaka results in blood vessel malformations, indicating an important role for this regulatory element in vivo. These findings highlight the crucial role of human regulatory elements and their function as a regulator and buffer of transcriptional regulation in cardiovascular development. Our studies further support the hypothesis that perturbations in human CREs are important contributors of developmental defects.

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

The heart is one of the first organs to develop in a vertebrate embryo. The intricate design of the asymmetric four-chambered organ is reflected in both its structures and mechanics. A healthy functional heart is an amalgamation of precisely aligned great vessels, continuous sheets of trilayered tissues that form the chamber walls and septum, a coordinated conduction system, and an uninterrupted supply of oxygenated blood from the coronary vasculature. At a cellular level, heart morphogenesis is a remarkable feat of tightly regulated cell proliferation, apoptosis, migration and differentiation. These events are driven by spatially and temporally coordinated gene expressions. Post-translational modifications of histone proteins such as acetylation and deacetylation are central to gene expression and repression. The genome site-specific activities of enzymes responsible for acetylation and/or deacetylation rely on protein domains that can recognize and bind to modified histone residues. These protein domains are present in either the histone modifying enzyme itself or adaptor proteins that are present within the histone modifying complex, known as histone ‘reader’ proteins. In this thesis, I have used knockout mice to investigate the roles of two histone reader proteins – ING4 and ING5, both present in histone acetyltransferase complexes in vivo, demonstrating the consequences of loss of these histone reader proteins in the patterning and structural development of the heart. In the first part of my thesis, I have demonstrated that loss of both Ing5 alleles alone did not incur any haematopoietic defects, unlike loss of the proposed histone acetyltransferase enzyme subunit present in the ING5 protein complex. Subsequently, I characterised the survival and appearances of mice with deletion of a combination of Ing4 and Ing5 alleles. I showed that ING4 and ING5 are functionally redundant, as mice died in mid-gestation in the absence of both proteins, whereas mice lacking o

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

Deoxyribonucleoside triphosphates (dNTPs) are precursors for DNA replication and repair. Mammalian cells have two distinct biosynthesis pathways to supply dNTPs: de novo and salvage pathways. These pathways are intimately coordinated to maintain optimal dNTP concentrations throughout different phases of the cell cycle, and perturbations in the production of dNTPs could lead to increased, decreased, or imbalanced dNTP pools. In yeasts, changes in both the level and balance of dNTPs increase mutation rates and genome instability. In mammals, elevated mutation rates and genome instability predispose to numerous diseases, including cancer. However, the correlation of dNTP changes with pathology has not been well established in mammals. In this thesis, I present how we addressed this issue using three separate mouse models – one with an increased dNTP pool, one with a decreased dNTP pool, and one with an imbalanced dNTP pool. To modulate dNTP levels in the mice, we deleted or mutated either sterile alpha motif and histidine-aspartic domain containing protein 1 (SAMHD1) or ribonucleotide reductase (RNR) proteins, which are involved in the salvage and de novo pathways, respectively. In the first model, mouse embryos without the SAMHD1 gene showed a slight increase in dNTP levels. A similar increase in dNTPs conferred moderately elevated mutation rates in cultured cancer cells. In the second model, we created a mouse strain carrying a modified allosteric specificity site in a subunit of RNR. Embryos with a heterozygous mutation had a mildly imbalanced dNTP pool. Heterozygous mutant mice showed a shorter lifespan and increased incidence and earlier onset of cancer. In the third model, the de novo dNTP production was inactivated in cardiac and skeletal muscles through the deletion of a gene encoding RNR. The hearts of knockout pups showed significant depletion of dNTPs, leading to aberrant DNA replication. In addition, knockout pups developed anatomic and histologic cardiac a

MEF2C is a member of the highly conserved MEF2 family of transcription factors and is a key regulator of cardiovascular development. In mice, Mef2c is expressed in the developing heart and vasculature, including the endothelium. Loss of Mef2c function in germline knockout mice leads to early embryonic demise and profound developmental abnormalities in the cardiovascular system. Previous attempts to uncover the cause of embryonic lethality by specifically disrupting Mef2c function in the heart or vasculature failed to recapitulate the global Mef2c knockout phenotype and instead resulted in relatively minor defects that did not compromise viability or result in significant cardiovascular defects. However, previous studies examined the requirement of Mef2c in the myocardial and endothelial lineages using Cre lines that begin to be expressed after the expression of Mef2c has already commenced. Here, we tested the requirement of Mef2c in the myocardial and endothelial lineages using conditional knockout approaches in mice with Cre lines that deleted Mef2c prior to onset of its expression in embryonic development. We found that deletion of Mef2c in the early myocardial lineage using Nkx2-5Cre resulted in cardiac and vascular abnormalities that were indistinguishable from the defects in the global Mef2c knockout. In contrast, early deletion of Mef2c in the vascular endothelium using an Etv2::Cre line active prior to the onset of Mef2c expression resulted in viable offspring that were indistinguishable from wild type controls with no overt defects in vascular development, despite nearly complete early deletion of Mef2c in the vascular endothelium. Thus, these studies support the idea that the requirement of MEF2C for vascular development is secondary to its requirement in the heart and suggest that the observed failure in vascular remodeling in Mef2c knockout mice results from defective heart function.

Steroids are complex lipophilic molecules that have many actions in the body to regulate cellular, tissue and organ functions across the life-span. Steroid hormones such as cortisol, aldosterone, estradiol and testosterone are synthesised from cholesterol in specialised endocrine cells in the adrenal gland, ovary and testis, and released into the circulation when required. Steroid hormones move freely into cells to activate intracellular nuclear receptors that function as multi-domain ligand-dependent transcriptional regulators in the cell nucleus. Activated nuclear receptors modify expression of hundreds to thousands of specific target genes in the genome. Steroid hormone actions in the fetus include developmental roles in the respiratory system, brain, and cardiovascular system. The synthetic glucocorticoid steroid betamethasone is used antenatally to reduce the complications of preterm birth. Development of novel selective partial glucocorticoid receptor agonists may provide improved therapies to treat the respiratory complications of preterm birth and spare the deleterious effects of postnatal glucocorticoids in other organs.Copyright © 2019 Elsevier Ltd

Introduction: Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages. In this study, we used mice genetically deficient in myeloid MR to evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice. Method(s): Transgenic mice with intact myeloid MR (MRflox/flox) or myeloid MR deficiency (MRflox/flox LysMCre) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with multiple low dose injections of streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (albuminuria, renal function impairment and kidney damage) and cardiomyopathy (cardiac dysfunction-echocardiography and cardiac damage). Result(s): MR intact Nos3-/- mice with diabetes developed hypertension, kidney damage (macrophage accumulation, albuminuria, renal function impairment, hypertrophy, tubular injury, fibrosis), and cardiac damage (Left ventricular (LV) fractional shortening, macrophage accumulation, fibrosis), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice had equivalent diabetes, hypertension and albuminuria, but showed protection against loss of renal function (plasma cystatin-C reduced 48% vs MR intact mice with diabetes, p<0.01) and cardiac dysfunction (LV fractional shortening reduced 26% in MR intact mice with diabetes, p<0.01, but not in myeloid MR deficient mice with diabetes). The cardiac protection resulting from myeloid MR deficiency included the prevention of a diabetes-ind

Steroids are complex lipophilic molecules that have many actions in the body to regulate cellular, tissue and organ functions across the life-span. Steroid hormones such as cortisol, aldosterone, estradiol and testosterone are synthesised from cholesterol in specialised endocrine cells in the adrenal gland, ovary and testis, and released into the circulation when required. Steroid hormones move freely into cells to activate intracellular nuclear receptors that function as multi-domain ligand-dependent transcriptional regulators in the cell nucleus. Activated nuclear receptors modify expression of hundreds to thousands of specific target genes in the genome. Steroid hormone actions in the fetus include developmental roles in the respiratory system, brain, and cardiovascular system. The synthetic glucocorticoid steroid betamethasone is used antenatally to reduce the complications of preterm birth. Development of novel selective partial glucocorticoid receptor agonists may provide improved therapies to treat the respiratory complications of preterm birth and spare the deleterious effects of postnatal glucocorticoids in other organs.Copyright © 2019 Elsevier Ltd

Introduction: Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages. In this study, we used mice genetically deficient in myeloid MR to evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice. Method(s): Transgenic mice with intact myeloid MR (MRflox/flox) or myeloid MR deficiency (MRflox/flox LysMCre) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with multiple low dose injections of streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (albuminuria, renal function impairment and kidney damage) and cardiomyopathy (cardiac dysfunction-echocardiography and cardiac damage). Result(s): MR intact Nos3-/- mice with diabetes developed hypertension, kidney damage (macrophage accumulation, albuminuria, renal function impairment, hypertrophy, tubular injury, fibrosis), and cardiac damage (Left ventricular (LV) fractional shortening, macrophage accumulation, fibrosis), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice had equivalent diabetes, hypertension and albuminuria, but showed protection against loss of renal function (plasma cystatin-C reduced 48% vs MR intact mice with diabetes, p<0.01) and cardiac dysfunction (LV fractional shortening reduced 26% in MR intact mice with diabetes, p<0.01, but not in myeloid MR deficient mice with diabetes). The cardiac protection resulting from myeloid MR deficiency included the prevention of a diabetes-ind

Myc is considered an essential transcription factor for heart development, but cardiac defects have only been studied in global Myc loss-of-function models. Here, we eliminated Myc by recombining a Myc floxed allele with the Nkx2.5Cre driver. We observed no anatomical, cellular or functional alterations in either fetuses or adult cardiac Myc-deficient mice. We re-examined Myc expression during development and found no expression in developing cardiomyocytes. In contrast, we confirmed that Mycn is essential for cardiomyocyte proliferation and cardiogenesis. Mosaic Myc overexpression in a Mycn-deficient background shows that Myc can replace Mycn function, recovering heart development. We further show that this recovery involves the elimination of Mycn-deficient cells by cell competition. Our results indicate that Myc is dispensable in cardiomyocytes both during cardiogenesis and for adult heart homeostasis, and that Mycn is exclusively responsible for cardiomyocyte proliferation during heart development. Nonetheless, our results show that Myc can functionally replace Mycn We also show that cardiomyocytes compete according to their combined Myc and Mycn levels and that cell competition eliminates flawed cardiomyocytes, suggesting its relevance as a quality control mechanism in cardiac development.

The building blocks of DNA, dNTPs, can be produced de novo or can be salvaged from deoxyribonucleosides. However, to what extent the absence of de novo dNTP production can be compensated for by the salvage pathway is unknown. Here, we eliminated de novo dNTP synthesis in the mouse heart and skeletal muscle by inactivating ribonucleotide reductase (RNR), a key enzyme for the de novo production of dNTPs, at embryonic day 13. All other tissues had normal de novo dNTP synthesis and theoretically could supply heart and skeletal muscle with deoxyribonucleosides needed for dNTP production by salvage. We observed that the dNTP and NTP pools in wild-type postnatal hearts are unexpectedly asymmetric, with unusually high dGTP and GTP levels compared with those in whole mouse embryos or murine cell cultures. We found that RNR inactivation in heart led to strongly decreased dGTP and increased dCTP, dTTP, and dATP pools; aberrant DNA replication; defective expression of muscle-specific proteins; progressive heart abnormalities; disturbance of the cardiac conduction system; and lethality between the second and fourth weeks after birth. We conclude that dNTP salvage cannot substitute for de novo dNTP synthesis in the heart and that cardiomyocytes and myocytes initiate DNA replication despite an inadequate dNTP supply. We discuss the possible reasons for the observed asymmetry in dNTP and NTP pools in wildtype hearts.

Adenovirus protein, Gam1, triggers the proteolytic destruction of the E1 SUMO-activating enzyme. Microinjection of an empirically determined amount of Gam1 mRNA into one-cell Xenopus embryos can reduce SUMOylation activity to undetectable, but nonlethal, levels, enabling an examination of the role of this post-translational modification during early vertebrate development.

The molecular machinery underlying heart development out of primordial anterior mesoderm remains incompletely understood. This is in part due to a restricted research emphasis on canonical genomic elements and the inability to precisely model the timing of cardiac lineage commitment. The genome is pervasively transcribed far beyond the coding exome. Therefore, we hypothesized that the transcriptome as a whole, including all noncoding and splice isoforms would most precisely define the identity of the cell as it differentiated into a cardiomyocyte. Using an enhancer fragment of the heart-critical Smarcd3 Brg1/Brm associated factor (BAF) subunit, we developed an in vitro reporter differentiation system that precisely delineated the first heart specification from murine embryonic stem cells (mESCs). From this, de novo total RNA isoform coexpression networks were generated to reveal a staged progression by which hundreds of thousands of gene isoforms were organized into hundreds of subnetwork modules that dynamically programmed nascent mesoderm to became restricted to a cardiovascular fate. Many transcripts within the nucleus bind and influence the genomic regulation of chromatin modifying complexes. Therefore, we next aimed to elucidate the BRG1/BAF RNA interactome during its key role at cardiac fate commitment. Using targeting immunoprecipitations coupled to molecular techniques to isolate and identify protein:RNA adducts, we found at least 7 subunits engaged RNA molecules via previously unrealized discrete domains. Furthermore, these subunits interfaced RNA through tens of thousands of binding events that frequently coincided the defining transcript isoform transitions of this developmental window.These interrogations into the transcriptional basis for cardiac lineage differentiation also identified 6 annotated long intergenic noncoding RNAs (lincRNA) with discrete gene structure, epigenetic regulation, and cardiac progenitor specificity in vivo. We ablated these lin

Aim: To evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice. Background(s): Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages. Method(s): Transgenic mice with intact myeloid MR (MRflox/flox =WT) or myeloid MR deficiency (MRflox/flox LysMCre = MyMRKO) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with low dose streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (renal function impairment, albuminuria, renal injury) and cardiomyopathy (cardiac dysfunction-echocardiography). Result(s): MR intact Nos3-/- mice with diabetes developed hypertension, albuminuria, renal function impairment (elevated cystatin-C), tubular injury (increased KIM-1 gene expression), and reduced cardiac function (LV fractional shortening: Diabetic 21% versus non-diabetic 31%), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice with similar diabetes had equivalent levels of hypertension, albuminuria and tubular injury, but showed improved renal function (plasma cystatin-C 48%#, p<0.01) and improved cardiac function (LV fractional shortening 28%, p<0.01). Conclusion(s): MR signaling in macrophages promotes injury in the diabetic kidneys and hearts of Nos3-/- mice without affecting hypertension. Therefore, MRAs can suppress diabetic nephropathy and diabetic cardiomyopathy by reducing macrophage-mediated injury independent of their ability to lower blood pressure

Aim: To investigate the effects of foetal growth restriction (FGR) and prematurity on cardiac morphology and function in infancy. We hypothesised that FGR and prematurity would both alter cardiac development. Method(s): Cardiac morphology and function were evaluated in 24 preterm FGR infants (p-FGR) and 23 preterm and 19 term appropriately grown for gestational age infants (p-AGA and t-AGA, respectively) by conventional echocardiography and Tissue Doppler Imaging. p-FGR and p-AGA infants were studied on postnatal day 1 and all groups were studied at one-and six-months post-term age. Result(s): p-FGR infants demonstrated increased cardiac sphericity compared to AGA peers on postnatal day 1 (p = 0.004) and at one-month post-term age (p = 0.004). Posterior and relative wall thickness increased overtime in the p-FGR group only (p < 0.05). Systolic function was not different between groups. E/e' ratio was higher in both preterm groups compared to the term group at one-month post-term age (p = 0.01). No statistically significant group differences were found at six-months post-term age. Conclusion(s): Foetal growth restriction was associated with subtle cardiac morphological changes, whereas both prematurity and FGR were associated with subclinical alterations in diastolic function.Copyright ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd

Aim: To evaluate whether macrophage mineralocorticoid receptor signaling contributes to the development of kidney and cardiac disease in hypertensive diabetic mice. Background(s): Mineralocorticoid receptor antagonists (MRAs) reduce hypertension, inflammation and tissue injury in human and experimental diabetes. Furthermore, inflammation and injury in diabetic kidneys and hearts is dependent on infiltrating macrophages. Therefore, we hypothesized that the tissue protective effects of MRAs in diabetes is partially due to inhibition of MR signaling in macrophages. Method(s): Transgenic mice with intact myeloid MR (MRflox/flox =WT) or myeloid MR deficiency (MRflox/flox LysMCre = MyMRKO) were developed on the hypertensive endothelial nitric oxide synthase deficient (Nos3-/-) mouse strain. Groups of these mice (n=13) were made diabetic with low dose streptozotocin (STZ) and were assessed after 15 weeks for the development of hypertension (systolic blood pressure), nephropathy (renal function impairment, albuminuria, renal injury) and cardiomyopathy (cardiac dysfunction-echocardiography). Result(s): MR intact Nos3-/- mice with diabetes developed hypertension, albuminuria, renal function impairment (elevated cystatin-C), tubular injury (increased KIM-1 gene expression), and reduced cardiac function (LV fractional shortening: Diabetic 21% versus non-diabetic 31%), but no neutrophil infiltrate. In comparison, myeloid MR deficient Nos3-/- mice with similar diabetes had equivalent levels of hypertension, albuminuria and tubular injury, but showed improved renal function (plasma cystatin-C 48%#, p<0.01) and improved cardiac function (LV fractional shortening 28%, p<0.01). Conclusion(s): MR signaling in macrophages promotes injury in the diabetic kidneys and hearts of Nos3-/- mice without affecting hypertension. Therefore, MRAs can suppress diabetic nephropathy and diabetic cardiomyopathy by reducing macrophage-mediated injury independent of their ability to lower blood pressure

Aim: To investigate the effects of foetal growth restriction (FGR) and prematurity on cardiac morphology and function in infancy. We hypothesised that FGR and prematurity would both alter cardiac development. Method(s): Cardiac morphology and function were evaluated in 24 preterm FGR infants (p-FGR) and 23 preterm and 19 term appropriately grown for gestational age infants (p-AGA and t-AGA, respectively) by conventional echocardiography and Tissue Doppler Imaging. p-FGR and p-AGA infants were studied on postnatal day 1 and all groups were studied at one-and six-months post-term age. Result(s): p-FGR infants demonstrated increased cardiac sphericity compared to AGA peers on postnatal day 1 (p = 0.004) and at one-month post-term age (p = 0.004). Posterior and relative wall thickness increased overtime in the p-FGR group only (p < 0.05). Systolic function was not different between groups. E/e' ratio was higher in both preterm groups compared to the term group at one-month post-term age (p = 0.01). No statistically significant group differences were found at six-months post-term age. Conclusion(s): Foetal growth restriction was associated with subtle cardiac morphological changes, whereas both prematurity and FGR were associated with subclinical alterations in diastolic function.Copyright ©2017 Foundation Acta Paediatrica. Published by John Wiley & Sons Ltd

The ultimate formation of a four-chambered heart allowing the separation of the pulmonary and systemic circuits was key for the evolutionary success of tetrapods. Complex processes of cell diversification and tissue morphogenesis allow the left and right cardiac compartments to become distinct but remain poorly understood. Here, we describe an unexpected laterality in the single zebrafish atrium analogous to that of the two atria in amniotes, including mammals. This laterality appears to derive from an embryonic antero-posterior asymmetry revealed by the expression of the transcription factor gene meis2b. In adult zebrafish hearts, meis2b expression is restricted to the left side of the atrium where it controls the expression of pitx2c, a regulator of left atrial identity in mammals. Altogether, our studies suggest that the multi-chambered atrium in amniotes arose from a molecular blueprint present before the evolutionary emergence of cardiac septation and provide insights into the establishment of atrial asymmetry., SCOPUS: ar.j, info:eu-repo/semantics/published

Tbx20 is a T-box transcription factor that plays essential roles in the development and maintenance of the heart. Although it is expressed by cardiac progenitors in all species examined, an involvement of Tbx20 in cardiac progenitor formation in vertebrates has not been previously described. Here we report the identification of a zebrafish tbx20 mutation that results in an inactive, truncated protein lacking any functional domains. The cardiac progenitor population is strongly diminished in this mutant, leading to the formation of a small, stretched-out heart. We found that overexpression of Tbx20 results in an enlarged heart with significantly more cardiomyocytes. Interestingly, this increase in cell number is caused by both enhanced cardiac progenitor cell formation and the proliferation of differentiated cardiomyocytes, and is dependent upon the activity of Tbx20's T-box and transcription activation domains. Together, our findings highlight a previously unappreciated role for Tbx20 in promoting cardiac progenitor formation in vertebrates and reveal a novel function for its activation domain in cardiac cell proliferation during embryogenesis.

Subtype-specific human cardiomyocytes (CMs) are valuable for basic and applied research. Induction of cardiomyogenesis and enrichment of nodal-like CMs was described for mouse pluripotent stem cells (mPSCs) in response to 1-ethyl-2-benzimidazolinone (EBIO), a chemical modulator of small-/intermediate-conductance Ca2+-activated potassium channels (SKs 1–4). Investigating EBIO in human pluripotent stem cells (PSCs), we have applied three independent differentiation protocols of low to high cardiomyogenic efficiency. Equivalent to mPSCs, timed EBIO supplementation during hPSC differentiation resulted in dose-dependent enrichment of up to 80% CMs, including an increase in nodal- and atrial-like phenotypes. However, our study revealed extensive EBIO-triggered cell loss favoring cardiac progenitor preservation and, subsequently, CMs with shortened action potentials. Proliferative cells were generally more sensitive to EBIO, presumably via an SK-independent mechanism. Together, EBIO did not promote cardiogenic differentiation of PSCs, opposing previous findings, but triggered lineage-selective survival at a cardiac progenitor stage, which we propose as a pharmacological strategy to modulate CM subtype composition.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Mutation of highly conserved residues in transcription factors may affect protein-protein or protein-DNA interactions, leading to gene network dysregulation and human disease. Human mutations in GATA4, a cardiogenic transcription factor, cause cardiac septal defects and cardiomyopathy. Here, iPS-derived cardiomyocytes from subjects with a heterozygous GATA4-G296S missense mutation showed impaired contractility, calcium handling, and metabolic activity. In human cardiomyocytes, GATA4 broadly co-occupied cardiac enhancers with TBX5, another transcription factor that causes septal defects when mutated. The GATA4-G296S mutation disrupted TBX5 recruitment, particularly to cardiac super-enhancers, concomitant with dysregulation of genes related to the phenotypic abnormalities, including cardiac septation. Conversely, the GATA4-G296S mutation led to failure of GATA4 and TBX5-mediated repression at non-cardiac genes and enhanced open chromatin states at endothelial/endocardial promoters. These results reveal how disease-causing missense mutations can disrupt transcriptional cooperativity, leading to aberrant chromatin states and cellular dysfunction, including those related to morphogenetic defects.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

The study of the mechanisms leading to cardiac hypertrophy is essential to better understand cardiac development and regeneration. Pathological conditions such as ischemia or pressure overload can induce a release of extracellular nucleotides within the heart. We recently investigated the potential role of nucleotide P2Y receptors in cardiac development. We showed that adult P2Y4-null mice displayed microcardia resulting from defective cardiac angiogenesis. Here we show that loss of another P2Y subtype called P2Y6, a UDP receptor, was associated with a macrocardia phenotype and amplified pathological cardiac hypertrophy. Cardiomyocyte proliferation and size were increased in vivo in hearts of P2Y6-null neonates, resulting in enhanced post-natal heart growth. We then observed that loss of P2Y6 receptor enhanced pathological cardiac hypertrophy induced after isoproterenol injection. We identified an inhibitory effect of UDP on in vitro isoproterenol-induced cardiomyocyte hyperplasia and hypertrophy. The present study identifies mouse P2Y6 receptor as a regulator of cardiac development and cardiomyocyte function. P2Y6 receptor could constitute a therapeutic target to regulate cardiac hypertrophy.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Intrauterine growth restriction (IUGR) refers to the situation where a fetus does not grow according to its genetic growth potential. One of the main causes of IUGR is uteroplacental vascular insufficiency. Under these circumstances of chronic oxygen and nutrient deprivation, the growth-restricted fetus often displays typical circulatory changes, which in part represent adaptations to the suboptimal intrauterine environment. These fetal adaptations aim to preserve oxygen and nutrient supply to vital organs such as the brain, the heart, and the adrenals. These prenatal circulatory adaptations are thought to lead to an altered development of the cardiovascular system and "program" the fetus for life long cardiovascular morbidities. In this review, we discuss the alterations to cardiovascular structure, function, and control that have been observed in growth-restricted fetuses, neonates, and infants following uteroplacental vascular insufficiency. We also discuss the current knowledge on early life surveillance and interventions to prevent progression into chronic disease.Copyright © 2016 International Pediatric Research Foundation, Inc.

Introduction: Reduced blood flow into the fetal left ventricle (LV) may underlie development of Hypoplastic Left Heart Syndrome (HLHS), for which palliative surgery is the only treatment option. Currently there is no HLHS model in a large fetal mammalian animal for testing and clinical translation of new therapy. We have recently published a percutaneous trans-hepatic technique to catheterise the fetal heart, which could be utilised to produce an animal model of HLHS. Using a percutaneous trans-hepatic technique to deliver an occluder into the fetal heart, we aimed to create a fetal lamb model of HLHS by occluding the foramen ovale (FO). Method(s): Three fetal lambs (110-117days, term=147days) under maternal general anaesthesia were used. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured through the maternal abdomen using a 14GA IV-cannula. A coronary catheter and guidewire were inserted and advanced into the fetal IVC and right atrium. An Amplatzer Duct Occluder II (ADO II 4x4 mm, St Jude Medical) was delivered to occlude the fetal FO. In 2 fetuses, a self-expandable stent (8x12mm Superflex-DS 4Fr deliverable stent, Opti-Med Inc. Germany) was positioned across the FO first, and the ADO II was anchored within the stent. Serial ultrasounds were performed to monitor cardiac development. Euthanasia and post-mortem examination was performed 3 weeks after. A twin fetus which did not undergo the procedure was used as control for comparison. Morphometric measurements were made on digital images (Image J1.48b, NIH) of transverse sections of the heart at the level of atrioventricular valves. Result(s): All 3 fetal lambs survived well to 3 weeks without any fetal compromise. At post-mortem examination, the LV lumen was markedly reduced (Fig 1a) with lower LV/right ventricular (RV) chamber volume ratio, LV/RV lumen area ratio and increased septal thickness at level of atrioventricular valves (Table), compared with the control fetal heart (Fig 1b).

Intrauterine growth restriction (IUGR) refers to the situation where a fetus does not grow according to its genetic growth potential. One of the main causes of IUGR is uteroplacental vascular insufficiency. Under these circumstances of chronic oxygen and nutrient deprivation, the growth-restricted fetus often displays typical circulatory changes, which in part represent adaptations to the suboptimal intrauterine environment. These fetal adaptations aim to preserve oxygen and nutrient supply to vital organs such as the brain, the heart, and the adrenals. These prenatal circulatory adaptations are thought to lead to an altered development of the cardiovascular system and "program" the fetus for life long cardiovascular morbidities. In this review, we discuss the alterations to cardiovascular structure, function, and control that have been observed in growth-restricted fetuses, neonates, and infants following uteroplacental vascular insufficiency. We also discuss the current knowledge on early life surveillance and interventions to prevent progression into chronic disease.Copyright © 2016 International Pediatric Research Foundation, Inc.

Introduction: Reduced blood flow into the fetal left ventricle (LV) may underlie development of Hypoplastic Left Heart Syndrome (HLHS), for which palliative surgery is the only treatment option. Currently there is no HLHS model in a large fetal mammalian animal for testing and clinical translation of new therapy. We have recently published a percutaneous trans-hepatic technique to catheterise the fetal heart, which could be utilised to produce an animal model of HLHS. Using a percutaneous trans-hepatic technique to deliver an occluder into the fetal heart, we aimed to create a fetal lamb model of HLHS by occluding the foramen ovale (FO). Method(s): Three fetal lambs (110-117days, term=147days) under maternal general anaesthesia were used. Under ultrasound guidance, the fetal hepatic vein was percutaneously punctured through the maternal abdomen using a 14GA IV-cannula. A coronary catheter and guidewire were inserted and advanced into the fetal IVC and right atrium. An Amplatzer Duct Occluder II (ADO II 4x4 mm, St Jude Medical) was delivered to occlude the fetal FO. In 2 fetuses, a self-expandable stent (8x12mm Superflex-DS 4Fr deliverable stent, Opti-Med Inc. Germany) was positioned across the FO first, and the ADO II was anchored within the stent. Serial ultrasounds were performed to monitor cardiac development. Euthanasia and post-mortem examination was performed 3 weeks after. A twin fetus which did not undergo the procedure was used as control for comparison. Morphometric measurements were made on digital images (Image J1.48b, NIH) of transverse sections of the heart at the level of atrioventricular valves. Result(s): All 3 fetal lambs survived well to 3 weeks without any fetal compromise. At post-mortem examination, the LV lumen was markedly reduced (Fig 1a) with lower LV/right ventricular (RV) chamber volume ratio, LV/RV lumen area ratio and increased septal thickness at level of atrioventricular valves (Table), compared with the control fetal heart (Fig 1b).

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

The cardiovascular system in adult organisms forms a network of interconnected endothelial cells, supported by mural cells and displaying a high degree of hierarchy: arteries emerging from the heart ramify into arterioles and then capillaries, which return to the venous systems through venules and veins. The cardiovascular system allows blood circulation, which in turn is essential for hemostasis through gas diffusion, nutrient distribution, and cell trafficking. In this chapter, we have summarized the current knowledge on how adhesion GPCRs (aGPCRs) impact heart development, followed by their role in modulating vascular angiogenesis.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHFrestricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

Congenital heart defects are the most common birth defects in humans, and those that affect the proper alignment of the outflow tracts and septation of the ventricles are a highly significant cause of morbidity and mortality in infants. A late differentiating population of cardiac progenitors, referred to as the anterior second heart field (AHF), gives rise to the outflow tract and the majority of the right ventricle and provides an embryological context for understanding cardiac outflow tract alignment and membranous ventricular septal defects. However, the transcriptional pathways controlling AHF development and their roles in congenital heart defects remain incompletely elucidated. Here, we inactivated the gene encoding the transcription factor MEF2C in the AHF in mice. Loss of Mef2c function in the AHF results in a spectrum of outflow tract alignment defects ranging from overriding aorta to double-outlet right ventricle and dextro-transposition of the great arteries. We identify Tdgf1, which encodes a Nodal co-receptor (also known as Cripto), as a direct transcriptional target of MEF2C in the outflow tract via an AHF-restricted Tdgf1 enhancer. Importantly, both the MEF2C and TDGF1 genes are associated with congenital heart defects in humans. Thus, these studies establish a direct transcriptional pathway between the core cardiac transcription factor MEF2C and the human congenital heart disease gene TDGF1. Moreover, we found a range of outflow tract alignment defects resulting from a single genetic lesion, supporting the idea that AHF-derived outflow tract alignment defects may constitute an embryological spectrum rather than distinct anomalies.

The development of cardiac and somatic muscle is coordinated by many factors that are highly conserved across species. Mutations in the coding or regulatory sequences of a gene can alter the structure, function or levels of expression of the protein it encodes. We have identified the genetic mechanism underlying the temperature sensitive mutation in MEF2 (myocyte enhancer factor 2), which is required for the differentiation of all muscle lineages. In addition, we have identified a regulatory element upstream of the Mef2 gene that directs its expression from stage fourteen through the end of embryogenesis which is activated by the NK-homeobox transcription factor Tinman and the GATA transcription factor Pannier. Additionally, we have found that Tinman and Pannier work in collaboration with MEF2 to activate the entire cardiac program. Finally, we have purified cardiac cells from Drosophila embryos and have paired-end sequenced their mRNA in order to identify novel factors that contribute to heart development.

Impaired heparan sulfate (HS) synthesis in vertebrate development causes complex malformations due to the functional disruption of multiple HS-binding growth factors and morphogens. Here, we report developmental heart defects in mice bearing a targeted disruption of the HS-generating enzyme GlcNAc N-deacetylase/GlcN N-sulfotransferase 1 (NDST1), including ventricular septal defects (VSD), persistent truncus arteriosus (PTA), double outlet right ventricle (DORV), and retroesophageal right subclavian artery (RERSC). These defects closely resemble cardiac anomalies observed in mice made deficient in the cardiogenic regulator fibroblast growth factor 8 (FGF8). Consistent with this, we show that HS-dependent FGF8/FGF-receptor2C assembly and FGF8-dependent ERK-phosphorylation are strongly reduced in NDST1(-/-) embryonic cells and tissues. Moreover, WNT1-Cre/LoxP-mediated conditional targeting of NDST function in neural crest cells (NCCs) revealed that their impaired HS-dependent development contributes strongly to the observed cardiac defects. These findings raise the possibility that defects in HS biosynthesis may contribute to congenital heart defects in humans that represent the most common type of birth defect.

With defined culture protocol, human embryonic stem cells (hESCs) are able to generate cardiomyocytes in vitro, therefore providing a great model for human heart development, and holding great potential for cardiac disease therapies. In this study, we successfully generated a highly pure population of human cardiomyocytes (hCMs) (>95% cTnT(+)) from hESC line, which enabled us to identify and characterize an hCM-specific signature, at both the gene expression and DNA methylation levels. Gene functional association network and gene-disease network analyses of these hCM-enriched genes provide new insights into the mechanisms of hCM transcriptional regulation, and stand as an informative and rich resource for investigating cardiac gene functions and disease mechanisms. Moreover, we show that cardiac-structural genes and cardiac-transcription factors have distinct epigenetic mechanisms to regulate their gene expression, providing a better understanding of how the epigenetic machinery coordinates to regulate gene expression in different cell types.