Integrative single-cell analysis of cardiogenesis identifies developmental trajectories and non-coding mutations in congenital heart disease.

To define the multi-cellular epigenomic and transcriptional landscape of cardiac cellular development, we generated single-cell chromatin accessibility maps of human fetal heart tissues. We identified eight major differentiation trajectories involving primary cardiac cell types, each associated with dynamic transcription factor (TF) activity signatures. We contrasted regulatory landscapes of iPSC-derived cardiac cell types and their in vivo counterparts, which enabled optimization of in vitro differentiation of epicardial cells. Further, we interpreted sequence based deep learning models of cell-type-resolved chromatin accessibility profiles to decipher underlying TF motif lexicons. De novo mutations predicted to affect chromatin accessibility in arterial endothelium were enriched in congenital heart disease (CHD) cases vs. controls. In vitro studies in iPSCs validated the functional impact of identified variation on the predicted developmental cell types. This work thus defines the cell-type-resolved cis-regulatory sequence determinants of heart development and identifies disruption of cell type-specific regulatory elements in CHD.

Transcription factor protein interactomes reveal genetic determinants in heart disease.

Congenital heart disease (CHD) is present in 1% of live births, yet identification of causal mutations remains challenging. We hypothesized that genetic determinants for CHDs may lie in the protein interactomes of transcription factors whose mutations cause CHDs. Defining the interactomes of two transcription factors haplo-insufficient in CHD, GATA4 and TBX5, within human cardiac progenitors, and integrating the results with nearly 9,000 exomes from proband-parent trios revealed an enrichment of de novo missense variants associated with CHD within the interactomes. Scoring variants of interactome members based on residue, gene, and proband features identified likely CHD-causing genes, including the epigenetic reader GLYR1. GLYR1 and GATA4 widely co-occupied and co-activated cardiac developmental genes, and the identified GLYR1 missense variant disrupted interaction with GATA4, impairing in vitro and in vivo function in mice. This integrative proteomic and genetic approach provides a framework for prioritizing and interrogating genetic variants in heart disease.

Radical S-Adenosylmethionine Enzymes in Human Health and Disease.

Radical S-adenosylmethionine (SAM) enzymes catalyze an astonishing array of complex and chemically challenging reactions across all domains of life. Of approximately 114,000 of these enzymes, 8 are known to be present in humans: MOCS1, molybdenum cofactor biosynthesis; LIAS, lipoic acid biosynthesis; CDK5RAP1, 2-methylthio-N(6)-isopentenyladenosine biosynthesis; CDKAL1, methylthio-N(6)-threonylcarbamoyladenosine biosynthesis; TYW1, wybutosine biosynthesis; ELP3, 5-methoxycarbonylmethyl uridine; and RSAD1 and viperin, both of unknown function. Aberrations in the genes encoding these proteins result in a variety of diseases. In this review, we summarize the biochemical characterization of these 8 radical S-adenosylmethionine enzymes and, in the context of human health, describe the deleterious effects that result from such genetic mutations.

Disease Model of GATA4 Mutation Reveals Transcription Factor Cooperativity in Human Cardiogenesis.

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.

Modeling congenital heart disease: lessons from mice, hPSC-based models, and organoids.

Congenital heart defects (CHDs) are among the most common birth defects, but their etiology has long been mysterious. In recent decades, the development of a variety of experimental models has led to a greater understanding of the molecular basis of CHDs. In this review, we contrast mouse models of CHD, which maintain the anatomical arrangement of the heart, and human cellular models of CHD, which are more likely to capture human-specific biology but lack anatomical structure. We also discuss the recent development of cardiac organoids, which are a promising step toward more anatomically informative human models of CHD.

Integrated multi-omic characterization of congenital heart disease.

The heart, the first organ to develop in the embryo, undergoes complex morphogenesis that when defective results in congenital heart disease (CHD). With current therapies, more than 90% of patients with CHD survive into adulthood, but many suffer premature death from heart failure and non-cardiac causes1. Here, to gain insight into this disease progression, we performed single-nucleus RNA sequencing on 157,273 nuclei from control hearts and hearts from patients with CHD, including those with hypoplastic left heart syndrome (HLHS) and tetralogy of Fallot, two common forms of cyanotic CHD lesions, as well as dilated and hypertrophic cardiomyopathies. We observed CHD-specific cell states in cardiomyocytes, which showed evidence of insulin resistance and increased expression of genes associated with FOXO signalling and CRIM1. Cardiac fibroblasts in HLHS were enriched in a low-Hippo and high-YAP cell state characteristic of activated cardiac fibroblasts. Imaging mass cytometry uncovered a spatially resolved perivascular microenvironment consistent with an immunodeficient state in CHD. Peripheral immune cell profiling suggested deficient monocytic immunity in CHD, in agreement with the predilection in CHD to infection and cancer2. Our comprehensive phenotyping of CHD provides a roadmap towards future personalized treatments for CHD.

STIGMA: Single-cell tissue-specific gene prioritization using machine learning.

Clinical exome and genome sequencing have revolutionized the understanding of human disease genetics. Yet many genes remain functionally uncharacterized, complicating the establishment of causal disease links for genetic variants. While several scoring methods have been devised to prioritize these candidate genes, these methods fall short of capturing the expression heterogeneity across cell subpopulations within tissues. Here, we introduce single-cell tissue-specific gene prioritization using machine learning (STIGMA), an approach that leverages single-cell RNA-seq (scRNA-seq) data to prioritize candidate genes associated with rare congenital diseases. STIGMA prioritizes genes by learning the temporal dynamics of gene expression across cell types during healthy organogenesis. To assess the efficacy of our framework, we applied STIGMA to mouse limb and human fetal heart scRNA-seq datasets. In a cohort of individuals with congenital limb malformation, STIGMA prioritized 469 variants in 345 genes, with UBA2 as a notable example. For congenital heart defects, we detected 34 genes harboring nonsynonymous de novo variants (nsDNVs) in two or more individuals from a set of 7,958 individuals, including the ortholog of Prdm1, which is associated with hypoplastic left ventricle and hypoplastic aortic arch. Overall, our findings demonstrate that STIGMA effectively prioritizes tissue-specific candidate genes by utilizing single-cell transcriptome data. The ability to capture the heterogeneity of gene expression across cell populations makes STIGMA a powerful tool for the discovery of disease-associated genes and facilitates the identification of causal variants underlying human genetic disorders.

Functional analysis of germline VANGL2 variants using rescue assays of vangl2 knockout zebrafish.

Developmental studies have shown that the evolutionarily conserved Wnt Planar Cell Polarity (PCP) pathway is essential for the development of a diverse range of tissues and organs including the brain, spinal cord, heart and sensory organs, as well as establishment of the left-right body axis. Germline mutations in the highly conserved PCP gene VANGL2 in humans have only been associated with central nervous system malformations, and functional testing to understand variant impact has not been performed. Here we report three new families with missense variants in VANGL2 associated with heterotaxy and congenital heart disease p.(Arg169His), non-syndromic hearing loss p.(Glu465Ala) and congenital heart disease with brain defects p.(Arg135Trp). To test the in vivo impact of these and previously described variants, we have established clinically-relevant assays using mRNA rescue of the vangl2 mutant zebrafish. We show that all variants disrupt Vangl2 function, although to different extents and depending on the developmental process. We also begin to identify that different VANGL2 missense variants may be haploinsufficient and discuss evidence in support of pathogenicity. Together, this study demonstrates that zebrafish present a suitable pipeline to investigate variants of unknown significance and suggests new avenues for investigation of the different developmental contexts of VANGL2 function that are clinically meaningful.

Autonomous and non-cell autonomous role of cilia in structural birth defects in mice.

Ciliopathies are associated with wide spectrum of structural birth defects (SBDs), indicating important roles for cilia in development. Here, we provide novel insights into the temporospatial requirement for cilia in SBDs arising from deficiency in Ift140, an intraflagellar transport (IFT) protein regulating ciliogenesis. Ift140-deficient mice exhibit cilia defects accompanied by wide spectrum of SBDs including macrostomia (craniofacial defects), exencephaly, body wall defects, tracheoesophageal fistula (TEF), randomized heart looping, congenital heart defects (CHDs), lung hypoplasia, renal anomalies, and polydactyly. Tamoxifen inducible CAGGCre-ER deletion of a floxed Ift140 allele between E5.5 to 9.5 revealed early requirement for Ift140 in left-right heart looping regulation, mid to late requirement for cardiac outflow septation and alignment, and late requirement for craniofacial development and body wall closure. Surprisingly, CHD were not observed with 4 Cre drivers targeting different lineages essential for heart development, but craniofacial defects and omphalocele were observed with Wnt1-Cre targeting neural crest and Tbx18-Cre targeting epicardial lineage and rostral sclerotome through which trunk neural crest cells migrate. These findings revealed cell autonomous role of cilia in cranial/trunk neural crest-mediated craniofacial and body wall closure defects, while non-cell autonomous multi-lineage interactions underlie CHD pathogenesis, revealing unexpected developmental complexity for CHD associated with ciliopathies.

Buffering Mechanism in Aortic Arch Artery Formation and Congenital Heart Disease.

BACKGROUND: The resiliency of embryonic development to genetic and environmental perturbations has been long appreciated; however, little is known about the mechanisms underlying the robustness of developmental processes. Aberrations resulting in neonatal lethality are exemplified by congenital heart disease arising from defective morphogenesis of pharyngeal arch arteries (PAAs) and their derivatives. METHODS: Mouse genetics, lineage tracing, confocal microscopy, and quantitative image analyses were used to investigate mechanisms of PAA formation and repair. RESULTS: The second heart field (SHF) gives rise to the PAA endothelium. Here, we show that the number of SHF-derived endothelial cells (ECs) is regulated by VEGFR2 (vascular endothelial growth factor receptor 2) and Tbx1. Remarkably, when the SHF-derived EC number is decreased, PAA development can be rescued by the compensatory endothelium. Blocking such compensatory response leads to embryonic demise. To determine the source of compensating ECs and mechanisms regulating their recruitment, we investigated 3-dimensional EC connectivity, EC fate, and gene expression. Our studies demonstrate that the expression of VEGFR2 by the SHF is required for the differentiation of SHF-derived cells into PAA ECs. The deletion of 1 VEGFR2 allele (VEGFR2SHF-HET) reduces SHF contribution to the PAA endothelium, while the deletion of both alleles (VEGFR2SHF-KO) abolishes it. The decrease in SHF-derived ECs in VEGFR2SHF-HET and VEGFR2SHF-KO embryos is complemented by the recruitment of ECs from the nearby veins. Compensatory ECs contribute to PAA derivatives, giving rise to the endothelium of the aortic arch and the ductus in VEGFR2SHF-KO mutants. Blocking the compensatory response in VEGFR2SHF-KO mutants results in embryonic lethality shortly after mid-gestation. The compensatory ECs are absent in Tbx1+/- embryos, a model for 22q11 deletion syndrome, leading to unpredictable arch artery morphogenesis and congenital heart disease. Tbx1 regulates the recruitment of the compensatory endothelium in an SHF-non-cell-autonomous manner. CONCLUSIONS: Our studies uncover a novel buffering mechanism underlying the resiliency of PAA development and remodeling.

Evolutionary history of MEK1 illuminates the nature of deleterious mutations.

Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in the MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the cardiofaciocutaneous syndrome and arteriovenous malformation. Evaluating variants associated with a disease is a challenge, and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1, we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We matched known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions and found that all known and many suspected disease-causing mutations are evolutionarily intolerable. We selected several variants that cannot be unambiguously assessed by automated prediction tools but that are confidently identified as "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation.

Pleiotropic role of TRAF7 in skull-base meningiomas and congenital heart disease.

While somatic variants of TRAF7 (Tumor necrosis factor receptor-associated factor 7) underlie anterior skull-base meningiomas, here we report the inherited mutations of TRAF7 that cause congenital heart defects. We show that TRAF7 mutants operate in a dominant manner, inhibiting protein function via heterodimerization with wild-type protein. Further, the shared genetics of the two disparate pathologies can be traced to the common origin of forebrain meninges and cardiac outflow tract from the TRAF7-expressing neural crest. Somatic and inherited mutations disrupt TRAF7-IFT57 interactions leading to cilia degradation. TRAF7-mutant meningioma primary cultures lack cilia, and TRAF7 knockdown causes cardiac, craniofacial, and ciliary defects in Xenopus and zebrafish, suggesting a mechanistic convergence for TRAF7-driven meningiomas and developmental heart defects.

Mutations in Bcl9 and Pygo genes cause congenital heart defects by tissue-specific perturbation of Wnt/ß-catenin signaling.

Bcl9 and Pygopus (Pygo) are obligate Wnt/ß-catenin cofactors in Drosophila, yet their contribution to Wnt signaling during vertebrate development remains unresolved. Combining zebrafish and mouse genetics, we document a conserved, ß-catenin-associated function for BCL9 and Pygo proteins during vertebrate heart development. Disrupting the ß-catenin-BCL9-Pygo complex results in a broadly maintained canonical Wnt response yet perturbs heart development and proper expression of key cardiac regulators. Our work highlights BCL9 and Pygo as selective ß-catenin cofactors in a subset of canonical Wnt responses during vertebrate development. Moreover, our results implicate alterations in BCL9 and BCL9L in human congenital heart defects.

Role of Epigenetics in Cardiac Development and Congenital Diseases.

The heart is the first organ to be functional in the fetus. Heart formation is a complex morphogenetic process regulated by both genetic and epigenetic mechanisms. Congenital heart diseases (CHD) are the most prominent congenital diseases. Genetics is not sufficient to explain these diseases or the impact of them on patients. Epigenetics is more and more emerging as a basis for cardiac malformations. This review brings the essential knowledge on cardiac biology of development. It further provides a broad background on epigenetics with a focus on three-dimensional conformation of chromatin. Then, we summarize the current knowledge of the impact of epigenetics on cardiac cell fate decision. We further provide an update on the epigenetic anomalies in the genesis of CHD.

Assigning pathogenicity for TAB2 variants using a novel scalable functional assay and expanding TAB2 disease spectrum.

Haploinsufficiency of TGF-beta-activated kinase 1 (MAP3K7) binding protein 2 (TAB2) has been associated with congenital heart disease and more recently multiorgan structural abnormalities. Missense variant represents a major proportion of non-synonymous TAB2 variants reported in gnomAD (295/576) and Clinvar (16/73), most of which are variants of uncertain significance (VUSs). However, interpretation of TAB2 missense variants remains challenging because of lack of functional assays. To address this issue, we established a cell-based luciferase assay that enables high-throughput screening of TAB2 variants to assess the functional consequence for predicting variant pathogenicity. Using this platform, we screened 47 TAB2 variants including five pathogenic controls and one benign control, and the results showed that the transcriptional activity of activator protein 1 (AP-1) but not nuclear factor kappa B predicts the TAB2 variant pathogenicity. This assay provides accurate functional readout for both loss-of-function (LOF) and gain-of-function variants, which are associated with distinct phenotypes. In all, 22 out of 32 tested VUSs were reclassified. Genotype-Phenotype association showed that most patients with partial LOF variants do not exhibit congenital heart disease but high frequency of developmental delay, hypotonia and dysmorphic features, which suggests that genetic testing for TAB2 is needed for a broader spectrum of patients with more diverse phenotypes. Molecular modeling with Npl4 zinc finger (NZF) domain variants revealed that the stability of the NZF domain in TAB2 protein is crucial for AP-1 activation. In conclusion, we developed a highly effective functional assay for TAB2 variant prediction and interpretation.

Congenital heart defects caused by FOXJ1.

FOXJ1 is expressed in ciliated cells of the airways, testis, oviduct, central nervous system and the embryonic left-right organizer. Ablation or targeted mutation of Foxj1 in mice, zebrafish and frogs results in loss of ciliary motility and/or reduced length and number of motile cilia, affecting the establishment of the left-right axis. In humans, heterozygous pathogenic variants in FOXJ1 cause ciliopathy leading to situs inversus, obstructive hydrocephalus and chronic airway disease. Here, we report a novel truncating FOXJ1 variant (c.784_799dup; p.Glu267Glyfs*12) identified by clinical exome sequencing from a patient with isolated congenital heart defects (CHD) which included atrial and ventricular septal defects, double outlet right ventricle (DORV) and transposition of the great arteries. Functional experiments show that FOXJ1 c.784_799dup; p.Glu267Glyfs*12, unlike FOXJ1, fails to induce ectopic cilia in frog epidermis in vivo or to activate the ADGB promoter, a downstream target of FOXJ1 in cilia, in transactivation assays in vitro. Variant analysis of patients with heterotaxy or heterotaxy-related CHD indicates that pathogenic variants in FOXJ1 are an infrequent cause of heterotaxy. Finally, we characterize embryonic-stage CHD in Foxj1 loss-of-function mice, demonstrating randomized heart looping. Abnormal heart looping includes reversed looping (dextrocardia), ventral looping and no looping/single ventricle hearts. Complex CHDs revealed by histological analysis include atrioventricular septal defects, DORV, single ventricle defects as well as abnormal position of the great arteries. These results indicate that pathogenic variants in FOXJ1 can cause isolated CHD.

SOX7 deficiency causes ventricular septal defects through its effects on endocardial-to-mesenchymal transition and the expression of Wnt4 and Bmp2.

SOX7 is a transcription factor-encoding gene located in a region on chromosome 8p23.1 that is recurrently deleted in individuals with ventricular septal defects (VSDs). We have previously shown that Sox7-/- embryos die of heart failure around E11.5. Here, we demonstrate that these embryos have hypocellular endocardial cushions with severely reduced numbers of mesenchymal cells. Ablation of Sox7 in the endocardium also resulted in hypocellular endocardial cushions, and we observed VSDs in rare E15.5 Sox7flox/-;Tie2-Cre and Sox7flox/flox;Tie2-Cre embryos that survived to E15.5. In atrioventricular explant studies, we showed that SOX7 deficiency leads to a severe reduction in endocardial-to-mesenchymal transition (EndMT). RNA-seq studies performed on E9.5 Sox7-/- heart tubes revealed severely reduced Wnt4 transcript levels. Wnt4 is expressed in the endocardium and promotes EndMT by acting in a paracrine manner to increase the expression of Bmp2 in the myocardium. Both WNT4 and BMP2 have been previously implicated in the development of VSDs in individuals with 46,XX sex reversal with dysgenesis of kidney, adrenals and lungs (SERKAL) syndrome and in individuals with short stature, facial dysmorphism and skeletal anomalies with or without cardiac anomalies 1 (SSFSC1) syndrome, respectively. We now show that Sox7 and Wnt4 interact genetically in the development of VSDs through their additive effects on endocardial cushion development with Sox7+/-;Wnt4+/- double heterozygous embryos having hypocellular endocardial cushions and perimembranous and muscular VSDs not seen in their Sox7+/- and Wnt4+/- littermates. These results provide additional evidence that SOX7, WNT4 and BMP2 function in the same pathway during mammalian septal development and that their deficiency can contribute to the development of VSDs in humans.

Neither cardiac mitochondrial DNA variation nor copy number contribute to congenital heart disease risk.

The well-established manifestation of mitochondrial mutations in functional cardiac disease (e.g., mitochondrial cardiomyopathy) prompted the hypothesis that mitochondrial DNA (mtDNA) sequence and/or copy number (mtDNAcn) variation contribute to cardiac defects in congenital heart disease (CHD). MtDNAcns were calculated and rare, non-synonymous mtDNA mutations were identified in 1,837 CHD-affected proband-parent trios, 116 CHD-affected singletons, and 114 paired cardiovascular tissue/blood samples. The variant allele fraction (VAF) of heteroplasmic variants in mitochondrial RNA from 257 CHD cardiovascular tissue samples was also calculated. On average, mtDNA from blood had 0.14 rare variants and 52.9 mtDNA copies per nuclear genome per proband. No variation with parental age at proband birth or CHD-affected proband age was seen. mtDNAcns in valve/vessel tissue (320 ± 70) were lower than in atrial tissue (1,080 ± 320, p = 6.8E-21), which were lower than in ventricle tissue (1,340 ± 280, p = 1.4E-4). The frequency of rare variants in CHD-affected individual DNA was indistinguishable from the frequency in an unaffected cohort, and proband mtDNAcns did not vary from those of CHD cohort parents. In both the CHD and the comparison cohorts, mtDNAcns were significantly correlated between mother-child, father-child, and mother-father. mtDNAcns among people with European (mean = 52.0), African (53.0), and Asian haplogroups (53.5) were calculated and were significantly different for European and Asian haplogroups (p = 2.6E-3). Variant heteroplasmic fraction (HF) in blood correlated well with paired cardiovascular tissue HF (r = 0.975) and RNA VAF (r = 0.953), which suggests blood HF is a reasonable proxy for HF in heart tissue. We conclude that mtDNA mutations and mtDNAcns are unlikely to contribute significantly to CHD risk.

Congenital septal defects in Karachi, Pakistan: an update of mutational screening by high-resolution melting (HRM) analysis of MTHFR C677T.

BACKGROUND: Congenital heart defects (CHDs) are the heart structural malformations present at birth. Septal defects account for 40% of CHD, including atrial, ventricular and atrioventricular septal defects. In Pakistan, the prevalence of CHD is 3.4 in 1000, and a study estimated that 60,000 babies are born with CHD annually. Methylenetetrahydrofolate reductase (MTHFR), a chief enzyme, involved in the folate metabolism. The missense mutation, C677T (rs1801133), exists in MTHFR gene, results in a MTHFR thermolabile variant having low enzymatic activity. The study is aim to identify the MTHFR C677T variant association with septal defects. METHODS: Samples of 194 CHD patients (age [Formula: see text]= 5.8 ± 5.1) and 50 normal echo controls (age [Formula: see text]= 6.0 ± 4.9), confirmed by pediatric consultant, were collected. Extracted DNA, quantified by agarose gel electrophoresis and nanodrop, was screened for SNP by high-resolution melting (HRM). Further, HRM results were confirmed using restriction analysis and sequencing. HRM was simply and precisely genotyped the samples within 3 h at low cost. RESULTS: Genotypic data suggested that heterozygous mutant (CT) was frequent in congenital septal defect patients (0.26) which was higher than controls (0.143), p > 0.05. Mutant (TT) genotype was not found in this study. CONCLUSIONS: rs1801133 has lack of significant association with congenital septal defects. The absence of TT genotype in this study suggesting the role of natural selection in targeted population. HRM is an easy, fast and next generation of PCR, which may be used for applied genomics.

Single-cell analysis of cardiogenesis reveals basis for organ-level developmental defects.

Organogenesis involves integration of diverse cell types; dysregulation of cell-type-specific gene networks results in birth defects, which affect 5% of live births. Congenital heart defects are the most common malformations, and result from disruption of discrete subsets of cardiac progenitor cells1, but the transcriptional changes in individual progenitors that lead to organ-level defects remain unknown. Here we used single-cell RNA sequencing to interrogate early cardiac progenitor cells as they become specified during normal and abnormal cardiogenesis, revealing how dysregulation of specific cellular subpopulations has catastrophic consequences. A network-based computational method for single-cell RNA-sequencing analysis that predicts lineage-specifying transcription factors2,3 identified Hand2 as a specifier of outflow tract cells but not right ventricular cells, despite the failure of right ventricular formation in Hand2-null mice4. Temporal single-cell-transcriptome analysis of Hand2-null embryos revealed failure of outflow tract myocardium specification, whereas right ventricular myocardium was specified but failed to properly differentiate and migrate. Loss of Hand2 also led to dysregulation of retinoic acid signalling and disruption of anterior-posterior patterning of cardiac progenitors. This work reveals transcriptional determinants that specify fate and differentiation in individual cardiac progenitor cells, and exposes mechanisms of disrupted cardiac development at single-cell resolution, providing a framework for investigating congenital heart defects.