Congenital heart defects (CHDs) are the most common developmental anomaly and are the leading non-infectious cause of mortality in newborns. Only one causative gene, NKX2-5, has been identified through genetic linkage analysis of pedigrees with non-syndromic CHDs. Here, we show that isolated cardiac septal defects in a large pedigree were linked to chromosome 8p22-23. A heterozygous G296S missense mutation of GATA4, a transcription factor essential for heart formation, was found in all available affected family members but not in any control individuals. This mutation resulted in diminished DNA-binding affinity and transcriptional activity of Gata4. Furthermore, the Gata4 mutation abrogated a physical interaction between Gata4 and TBX5, a T-box protein responsible for a subset of syndromic cardiac septal defects. Conversely, interaction of Gata4 and TBX5 was disrupted by specific human TBX5 missense mutations that cause similar cardiac septal defects. In a second family, we identified a frame-shift mutation of GATA4 (E359del) that was transcriptionally inactive and segregated with cardiac septal defects. These results implicate GATA4 as a genetic cause of human cardiac septal defects, perhaps through its interaction with TBX5.

Activation of complex molecular programs in specific cell lineages governs mammalian heart development, from a primordial linear tube to a four-chamber organ. To characterize lineage-specific, temporal-spatial developmental programs, we performed single-cell RNA sequencing of >1200 murine cells isolated at seven time points spanning E9.5 (primordial heart tube) to P21 (mature heart). Using unbiased transcriptional data we classified cardiomyocytes (CM), endothelial cells (EC), and fibroblast-enriched cells, thus identifying markers for temporal and chamber-specific developmental programs. Harnessing these datasets, we defined developmental ages of human and mouse pluripotent stem cell-derived CMs and characterized lineage-specific maturation defects in hearts of mice with heterozygous mutations in Nkx2.5 that cause human heart malformations. This spatial-temporal transcriptome analysis of heart development reveals lineage-specific gene programs underlying normal cardiac development and congenital heart disease.

Appropriate interactions between the epithelium and adjacent neural crest-derived mesenchyme are necessary for normal pharyngeal arch development. Disruption of pharyngeal arch development in humans underlies many of the craniofacial defects observed in the 22q11.2 deletion syndrome (del22q11), but the genes responsible remain unknown. Tbx1 is a T-box transcription factor that lies in the 22q11.2 locus. Tbx1 transcripts were found to be localized to the pharyngeal endoderm and the mesodermal core of the pharyngeal arches, but were not present in the neural crest-derived mesenchyme of the pharyngeal arches. Sonic hedgehog (Shh) is also expressed in the pharyngeal arches and is necessary for normal craniofacial development. We found that Tbx1 expression was dependent upon Shh signaling in mouse embryos, consistent with their overlapping expression in the pharyngeal arches. Furthermore, Shh was sufficient to induce Tbx1 expression when misexpressed in selected regions of chick embryos. These studies reveal a Shh-mediated pathway that regulates Tbx1 during pharyngeal arch development.

Human mutations in TBX5, a gene encoding a T-box transcription factor, and SALL4, a gene encoding a zinc-finger transcription factor, cause similar upper limb and heart defects. Here we show that Tbx5 regulates Sall4 expression in the developing mouse forelimb and heart; mice heterozygous for a gene trap allele of Sall4 show limb and heart defects that model human disease. Tbx5 and Sall4 interact both positively and negatively to finely regulate patterning and morphogenesis of the anterior forelimb and heart. Thus, a positive and negative feed-forward circuit between Tbx5 and Sall4 ensures precise patterning of embryonic limb and heart and provides a unifying mechanism for heart/hand syndromes.