The vertebrate heart is formed from diverse embryonic territories, including the first and second heart fields. The second heart field (SHF) gives rise to the right ventricle and outflow tract, yet its evolutionary origins are unclear. We found that heart progenitor cells of the simple chordate Ciona intestinalis also generate precursors of the atrial siphon muscles (ASMs). These precursors express Islet and Tbx1/10, evocative of the splanchnic mesoderm that produces the lower jaw muscles and SHF of vertebrates. Evidence is presented that the transcription factor COE is a critical determinant of ASM fate. We propose that the last common ancestor of tunicates and vertebrates possessed multipotent cardiopharyngeal muscle precursors, and that their reallocation might have contributed to the emergence of the SHF.

To elucidate the function of the T-box transcription factor Tbx20 in mammalian development, we generated a graded loss-of-function series by transgenic RNA interference in entirely embryonic stem cell-derived mouse embryos. Complete Tbx20 knockdown resulted in defects in heart formation, including hypoplasia of the outflow tract and right ventricle,which derive from the anterior heart field (AHF), and decreased expression of Nkx2-5 and Mef2c, transcription factors required for AHF formation. A mild knockdown led to persistent truncus arteriosus (unseptated outflow tract) and hypoplastic right ventricle, entities similar to human congenital heart defects, and demonstrated a critical requirement for Tbx20 in valve formation. Finally, an intermediate knockdown revealed a role for Tbx20 in motoneuron development, specifically in the regulation of the transcription factors Isl2 and Hb9, which are important for terminal differentiation of motoneurons. Tbx20 could activate promoters/enhancers of several genes in cultured cells, including the Mef2c AHF enhancer and the Nkx2-5 cardiac enhancer. The Mef2c AHF enhancer relies on Isl1- and Gata-binding sites. We identified a similar Isl1 binding site in the Nkx2-5 AHF enhancer,which in transgenic mouse embryos was essential for activity in a large part of the heart, including the outflow tract. Tbx20 synergized with Isl1 and Gata4 to activate both the Mef2c and Nkx2-5 enhancers, thus providing a unifying mechanism for gene activation by Tbx20 in the AHF. We conclude that Tbx20 is positioned at a critical node in transcription factor networks required for heart and motoneuron development where it dose-dependently regulates gene expression.

Dominant mutations in the T-box transcription factor gene TBX5 cause Holt-Oram syndrome (HOS), an inherited human disease characterized by upper limb malformations and congenital heart defects (CHDs) of variable severity. We hypothesize that minor alterations in the dosage of Tbx5 directly influences severity of CHDs. Using a mouse allelic series, we show a sensitive inverse correlation between Tbx5 dosage and abnormal cardiac morphogenesis and gene expression. The CHDs found in mice harbouring a hypomorphic allele of Tbx5 (Tbx5(lox/+) mice) are less pronounced than those found in Tbx5 haploinsufficient mice (Tbx5(del/+)), and homozygous hypomorphic (Tbx5(lox/lox)) embryos have noticeably more advanced cardiac development than Tbx5 null (Tbx5(del/del)) embryos. Examination of target gene expression across the allelic series uncovers very fine sensitivity across the range of Tbx5 dosages, in which some genes respond dramatically differently to only 15% differences in Tbx5 mRNA levels. This analysis was expanded to a genome-wide level, which uncovered a Tbx5 dosage-sensitive genetic program involving a network of cardiac transcription factors, developmentally important cell-cell signaling molecules, and ion channel proteins. These results indicate an exquisite sensitivity of the developing heart to Tbx5 dosage and provide significant insight into the transcriptional and cellular mechanisms that are disrupted in CHDs.

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

The emergence of terrestrial life witnessed the need for more sophisticated circulatory systems. This has evolved in birds, mammals, and crocodilians into complete septation of the heart into left and right sides, allowing separate pulmonary and systemic circulatory systems, a key requirement for the evolution of endothermy1–3. However, the evolution of the amniote heart is poorly understood. Reptilian hearts have been the subject of debate in the context of the evolution of cardiac septation: do they possess a single ventricular chamber or two incompletely septated ventricles4–7? We examined heart development in the red-eared slider turtle, Trachemys scripta elegans (a chelonian), and the green anole, Anolis carolinensis (a squamate), focusing on gene expression in the developing ventricles. Both reptiles initially form a ventricular chamber that homogenously expresses the T-box transcription factor gene Tbx5. In contrast, in birds and mammals, Tbx5 is restricted to left ventricle precursors8,9. In later stages, Tbx5 expression in the turtle (but not anole) heart is gradually restricted to a distinct left ventricle, forming a left-right gradient. This suggests that Tbx5 expression was refined during evolution to pattern the ventricles. In support of this hypothesis, we show that loss of Tbx5 in the mouse ventricle results in a single chamber lacking distinct identity, indicating a requirement for Tbx5 in septation. Importantly, misexpression of Tbx5 throughout the developing myocardium to mimic the reptilian expression pattern also results in a single mispatterned ventricular chamber lacking septation. Thus, ventricular septation is established by a steep and correctly positioned Tbx5 gradient. Our findings provide a molecular mechanism for the evolution of the amniote ventricle, and support the concept that altered expression of developmental regulators is a key mechanism of vertebrate evolution.

Genetic analysis of patient populations and the biochemical characterization of the mutated proteins have provided considerable insight into the function of TBX5 in cardiac development and disease pathology. Novel discoveries await as these two paradigms merge.

Heart formation requires a highly balanced network of transcriptional activation of genes. The homeodomain transcription factor, Shox2, is essential for the formation of the sinoatrial valves and for the development of the pacemaking system. The elucidation of molecular mechanisms underlying the development of pacemaker tissue has gained clinical interest as defects in its patterning can be related to atrial arrhythmias. We have analyzed putative targets of Shox2 and identified the Bmp4 gene as a direct target. Shox2 interacts directly with the Bmp4 promoter in chromatin immunoprecipitation assays and activates transcription in luciferase-reporter assays. In addition, ectopic expression of Shox2 in Xenopus embryos stimulates transcription of the Bmp4 gene, and silencing of Shox2 in cardiomyocytes leads to a reduction in the expression of Bmp4. In Tbx5del/+ mice, a model for Holt-Oram syndrome, and Shox2−/− mice, we show that the T-box transcription factor Tbx5 is a regulator of Shox2 expression in the inflow tract and that Bmp4 is regulated by Shox2 in this compartment of the embryonic heart. In addition, we could show that Tbx5 acts cooperatively with Nkx2.5 to regulate the expression of Shox2 and Bmp4. This work establishes a link between Tbx5, Shox2 and Bmp4 in the pacemaker region of the developing heart and thus contributes to the unraveling of the intricate interplay between the heart-specific transcriptional machinery and developmental signaling pathways.

A simple remote ischemic preconditioning stimulus modifies myocardial gene expression by upregulating cardioprotective genes and suppressing genes potentially involved in the pathogenesis of ischemia-reperfusion injury.