The vertebrate heart arises from the fusion of bilateral regions of anterior mesoderm to form a linear heart tube. Recent studies in mouse and chick have demonstrated that a second cardiac progenitor population, known as the anterior or secondary heart field, is progressively added to the heart at the time of cardiac looping. While it is clear that this second field contributes to the myocardium, its precise boundaries, other lineages derived from this population, and its contributions to the postnatal heart remain unclear. In this study, we used regulatory elements from the mouse mef2c gene to direct the expression of Cre recombinase exclusively in the anterior heart field and its derivatives in transgenic mice. By crossing these mice, termed mef2c-AHF-Cre, to Cre-dependent lacZ reporter mice, we generated a fate map of the embryonic, fetal, and postnatal heart. These studies show that the endothelial and myocardial components of the outflow tract, right ventricle, and ventricular septum are derivatives of mef2c-AHF-Cre expressing cells within the anterior heart field and its derivatives. These studies also show that the atria, epicardium, coronary vessels, and the majority of outflow tract smooth muscle are not derived from this anterior heart field population. Furthermore, a transgene marker specific for the anterior heart field is expressed in the common ventricular chamber in mef2c mutant mice, suggesting that the cardiac looping defect in these mice is not due to a failure in anterior heart field addition to the heart. Finally, the Cre transgenic mice described here will be a crucial tool for conditional gene inactivation exclusively in the anterior heart field and its derivatives.

Accurate control of tissue-specific gene expression plays a pivotal role in heart development, but few cardiac transcriptional enhancers have thus far been identified. Extreme non-coding sequence conservation successfully predicts enhancers active in many tissues, but fails to identify substantial numbers of heart enhancers. Here we used ChIP-seq with the enhancer-associated protein p300 from mouse embryonic day 11.5 heart tissue to identify over three thousand candidate heart enhancers genome-wide. Compared to other tissues studied at this time-point, most candidate heart enhancers are less deeply conserved in vertebrate evolution. Nevertheless, the testing of 130 candidate regions in a transgenic mouse assay revealed that most of them reproducibly function as enhancers active in the heart, irrespective of their degree of evolutionary constraint. These results provide evidence for a large population of poorly conserved heart enhancers and suggest that the evolutionary constraint of embryonic enhancers can vary depending on tissue type.

Development and function of the human heart depend on the dynamic control of tissue-specific gene expression by distant-acting transcriptional enhancers. To generate an accurate genome-wide map of human heart enhancers, we used an epigenomic enhancer discovery approach and identified ∼6,200 candidate enhancer sequences directly from fetal and adult human heart tissue. Consistent with their predicted function, these elements were markedly enriched near genes implicated in heart development, function and disease. To further validate their in vivo enhancer activity, we tested 65 of these human sequences in a transgenic mouse enhancer assay and observed that 43 (66%) drove reproducible reporter gene expression in the heart. These results support the discovery of a genome-wide set of non-coding sequences highly enriched in human heart enhancers which is likely to facilitate down-stream studies of the role of enhancers in development and pathological conditions of the heart.

Congenital heart disease is a major cause of morbidity and mortality throughout life. Mutations in numerous transcription factors have been identified in patients and families with some of the most common forms of cardiac malformations and arrhythmias. This review discusses factor pathways known to be important for normal heart development and how abnormalities in these pathways have been linked to morphological and functional forms of congenital heart defects. A comprehensive, current list of known transcription factor mutations associated with congenital heart disease is provided, but the review focuses primarily on three key transcription factors, Nkx2-5, GATA4, and Tbx5, and their known biochemical and genetic partners. By understanding the interaction partners, transcriptional targets, and upstream activators of these core cardiac transcription factors, additional information about normal heart formation and further insight into genes and pathways affected in congenital heart disease should result.

The genetic hierarchies guiding lineage specification and morphogenesis of the mammalian embryonic heart are poorly understood. We now show by gene targeting that murine T-box transcription factor Tbx20 plays a central role in these pathways, and has important activities in both cardiac development and adult function. Loss of Tbx20 results in death of embryos at mid-gestation with grossly abnormal heart morphogenesis. Underlying these disturbances was a severely compromised cardiac transcriptional program,defects in the molecular pre-pattern, reduced expansion of cardiac progenitors and a block to chamber differentiation. Notably, Tbx20-null embryos showed ectopic activation of Tbx2 across the whole heart myogenic field. Tbx2 encodes a transcriptional repressor normally expressed in non-chamber myocardium, and in the atrioventricular canal it has been proposed to inhibit chamber-specific gene expression through competition with positive factor Tbx5. Our data demonstrate a repressive activity for Tbx20 and place it upstream of Tbx2 in the cardiac genetic program. Thus, hierarchical,repressive interactions between Tbx20 and other T-box genes and factors underlie the primary lineage split into chamber and non-chamber myocardium in the forming heart, an early event upon which all subsequent morphogenesis depends. Additional roles for Tbx20 in adult heart integrity and contractile function were revealed by in-vivo cardiac functional analysis of Tbx20 heterozygous mutant mice. These data suggest that mutations in human cardiac transcription factor genes, possibly including TBX20,underlie both congenital heart disease and adult cardiomyopathies.

Alveologenesis is the final step of lung maturation, which subdivides the alveolar region of the lung into smaller units called alveoli. Each of the nascent dividers serves as a new gas-exchange surface, and collectively they drastically increase the surface area for breathing. Disruption of alveologenesis results in simplification of alveoli, as is seen in premature infants diagnosed with bronchopulmonary dysplasia (BPD), a prevalent lung disease that is often associated with lifelong breathing deficiencies. To date, a majority of studies of alveologenesis rely on two-dimensional (2D) analysis of tissue sections. Given that an overarching theme of alveologenesis is thinning and extension of the epithelium and mesenchyme to facilitate gas exchange, often only a small portion of a cell or a cellular structure is represented in a single 2D plane. Here, we use a three-dimensional (3D) approach to examine the structural architecture and cellular composition of myofibroblasts, alveolar type 2 cells, elastin and lipid droplets in normal as well as BPD-like mouse lung. We found that 2D finger-like septal crests, commonly used to depict growing alveolar septae, are often artifacts of sectioning through fully established alveolar walls. Instead, a more accurate representation of growing septae are 3D ridges that are lined by platelet-derived growth factor receptor alpha (PDGFRA) and alpha smooth muscle actin (α-SMA)-expressing myofibroblasts, as well as the elastin fibers that they produce. Accordingly in 3D, both α-SMA and elastin were each found in connected networks underlying the 3D septal ridges rather than as isolated dots at the tip of 2D septal crests. Analysis through representative stages of alveologenesis revealed unappreciated dynamic changes in these patterns. PDGFRA-expressing cells are only α-SMA-positive during the first phase of alveologenesis, but not in the second phase, suggesting that the two phases of septae formation may be driven by distinct mechanisms. Thin elastin fibers are already present in the alveolar region prior to alveologenesis, suggesting that during alveologenesis, there is not only new elastin deposition, but also extensive remodeling to transform thin and uniformly distributed fibers into thick cables that rim the nascent septae. Analysis of several genetic as well as hyperoxia-induced models of BPD revealed that the myofibroblast organization is perturbed in all, regardless of whether the origin of defect is epithelial, mesenchymal, endothelial or environmental. Finally, analysis of relative position of PDGFRA-positive cells and alveolar type 2 cells reveal that during alveologenesis, these two cell types are not always adjacent to one another. This result suggests that the niche and progenitor relationship afforded by their close juxtaposition in the adult lung may be a later acquired property. These insights revealed by 3D reconstruction of the septae set the foundation for future investigations of the mechanisms driving normal alveologenesis, as well as causes of alveolar simpli...

Congenital diaphragmatic hernias (CDHs) and structural anomalies of the diaphragm are a common class of congenital birth defects that are associated with significant morbidity and mortality due to associated pulmonary hypoplasia, pulmonary hypertension and heart failure. In ∼30% of CDH patients, genomic analyses have identified a range of genetic defects, including chromosomal anomalies, copy number variants and sequence variants. The affected genes identified in CDH patients include transcription factors, such as GATA4, ZFPM2, NR2F2 and WT1, and signaling pathway components, including members of the retinoic acid pathway. Mutations in these genes affect diaphragm development and can have pleiotropic effects on pulmonary and cardiac development. New therapies, including fetal endoscopic tracheal occlusion and prenatal transplacental fetal treatments, aim to normalize lung development and pulmonary vascular tone to prevent and treat lung hypoplasia and pulmonary hypertension, respectively. Studies of the association between particular genetic mutations and clinical outcomes should allow us to better understand the origin of this birth defect and to improve our ability to predict and identify patients most likely to benefit from specialized treatment strategies.

MEF2 transcription factors are well-established regulators of muscle development. We have discovered an unanticipated role for MEF2C in the neural crest, where tissue-specific inactivation results in neonatal lethality due to severe craniofacial defects. We show that MEF2C is required for expression of the Dlx5, Dlx6, and Hand2 transcription factor genes in the branchial arches, and we identify a branchial arch-specific enhancer in the Dlx5/6 locus, which is activated synergistically by MEF2C and Dlx5, demonstrating that these factors interact to induce transcription. Mef2c and Dlx5/6 also interact genetically. Mice heterozygous for either Dlx5/6 or Mef2c are normal at birth and survive to weaning. By contrast, heterozygosity for both Mef2c and Dlx5/6 results in defective palate development and neonatal lethality. Taken together, the studies presented here define a feed-forward transcriptional circuit between the MADS-box transcription factor MEF2C and the homeodomain transcription factors Dlx5 and Dlx6 in craniofacial development.