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Background The reactivation of genetic programs from early development is a common mechanism for injury-induced organ regeneration. T-box 3 (TBX3) is a member of the T-box family of transcription factors previously shown to regulate pluripotency and subsequent lineage commitment in a number of tissues, including limb and lung. TBX3 is also involved in lung and heart organogenesis. Here, we provide a comprehensive and thorough characterization of TBX3 and its role during pancreatic organogenesis and regeneration. Results We interrogated the level and cell specificity of TBX3 in the developing and adult pancreas at mRNA and protein levels at multiple developmental stages in mouse and human pancreas. We employed conditional mutagenesis to determine its role in murine pancreatic development and in regeneration after the induction of acute pancreatitis. We found that Tbx3 is dynamically expressed in the pancreatic mesenchyme and epithelium. While Tbx3 is expressed in the developing pancreas, its absence is likely compensated by other factors after ablation from either the mesenchymal or epithelial compartments. In an adult model of acute pancreatitis, we found that a lack of Tbx3 resulted in increased proliferation and fibrosis as well as an enhanced inflammatory gene programs, indicating that Tbx3 has a role in tissue homeostasis and regeneration. Conclusions TBX3 demonstrates dynamic expression patterns in the pancreas. Although TBX3 is dispensable for proper pancreatic development, its absence leads to altered organ regeneration after induction of acute pancreatitis.

Aims In the heart, splicing factors orchestrate the functional properties of cardiomyocytes by regulating the alternative splicing of multiple genes. Work in embryonic stem cells has shown that the splicing factor Quaking (QKI) regulates alternative splicing during cardiomyocyte differentiation. However, the relevance and function of QKI in adult cardiomyocytes remains unknown. In this study, we aim to identify the in vivo function of QKI in the adult mouse heart. Methods and results We generated mice with conditional deletion of QKI in cardiomyocytes by the Cre-Lox system. Mice with cardiomyocyte-specific deletion of QKI died during the foetal period (E14.5), without obvious anatomical abnormalities of the heart. Adult mice with tamoxifen-inducible QKI deletion rapidly developed heart failure associated with severe disruption of sarcomeres, already 7 days after knocking out QKI. RNA sequencing revealed that QKI regulates the alternative splicing of more than 1000 genes, including sarcomere and cytoskeletal components, calcium-handling genes, and (post-)transcriptional regulators. Many of these splicing changes corresponded to the loss of muscle-specific isoforms in the heart. Forced overexpression of QKI in cultured neonatal rat ventricular myocytes directed these splicing events in the opposite direction and enhanced contractility of cardiomyocytes. Conclusion Altogether, our findings show that QKI is an important regulator of the muscle-specific alternative splicing program that builds the contractile apparatus of cardiomyocytes.

The sinoatrial node (SAN) is the primary pacemaker of the mammalian heart, initiating its electrical activation and ensuring that the heart's functional cardiac output meets physiological demand. SAN dysfunction (SND) can cause complex cardiac arrhythmias that can manifest as severe sinus bradycardia, sinus arrest, chronotropic incompetence and increased susceptibility to atrial fibrillation, among other cardiac conditions. SND has a complex aetiology, with both pre-existing disease and heritable genetic variation predisposing individuals to this pathology. In this Review, we summarize the current understanding of the genetic contributions to SND and the insights that they provide into this disorder's underlying molecular mechanisms. With an improved understanding of these molecular mechanisms, we can improve treatment options for SND patients and develop new therapeutics.

Background: The pathogenic missense variant p.G125R in TBX5 (T-box transcription factor 5) causes Holt–Oram syndrome (also known as hand–heart syndrome) and early onset of atrial fibrillation. Revealing how an altered key developmental transcription factor modulates cardiac physiology in vivo will provide unique insights into the mechanisms underlying atrial fibrillation in these patients. Methods: We analyzed ECGs of an extended family pedigree of Holt–Oram syndrome patients. Next, we introduced the TBX5-p.G125R variant in the mouse genome ( Tbx5 G125R ) and performed electrophysiologic analyses (ECG, optical mapping, patch clamp, intracellular calcium measurements), transcriptomics (single-nuclei and tissue RNA sequencing), and epigenetic profiling (assay for transposase-accessible chromatin using sequencing, H3K27ac [histone H3 lysine 27 acetylation] CUT&RUN [cleavage under targets and release under nuclease sequencing]). Results: We discovered high incidence of atrial extra systoles and atrioventricular conduction disturbances in Holt–Oram syndrome patients. Tbx5 G125R/+ mice were morphologically unaffected and displayed variable RR intervals, atrial extra systoles, and susceptibility to atrial fibrillation, reminiscent of TBX5-p.G125R patients. Atrial conduction velocity was not affected but systolic and diastolic intracellular calcium concentrations were decreased and action potentials were prolonged in isolated cardiomyocytes of Tbx5 G125R/+ mice compared with controls. Transcriptional profiling of atria revealed the most profound transcriptional changes in cardiomyocytes versus other cell types, and identified over a thousand coding and noncoding transcripts that were differentially expressed. Epigenetic profiling uncovered thousands of TBX5-p.G125R-sensitive, putative regulatory elements (including enhancers) that gained accessibility in atrial cardiomyocytes. The majority of sites with increased accessibility were occupied by Tbx5. The small group of sites with reduced accessibility was enriched for DNA-binding motifs of members of the SP (specificity protein) and KLF (Krüppel-like factor) families of transcription factors. These data show that Tbx5-p.G125R induces changes in regulatory element activity, alters transcriptional regulation, and changes cardiomyocyte behavior, possibly caused by altered DNA binding and cooperativity properties. Conclusions: Our data reveal that a disease-causing missense variant in TBX5 induces profound changes in the atrial transcriptional regulatory network and epigenetic state in vivo, leading to arrhythmia reminiscent of those seen in human TBX5-p.G125R variant carriers.

The auditory function of the mammalian cochlea relies on two types of mechanosensory hair cells and various non-sensory supporting cells. Recent studies identified the transcription factors INSM1 and IKZF2 as regulators of outer hair cell (OHC) fate. However, the transcriptional regulation of the differentiation of inner hair cells (IHCs) and their associated inner supporting cells (ISCs) has remained enigmatic. Here, we show that the expression of the transcription factor TBX2 is restricted to IHCs and ISCs from the onset of differentiation until adulthood and examine its function using conditional deletion and misexpression approaches in the mouse. We demonstrate that TBX2 acts in prosensory progenitors as a patterning factor by specifying the inner compartment of the sensory epithelium that subsequently gives rise to IHCs and ISCs. Hair cell-specific inactivation or misexpression causes transdifferentiation of hair cells indicating a cell-autonomous function of TBX2 in inducing and maintaining IHC fate.

Background: The sinoatrial node (SAN) functions as the pacemaker of the heart, initiating rhythmic heartbeats. Despite its importance, the SAN is one of the most poorly understood cardiac entities because of its small size and complex composition and function. The Hippo signaling pathway is a molecular signaling pathway fundamental to heart development and regeneration. Although abnormalities of the Hippo pathway are associated with cardiac arrhythmias in human patients, the role of this pathway in the SAN is unknown. Methods: We investigated key regulators of the Hippo pathway in SAN pacemaker cells by conditionally inactivating the Hippo signaling kinases Lats1 and Lats2 using the tamoxifen-inducible, cardiac conduction system–specific Cre driver Hcn4 CreERT2 with Lats1 and Lats2 conditional knockout alleles. In addition, the Hippo-signaling effectors Yap and Taz were conditionally inactivated in the SAN. To determine the function of Hippo signaling in the SAN and other cardiac conduction system components, we conducted a series of physiological and molecular experiments, including telemetry ECG recording, echocardiography, Masson Trichrome staining, calcium imaging, immunostaining, RNAscope, cleavage under targets and tagmentation sequencing using antibodies against Yap1 or H3K4me3, quantitative real-time polymerase chain reaction, and Western blotting. We also performed comprehensive bioinformatics analyses of various datasets. Results: We found that Lats1/2 inactivation caused severe sinus node dysfunction. Compared with the controls, Lats1/2 conditional knockout mutants exhibited dysregulated calcium handling and increased fibrosis in the SAN, indicating that Lats1/2 function through both cell-autonomous and non–cell-autonomous mechanisms. It is notable that the Lats1/2 conditional knockout phenotype was rescued by genetic deletion of Yap and Taz in the cardiac conduction system. These rescued mice had normal sinus rhythm and reduced fibrosis of the SAN, indicating that Lats1/2 function through Yap and Taz. Cleavage Under Targets and Tagmentation sequencing data showed that Yap potentially regulates genes critical for calcium homeostasis such as Ryr2 and genes encoding paracrine factors important in intercellular communication and fibrosis induction such as Tgfb1 and Tgfb3 . Consistent with this, Lats1/2 conditional knockout mutants had decreased Ryr2 expression and increased Tgfb1 and Tgfb3 expression compared with control mice. Conclusions: We reveal, for the first time to our knowledge, that the canonical Hippo-Yap pathway plays a pivotal role in maintaining SAN homeostasis.

Trabecular myocardium makes up most of the ventricular wall of the human embryo. A process of compaction in the fetal period presumably changes ventricular wall morphology by converting ostensibly weaker trabecular myocardium into stronger compact myocardium. Using developmental series of embryonic and fetal humans, mice and chickens, we show ventricular morphogenesis is driven by differential rates of growth of trabecular and compact layers rather than a process of compaction. In mouse, fetal cardiomyocytes are relatively weak but adult cardiomyocytes from the trabecular and compact layer show an equally large force generating capacity. In fetal and adult humans, trabecular and compact myocardium are not different in abundance of immunohistochemically detected vascular, mitochondrial and sarcomeric proteins. Similar findings are made in human excessive trabeculation, a congenital malformation. In conclusion, trabecular and compact myocardium is equally equipped for force production and their proportions are determined by differential growth rates rather than by compaction.

The ventricular walls of the human heart comprise an outer compact layer and an inner trabecular layer. In the context of an increased pre‐test probability, diagnosis left ventricular noncompaction cardiomyopathy is given when the left ventricle is excessively trabeculated in volume (trabecular vol >25% of total LV wall volume) or thickness (trabecular/compact (T/C) >2.3). Here, we investigated whether higher spatial resolution affects the detection of trabeculation and thus the assessment of normal and excessively trabeculated wall morphology. First, we screened left ventricles in 1112 post‐natal autopsy hearts. We identified five excessively trabeculated hearts and this low prevalence of excessive trabeculation is in agreement with pathology reports but contrasts the prevalence of approximately 10% of the population found by in vivo non‐invasive imaging. Using macroscopy, histology and low‐ and high‐resolution MRI, the five excessively trabeculated hearts were compared with six normal hearts and seven abnormally trabeculated and excessive trabeculation‐negative hearts. Some abnormally trabeculated hearts could be considered excessively trabeculated macroscopically because of a trabecular outflow or an excessive number of trabeculations, but they were excessive trabeculation‐negative when assessed with MRI‐based measurements (T/C, Jensen et al. show that higher spatial resolution may affect the sensitivity of diagnostic measurements of noncompaction and in addition could allow for novel measurements such as counting of trabeculations.

Rationale: Atrial fibrillation (AF) is the most common cardiac arrhythmia diagnosed in clinical practice. Genome-wide association studies have identified AF-associated common variants across 100+ genomic loci, but the mechanism underlying the impact of these variant loci on AF susceptibility in vivo has remained largely undefined. One such variant region, highly associated with AF, is found at 1q24, close to PRRX1 , encoding the paired-related homeobox 1 transcription factor. Objective: To identify the mechanistic link between the variant region at 1q24 and AF predisposition. Methods and Results: The mouse orthologue of the noncoding variant genomic region ( R1A ) at 1q24 was deleted using CRISPR genome editing. Among the genes sharing the topologically associated domain with the deleted R1A region ( Kifap3 , Prrx1 , Fmo2 , and Prrc2c ), only the broadly expressed gene Prrx1 was downregulated in mutants, and only in cardiomyocytes. Expression and epigenetic profiling revealed that a cardiomyocyte lineage-specific gene program ( Mhrt , Myh6 , Rbm20 , Tnnt2 , Ttn , and Ckm ) was upregulated in R1A −/− atrial cardiomyocytes and that Mef2 (myocyte enhancer factor)-binding motifs were significantly enriched at differentially accessible chromatin sites. Consistently, Prrx1 suppressed Mef2-activated enhancer activity in HL-1 cells. Mice heterozygous or homozygous for the R1A deletion were susceptible to atrial arrhythmia induction, had atrial conduction slowing and more irregular RR intervals. Isolated R1A −/− mouse left atrial cardiomyocytes showed lower action potential upstroke velocities and sodium current, as well as increased systolic and diastolic calcium concentrations compared with controls. Conclusions: The noncoding AF variant region at 1q24 modulates Prrx1 expression in cardiomyocytes. Cardiomyocyte-specific reduction of Prrx1 expression upon deletion of the noncoding region leads to a profound induction of a cardiac lineage-specific gene program and to propensity for AF. These data indicate that AF-associated variants in humans may exert AF predisposition through reduced PRRX1 expression in cardiomyocytes.

Heart development and rhythm control are highly Tbx5 dosage-sensitive. TBX5 haploinsufficiency causes congenital conduction disorders, whereas increased expression levels of TBX5 in human heart samples has been associated with atrial fibrillation. We deleted the conserved mouse orthologues of two independent AF-associated genomic regions in the Tbx5 locus, one intronic (RE(int)-/-) and one downstream of Tbx5 (RE(down)-/-). In both lines we observed a modest (30%) increase of Tbx5 in the postnatal atria. To gain insight into the effects of slight dosage increase in vivo, we investigated the atrial transcriptional, epigenetic and electrophysiological properties of both lines. We observed induction of genes involved in development, ion transport and conduction, increased action potential duration and increased susceptibility to atrial arrhythmias. We identified an AF-associated variant in the human intronic regulatory region that increases transcriptional activity. Expression of the AF-associated transcription factor Prrx1 was induced in RE(int)-/- cardiomyocytes. We found that some of the transcriptional and functional changes in the atria caused by increased Tbx5 expression were normalized when reducing cardiac Prrx1 expression in RE(int)-/- mice, indicating an interaction between these two AF genes. We conclude that modest increases in expression of dose-dependent transcription factors, caused by common regulatory variants, significantly impact on the cardiac gene regulatory network and disease susceptibility.

Tricuspid valve agenesis/atresia (TVA) is a congenital cardiac malformation where the tricuspid valve is not formed. It is hypothesized that TVA results from a failure of the normal rightward expansion of the atrioventricular canal (AVC). We tested predictions of this hypothesis by morphometric analyses of the AVC in fetal hearts. We used high-resolution MRI and ultrasonography on a post-mortem fetal heart with TVA and with tricuspid valve stenosis (TVS) to validate the position of measurement landmarks that were to be applied to clinical echocardiograms. This revealed a much deeper right atrioventricular sulcus in TVA than in TVS. Subsequently, serial echocardiograms of in utero fetuses between 12 and 38 weeks of gestation were included (n = 23 TVA, n = 16 TVS, and n = 74 controls) to establish changes in AVC width and ventricular dimensions over time. Ventricular length and width and estimated fetal weight all increased significantly with age, irrespective of diagnosis. Heart rate did not differ between groups. However, in the second trimester, in TVA, the ratio of AVC to ventricular width was significantly lower compared to TVS and controls. This finding supports the hypothesis that TVA is due to a failed rightward expansion of the AVC. Notably, we found in the third trimester that the AVC to ventricular width normalized in TVA fetuses as their mitral valve area was greater than in controls. Hence, TVA associates with a quantifiable under-development of the AVC. This under-development is obscured in the third trimester, likely because of adaptational growth that allows for increased stroke volume of the left ventricle.

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3(+/−)) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3(+/−) mutants. Notably, Tbx3(+/−) atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.

Genome-wide association studies have uncovered over a 100 genetic loci associated with atrial fibrillation (AF), the most common arrhythmia. Many of the top AF-associated loci harbor key cardiac transcription factors, including PITX2, TBX5, PRRX1, and ZFHX3. Moreover, the vast majority of the AF-associated variants lie within noncoding regions of the genome where causal variants affect gene expression by altering the activity of transcription factors and the epigenetic state of chromatin. In this review, we discuss a transcriptional regulatory network model for AF defined by effector genes in Genome-wide association studies loci. We describe the current state of the field regarding the identification and function of AF-relevant gene regulatory networks, including variant regulatory elements, dose-sensitive transcription factor functionality, target genes, and epigenetic states. We illustrate how altered transcriptional networks may impact cardiomyocyte function and ionic currents that impact AF risk. Last, we identify the need for improved tools to identify and functionally test transcriptional components to define the links between genetic variation, epigenetic gene regulation, and atrial function.

Paucity of physiologically relevant cardiac models has limited the widespread application of human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes in drug development. Here, we performed comprehensive characterization of hiPSC-derived cardiomyocyte subtypes from 2D and 3D cultures and established a novel 3D model to study impulse initiation and propagation. Directed differentiation approaches were used to generate sinoatrial nodal (SANCM), atrial (ACM) and ventricular cardiomyocytes (VCM). Single cell RNA sequencing established that the protocols yield distinct cell populations in line with expected identities, which was also confirmed by electrophysiological characterization. In 3D EHT cultures of all subtypes, we observed prominent expression of stretch-responsive genes such as NPPA. Response to rate modulating drugs noradrenaline, carbachol and ivabradine were comparable in single cells and EHTs. Differences in the speed of impulse propagation between the subtypes were more pronounced in EHTs compared with 2D monolayers owing to a progressive increase in conduction velocities in atrial and ventricular cardiomyocytes, in line with a more mature phenotype. In a novel binary EHT model of pacemaker-atrial interface, the SANCM end of the tissue consistently paced the EHTs under baseline conditions, which was inhibited by ivabradine. Taken together, our data provide comprehensive insights into molecular and electrophysiological properties of hiPSC-derived cardiomyocyte subtypes, facilitating the creation of next generation composite cardiac models for drug discovery, disease modeling and cell-based regenerative therapies.

Each heartbeat is triggered by the sinoatrial node, the natural pacemaker of the heart. Animal models have revealed that pacemaker cells share a common progenitor with the (pro)epicardium, and that the pacemaker cardiomyocytes further diversify into “transitional”, “tail” and “head” subtypes. However, the underlying molecular mechanisms are poorly understood. Here, we studied the differentiation of human induced pluripotent stem cells into pacemaker cardiomyocytes. Single cell RNA sequencing identified the presence of myocardial populations resembling subtypes present in the formed sinoatrial node, and in addition revealed a side population of (pro)epicardial cells. Time-course trajectory analysis uncovered a role for WNT signaling in determining myocardial versus proepicardial cell fate. We experimentally demonstrate that presence of WNT signaling prior to the branching point of a common progenitor enhances proepicardial cell differentiation at the expense of myocardial pacemaker cells. Furthermore, we uncover a role for TGFβ and WNT signaling in differentiation towards transitional and head pacemaker subtypes, respectively. Our findings provide new biological insights into human pacemaker differentiation, open avenues for complex disease modeling and inform regenerative approaches.

Background Tbx2 encodes a transcriptional repressor implicated in the development of numerous organs in mouse. During lung development TBX2 maintains the proliferation of mesenchymal progenitors, and hence, epithelial proliferation and branching morphogenesis. The pro-proliferative function was traced to direct repression of the cell-cycle inhibitor genes Cdkn1a and Cdkn1b, as well as of genes encoding WNT antagonists, Frzb and Shisa3, to increase pro-proliferative WNT signaling. Despite these important molecular insights, we still lack knowledge of the DNA occupancy of TBX2 in the genome, and of the protein interaction partners involved in transcriptional repression of target genes. Methods We used chromatin immunoprecipitation (ChIP)-sequencing and expression analyses to identify genomic DNA-binding sites and transcription units directly regulated by TBX2 in the developing lung. Moreover, we purified TBX2 containing protein complexes from embryonic lung tissue and identified potential interaction partners by subsequent liquid chromatography/mass spectrometry. The interaction with candidate proteins was validated by immunofluorescence, proximity ligation and individual co-immunoprecipitation analyses. Results We identified Il33 and Ccn4 as additional direct target genes of TBX2 in the pulmonary mesenchyme. Analyzing TBX2 occupancy data unveiled the enrichment of five consensus sequences, three of which match T-box binding elements. The remaining two correspond to a high mobility group (HMG)-box and a homeobox consensus sequence motif. We found and validated binding of TBX2 to the HMG-box transcription factor HMGB2 and the homeobox transcription factor PBX1, to the heterochromatin protein CBX3, and to various members of the nucleosome remodeling and deacetylase (NuRD) chromatin remodeling complex including HDAC1, HDAC2 and CHD4. Conclusion Our data suggest that TBX2 interacts with homeobox and HMG-box transcription factors as well as with the NuRD chromatin remodeling complex to repress transcription of anti-proliferative genes in the pulmonary mesenchyme.

Myocardial infarction causes ventricular muscle loss and formation of scar tissue. The surviving myocardium in the border zone, located adjacent to the infarct, undergoes profound changes in function, structure and composition. How and to what extent these changes of border zone cardiomyocytes are regulated epigenetically is not fully understood. Here, we obtained transcriptomes of PCM-1-sorted mouse cardiomyocyte nuclei of healthy left ventricle and 7 days post myocardial infarction border zone tissue. We validated previously observed downregulation of genes involved in fatty acid metabolism, oxidative phosphorylation and mitochondrial function in border zone-derived cardiomyocytes, and observed a modest induction of genes involved in glycolysis, including Slc2a1 (Glut1) and Pfkp. To gain insight into the underlying epigenetic regulatory mechanisms, we performed H3K27ac profiling of healthy and border zone cardiomyocyte nuclei. We confirmed the switch from Mef2- to AP-1 chromatin association in border zone cardiomyocytes, and observed, in addition, an enrichment of PPAR/RXR binding motifs in the sites with reduced H3K27ac signal. We detected downregulation and accompanying epigenetic state changes at several key PPAR target genes including Ppargc1a (PGC-1α), Cpt2, Ech1, Fabpc3 and Vldrl in border zone cardiomyocytes. These data indicate that changes in epigenetic state and gene regulation underlie the maintained metabolic switch in border zone cardiomyocytes.

Organ laterality refers to the left-right asymmetry in disposition and conformation of internal organs and is established during embryogenesis. The heart is the first organ to display visible left-right asymmetries through its left-sided positioning and rightward looping. Here, we present a new zebrafish loss-of-function allele for tbx5a, which displays defective rightward cardiac looping morphogenesis. By mapping individual cardiomyocyte behavior during cardiac looping, we establish that ventricular and atrial cardiomyocytes rearrange in distinct directions. As a consequence, the cardiac chambers twist around the atrioventricular canal resulting in torsion of the heart tube, which is compromised in tbx5a mutants. Pharmacological treatment and ex vivo culture establishes that the cardiac twisting depends on intrinsic mechanisms and is independent from cardiac growth. Furthermore, genetic experiments indicate that looping requires proper tissue patterning. We conclude that cardiac looping involves twisting of the chambers around the atrioventricular canal, which requires correct tissue patterning by Tbx5a.

BackgroundIn the embryonic mammalian lung, mesenchymal cells act both as a signaling center for epithelial proliferation, differentiation and morphogenesis as well as a source for a multitude of differentiated cell types that support the structure of the developing and mature organ. Whether the embryonic pulmonary mesenchyme is a homogenous precursor pool and how it diversifies into different cell lineages is poorly understood. We have previously shown that the T-box transcription factor geneTbx2is expressed in the pulmonary mesenchyme of the developing murine lung and is required therein to maintain branching morphogenesis.MethodsWe determined Tbx2/TBX2 expression in the developing murine lung by in situ hybridization and immunofluorescence analyses. We used a genetic lineage tracing approach with aCreline under the control of endogenousTbx2control elements (Tbx2cre), and theR26mTmGreporter line to trace TBX2-positive cells in the murine lung. We determined the fate of the TBX2 lineage by co-immunofluorescence analysis of the GFP reporter and differentiation markers in normal murine lungs and in lungs lacking or overexpressing TBX2 in the pulmonary mesenchyme.ResultsWe show that TBX2 is strongly expressed in mesenchymal progenitors in the developing murine lung. In differentiated smooth muscle cells and in fibroblasts, expression of TBX2 is still widespread but strongly reduced. In mesothelial and endothelial cells expression is more variable and scattered. All fetal smooth muscle cells, endothelial cells and fibroblasts derive from TBX2+progenitors, whereas half of the mesothelial cells have a different descent. The fate of TBX2-expressing cells is not changed inTbx2-deficient and inTBX2-constitutively overexpressing mice but the distribution and abundance of endothelial and smooth muscle cells is changed in the overexpression condition.ConclusionThe fate of pulmonary mesenchymal progenitors is largely independent of TBX2. Nevertheless, a successive and precisely timed downregulation of TBX2 is necessary to allow proper differentiation and functionality of bronchial smooth muscle cells and to limit endothelial differentiation. Our work suggests expression of TBX2 in an early pulmonary mesenchymal progenitor and supports a role of TBX2 in maintaining the precursor state of these cells.

The atrial septum enables efficient oxygen transport by separating the systemic and pulmonary venous blood returning to the heart. Only in placental mammals will the atrial septum form by the coming-together of the septum primum and the septum secundum. In up to one of four placental mammals, this complex morphogenesis is incomplete and yields patent foramen ovale. The incidence of incomplete atrial septum is unknown for groups with the septum primum only, such as birds and reptiles. We found a low incidence of incomplete atrial septum in 11 species of bird (0% of specimens) and 13 species of reptiles (3% of specimens). In reptiles, there was a trabecular interface between the atrial septum and the atrial epicardium which was without a clear boundary between left and right atrial cavities. In developing reptiles (four squamates and one crocodylian), the septum primum initiated as a sheet that acquired perforations and the trabecular interface developed late. We conclude that atrial septation from the septum primum only results in a low incidence of incompleteness. In reptiles, the atrial septum and atrial wall develop a trabecular interface, but previous studies on atrial hemodynamics suggest this interface has a very limited capacity for shunting.

Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that co-localize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations.

Rationale: The ventricular conduction system (VCS) rapidly propagates electrical impulses through the working myocardium of the ventricles to coordinate chamber contraction. GWAS (Genome-wide association studies) have associated nucleotide polymorphisms, most are located within regulatory intergenic or intronic sequences, with variation in VCS function. Two highly correlated polymorphisms (r 2 >0.99) associated with VCS functional variation (rs13165478 and rs13185595) occur 5′ to the gene encoding the basic helix–loop–helix transcription factor HAND1 (heart- and neural crest derivatives-expressed protein 1). Objective: Here, we test the hypothesis that these polymorphisms influence HAND1 transcription thereby influencing VCS development and function. Methods and Results: We employed transgenic mouse models to identify an enhancer that is sufficient for left ventricle (LV) cis -regulatory activity. Two evolutionarily conserved GATA transcription factor cis -binding elements within this enhancer are bound by GATA4 and are necessary for cis -regulatory activity, as shown by in vitro DNA binding assays. CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9-mediated deletion of this enhancer dramatically reduces Hand1 expression solely within the LV but does not phenocopy previously published mouse models of cardiac Hand1 loss-of-function. Electrophysiological and morphological analyses reveals that mice homozygous for this deleted enhancer display a morphologically abnormal VCS and a conduction system phenotype consistent with right bundle branch block. Using 1000 Genomes Project data, we identify 3 additional single nucleotide polymorphisms (SNPs), located within the Hand1 LV enhancer, that compose a haplotype with rs13165478 and rs13185595. One of these SNPs, rs10054375, overlaps with a critical GATA cis -regulatory element within the Hand1 LV enhancer. This SNP, when tested in electrophoretic mobility shift assays, disrupts GATA4 DNA-binding. Modeling 2 of these SNPs in mice causes diminished Hand1 expression and mice present with abnormal VCS function. Conclusions: Together, these findings reveal that SNP rs10054375, which is located within a necessary and sufficient LV-specific Hand1 enhancer, exhibits reduces GATA DNA-binding in electrophoretic mobility shift assay, and this enhancer in total, is required for VCS development and function in mice and perhaps humans.

The sinus venosus is a cardiac chamber upstream of the right atrium that harbours the dominant cardiac pacemaker. During human heart development, the sinus venosus becomes incorporated into the right atrium. However, from the literature it is not possible to deduce the characteristics and importance of this process of incorporation, due to inconsistent terminology and definitions in the description of multiple lines of evidence. We reviewed the literature regarding the incorporation of the sinus venosus and included novel electrophysiological data. Most mammals that have an incorporated sinus venosus show a loss of a functional valve guard of the superior caval vein together with a loss of the electrical sinuatrial delay between the sinus venosus and the right atrium. However, these processes are not necessarily intertwined and in a few species only the sinuatrial delay may be lost. Sinus venosus incorporation can be characterised as the loss of the sinuatrial delay of which the anatomical and molecular underpinnings are not yet understood.

Background: Surviving cells in the postinfarction border zone are subjected to intense fluctuations of their microenvironment. Recently, border zone cardiomyocytes have been specifically implicated in cardiac regeneration. Here, we defined their unique transcriptional and regulatory properties, and comprehensively validated new molecular markers, including Nppb , encoding B-type natriuretic peptide, after infarction. Methods: Transgenic reporter mice were used to identify the Nppb -positive border zone after myocardial infarction. Transcriptome analysis of remote, border, and infarct zones and of purified cardiomyocyte nuclei was performed using RNA-sequencing. Top candidate genes displaying border zone spatial specificity were histologically validated in ischemic human hearts. Mice in which Nppb was deleted by genome editing were subjected to myocardial infarction. Chromatin accessibility landscapes of border zone and control cardiomyocyte nuclei were assessed by using assay for transposase-accessible chromatin using sequencing. Results: We identified the border zone as a spatially confined region transcriptionally distinct from the remote myocardium. The transcriptional response of the border zone was much stronger than that of the remote ventricular wall, involving acute downregulation of mitochondrial oxidative phosphorylation, fatty acid metabolism, calcium handling, and sarcomere function, and the activation of a stress-response program. Analysis of infarcted human hearts revealed that the transcriptionally discrete border zone is conserved in humans, and led to the identification of novel conserved border zone markers including NPPB , ANKRD1 , DES , UCHL1, JUN , and FOXP1 . Homozygous Nppb mutant mice developed acute and lethal heart failure after myocardial infarction, indicating that B-type natriuretic peptide is required to preserve postinfarct heart function. Assay for transposase-accessible chromatin using sequencing revealed thousands of cardiomyocyte lineage-specific MEF2-occupied regulatory elements that lost accessibility in the border zone. Putative injury-responsive enhancers that gained accessibility were highly associated with AP-1 (activator protein 1) binding sites. Nuclear c-Jun, a component of AP-1, was observed specifically in border zone cardiomyocytes. Conclusions: Cardiomyocytes in a discrete zone bordering the infarct switch from a MEF2-driven homeostatic lineage-specific to an AP-1–driven injury-induced gene expression program. This program is conserved between mouse and human, and includes Nppb expression, which is required to prevent acute heart failure after infarction.