Identification and characterization of Hand2 upstream genomic enhancers active in developing stomach and limbs.

BACKGROUND: The bHLH transcription factor HAND2 plays important roles in the development of the embryonic heart, face, limbs, and sympathetic and enteric nervous systems. To define how and when HAND2 regulates these developmental systems, requires understanding the transcriptional regulation of Hand2. RESULTS: Remarkably, Hand2 is flanked by an extensive upstream gene desert containing a potentially diverse enhancer landscape. Here, we screened the regulatory interval 200 kb proximal to Hand2 for putative enhancers using evolutionary conservation and histone marks in Hand2-expressing tissues. H3K27ac signatures across embryonic tissues pointed to only two putative enhancer regions showing deep sequence conservation. Assessment of the transcriptional enhancer potential of these elements using transgenic reporter lines uncovered distinct in vivo enhancer activities in embryonic stomach and limb mesenchyme, respectively. Activity of the identified stomach enhancer was restricted to the developing antrum and showed expression within the smooth muscle and enteric neurons. Surprisingly, the activity pattern of the limb enhancer did not overlap Hand2 mRNA but consistently yielded a defined subectodermal anterior expression pattern within multiple transgenic lines. CONCLUSIONS: Together, these results start to uncover the diverse regulatory potential inherent to the Hand2 upstream regulatory interval.

Single cell evaluation of endocardial Hand2 gene regulatory networks reveals HAND2-dependent pathways that impact cardiac morphogenesis.

The transcription factor HAND2 plays essential roles during cardiogenesis. Hand2 endocardial deletion (H2CKO) results in tricuspid atresia or double inlet left ventricle with accompanying intraventricular septum defects, hypo-trabeculated ventricles and an increased density of coronary lumens. To understand the regulatory mechanisms of these phenotypes, single cell transcriptome analysis of mouse E11.5 H2CKO hearts was performed revealing a number of disrupted endocardial regulatory pathways. Using HAND2 DNA occupancy data, we identify several HAND2-dependent enhancers, including two endothelial enhancers for the shear-stress master regulator KLF2. A 1.8 kb enhancer located 50 kb upstream of the Klf2 TSS imparts specific endothelial/endocardial expression within the vasculature and endocardium. This enhancer is HAND2-dependent for ventricular endocardium expression but HAND2-independent for Klf2 vascular and valve expression. Deletion of this Klf2 enhancer results in reduced Klf2 expression within ventricular endocardium. These data reveal that HAND2 functions within endocardial gene regulatory networks including shear-stress response.

Neonatal Deletion of Hand1 and Hand2 within Murine Cardiac Conduction System Reveals a Novel Role for HAND2 in Rhythm Homeostasis.

The cardiac conduction system, a network of specialized cells, is required for the functioning of the heart. The basic helix loop helix factors Hand1 and Hand2 are required for cardiac morphogenesis and have been implicated in cardiac conduction system development and maintenance. Here we use embryonic and post-natal specific Cre lines to interrogate the role of Hand1 and Hand2 in the function of the murine cardiac conduction system. Results demonstrate that loss of HAND1 in the post-natal conduction system does not result in any change in electrocardiogram parameters or within the ventricular conduction system as determined by optical voltage mapping. Deletion of Hand2 within the post-natal conduction system results in sex-dependent reduction in PR interval duration in these mice, suggesting a novel role for HAND2 in regulating the atrioventricular conduction. Surprisingly, results show that loss of both HAND factors within the post-natal conduction system does not cause any consistent changes in cardiac conduction system function. Deletion of Hand2 in the embryonic left ventricle results in inconsistent prolongation of PR interval and susceptibility to atrial arrhythmias. Thus, these results suggest a novel role for HAND2 in homeostasis of the murine cardiac conduction system and that HAND1 loss potentially rescues the shortened HAND2 PR phenotype.

Hand2 delineates mesothelium progenitors and is reactivated in mesothelioma.

The mesothelium lines body cavities and surrounds internal organs, widely contributing to homeostasis and regeneration. Mesothelium disruptions cause visceral anomalies and mesothelioma tumors. Nonetheless, the embryonic emergence of mesothelia remains incompletely understood. Here, we track mesothelial origins in the lateral plate mesoderm (LPM) using zebrafish. Single-cell transcriptomics uncovers a post-gastrulation gene expression signature centered on hand2 in distinct LPM progenitor cells. We map mesothelial progenitors to lateral-most, hand2-expressing LPM and confirm conservation in mouse. Time-lapse imaging of zebrafish hand2 reporter embryos captures mesothelium formation including pericardium, visceral, and parietal peritoneum. We find primordial germ cells migrate with the forming mesothelium as ventral migration boundary. Functionally, hand2 loss disrupts mesothelium formation with reduced progenitor cells and perturbed migration. In mouse and human mesothelioma, we document expression of LPM-associated transcription factors including Hand2, suggesting re-initiation of a developmental program. Our data connects mesothelium development to Hand2, expanding our understanding of mesothelial pathologies.

GATA3 is essential for separating patterning domains during facial morphogenesis.

Neural crest cells (NCCs) within the mandibular and maxillary prominences of the first pharyngeal arch are initially competent to respond to signals from either region. However, mechanisms that are only partially understood establish developmental tissue boundaries to ensure spatially correct patterning. In the 'hinge and caps' model of facial development, signals from both ventral prominences (the caps) pattern the adjacent tissues whereas the intervening region, referred to as the maxillomandibular junction (the hinge), maintains separation of the mandibular and maxillary domains. One cap signal is GATA3, a member of the GATA family of zinc-finger transcription factors with a distinct expression pattern in the ventral-most part of the mandibular and maxillary portions of the first arch. Here, we show that disruption of Gata3 in mouse embryos leads to craniofacial microsomia and syngnathia (bony fusion of the upper and lower jaws) that results from changes in BMP4 and FGF8 gene regulatory networks within NCCs near the maxillomandibular junction. GATA3 is thus a crucial component in establishing the network of factors that functionally separate the upper and lower jaws during development.

Deletion of a Hand1 lncRNA-Containing Septum Transversum Enhancer Alters lncRNA Expression but Is Not Required for Hand1 Expression.

We have previously identified a Hand1 transcriptional enhancer that drives expression within the septum transversum, the origin of the cells that contribute to the epicardium. This enhancer directly overlaps a common exon of a predicted family of long non-coding RNAs (lncRNA) that are specific to mice. To interrogate the necessity of this Hand1 enhancer, as well as the importance of these novel lncRNAs, we deleted the enhancer sequences, including the common exon shared by these lncRNAs, using genome editing. Resultant homozygous Hand1 enhancer mutants (Hand1ΔST/ΔST) present with no observable phenotype. Assessment of lncRNA expression reveals that Hand1ΔST/ΔST mutants effectively eliminate detectable lncRNA expression. Expression analysis within Hand1ΔST/ΔST mutant hearts indicates higher levels of Hand1 than in controls. The generation of Hand1 compound heterozygous mutants with the Hand1LacZ null allele (Hand1ΔST/LacZ) also did not reveal any observable phenotypes. Together these data indicate that deletion of this Hand1 enhancer and by consequence a family of murine-specific lncRNAs does not impact embryonic development in observable ways.

Mechanisms Underlying Cardiomyocyte Development: Can We Exploit Them to Regenerate the Heart?

PURPOSE OF REVIEW: It is well established that the adult mammalian cardiomyocytes retain a low capacity for cell cycle activity; however, it is insufficient to effectively respond to myocardial injury and facilitate cardiac regenerative repair. Lessons learned from species in which cardiomyocytes do allow for proliferative regeneration/repair have shed light into the mechanisms underlying cardiac regeneration post-injury. Importantly, many of these mechanisms are conserved across species, including mammals, and efforts to tap into these mechanisms effectively within the adult heart are currently of great interest. RECENT FINDINGS: Targeting the endogenous gene regulatory networks (GRNs) shown to play roles in the cardiac regeneration of conducive species is seen as a strong approach, as delivery of a single or combination of genes has promise to effectively enhance cell cycle activity and CM proliferation in adult hearts post-myocardial infarction (MI). In situ re-induction of proliferative gene regulatory programs within existing, local, non-damaged cardiomyocytes helps overcome significant technical hurdles, such as successful engraftment of implanted cells or achieving complete cardiomyocyte differentiation from cell-based approaches. Although many obstacles currently exist and need to be overcome to successfully translate these approaches to clinical settings, the current efforts presented here show great promise.

Epigenetics and Heart Development.

Epigenetic control of gene expression during cardiac development and disease has been a topic of intense research in recent years. Advances in experimental methods to study DNA accessibility, transcription factor occupancy, and chromatin conformation capture technologies have helped identify regions of chromatin structure that play a role in regulating access of transcription factors to the promoter elements of genes, thereby modulating expression. These chromatin structures facilitate enhancer contacts across large genomic distances and function to insulate genes from cis-regulatory elements that lie outside the boundaries for the gene of interest. Changes in transcription factor occupancy due to changes in chromatin accessibility have been implicated in congenital heart disease. However, the factors controlling this process and their role in changing gene expression during development or disease remain unclear. In this review, we focus on recent advances in the understanding of epigenetic factors controlling cardiac morphogenesis and their role in diseases.

HAND transcription factors cooperatively specify the aorta and pulmonary trunk.

Congenital heart defects (CHDs) affecting the cardiac outflow tract (OFT) constitute a significant cause of morbidity and mortality. The OFT develops from migratory cell populations which include the cardiac neural crest cells (cNCCs) and secondary heart field (SHF) derived myocardium and endocardium. The related transcription factors HAND1 and HAND2 have been implicated in human CHDs involving the OFT. Although Hand1 is expressed within the OFT, Hand1 NCC-specific conditional knockout mice (H1CKOs) are viable. Here we show that these H1CKOs present a low penetrance of OFT phenotypes, whereas SHF-specific Hand1 ablation does not reveal any cardiac phenotypes. Further, HAND1 and HAND2 appear functionally redundant within the cNCCs, as a reduction/ablation of Hand2 on an NCC-specific H1CKO background causes pronounced OFT defects. Double conditional Hand1 and Hand2 NCC knockouts exhibit persistent truncus arteriosus (PTA) with 100% penetrance. NCC lineage-tracing and Sema3c in situ mRNA expression reveal that Sema3c-expressing cells are mis-localized, resulting in a malformed septal bridge within the OFTs of H1CKO;H2CKO embryos. Interestingly, Hand1 and Hand2 also genetically interact within the SHF, as SHF H1CKOs on a heterozygous Hand2 background exhibit Ventricular Septal Defects (VSDs) with incomplete penetrance. Previously, we identified a BMP, HAND2, and GATA-dependent Hand1 OFT enhancer sufficient to drive reporter gene expression within the nascent OFT and aorta. Using these transcription inputs as a probe, we identify a novel Hand2 OFT enhancer, suggesting that a conserved BMP-GATA dependent mechanism transcriptionally regulates both HAND factors. These findings support the hypothesis that HAND factors interpret BMP signaling within the cNCCs to cooperatively coordinate OFT morphogenesis.

The heart of the neural crest: cardiac neural crest cells in development and regeneration.

Cardiac neural crest cells (cNCCs) are a migratory cell population that stem from the cranial portion of the neural tube. They undergo epithelial-to-mesenchymal transition and migrate through the developing embryo to give rise to portions of the outflow tract, the valves and the arteries of the heart. Recent lineage-tracing experiments in chick and zebrafish embryos have shown that cNCCs can also give rise to mature cardiomyocytes. These cNCC-derived cardiomyocytes appear to be required for the successful repair and regeneration of injured zebrafish hearts. In addition, recent work examining the response to cardiac injury in the mammalian heart has suggested that cNCC-derived cardiomyocytes are involved in the repair/regeneration mechanism. However, the molecular signature of the adult cardiomyocytes involved in this repair is unclear. In this Review, we examine the origin, migration and fates of cNCCs. We also review the contribution of cNCCs to mature cardiomyocytes in fish, chick and mice, as well as their role in the regeneration of the adult heart.

SHROOM3 is downstream of the planar cell polarity pathway and loss-of-function results in congenital heart defects.

Congenital heart disease (CHD) is the most common birth defect, and the leading cause of death due to birth defects, yet causative molecular mechanisms remain mostly unknown. We previously implicated a novel CHD candidate gene, SHROOM3, in a patient with CHD. Using a Shroom3 gene trap knockout mouse (Shroom3gt/gt) we demonstrate that SHROOM3 is downstream of the noncanonical Wnt planar cell polarity signaling pathway (PCP) and loss-of-function causes cardiac defects. We demonstrate Shroom3 expression within cardiomyocytes of the ventricles and interventricular septum from E10.5 onward, as well as within cardiac neural crest cells and second heart field cells that populate the cardiac outflow tract. We demonstrate that Shroom3gt/gt mice exhibit variable penetrance of a spectrum of CHDs that include ventricular septal defects, double outlet right ventricle, and thin left ventricular myocardium. This CHD spectrum phenocopies what is observed with disrupted PCP. We show that during cardiac development SHROOM3 interacts physically and genetically with, and is downstream of, key PCP signaling component Dishevelled 2. Within Shroom3gt/gt hearts we demonstrate disrupted terminal PCP components, actomyosin cytoskeleton, cardiomyocyte polarity, organization, proliferation and morphology. Together, these data demonstrate SHROOM3 functions during cardiac development as an actomyosin cytoskeleton effector downstream of PCP signaling, revealing SHROOM3's novel role in cardiac development and CHD.

Mis-Expression of a Cranial Neural Crest Cell-Specific Gene Program in Cardiac Neural Crest Cells Modulates HAND Factor Expression, Causing Cardiac Outflow Tract Phenotypes.

Congenital heart defects (CHDs) occur with such a frequency that they constitute a significant cause of morbidity and mortality in both children and adults. A significant portion of CHDs can be attributed to aberrant development of the cardiac outflow tract (OFT), and of one of its cellular progenitors known as the cardiac neural crest cells (NCCs). The gene regulatory networks that identify cardiac NCCs as a distinct NCC population are not completely understood. Heart and neural crest derivatives (HAND) bHLH transcription factors play essential roles in NCC morphogenesis. The Hand1PA/OFT enhancer is dependent upon bone morphogenic protein (BMP) signaling in both cranial and cardiac NCCs. The Hand1PA/OFT enhancer is directly repressed by the endothelin-induced transcription factors DLX5 and DLX6 in cranial but not cardiac NCCs. This transcriptional distinction offers the unique opportunity to interrogate NCC specification, and to understand why, despite similarities, cranial NCC fate determination is so diverse. We generated a conditionally active transgene that can ectopically express DLX5 within the developing mouse embryo in a Cre-recombinase-dependent manner. Ectopic DLX5 expression represses cranial NCC Hand1PA/OFT-lacZ reporter expression more effectively than cardiac NCC reporter expression. Ectopic DLX5 expression induces broad domains of NCC cell death within the cranial pharyngeal arches, but minimal cell death in cardiac NCC populations. This study shows that transcription control of NCC gene regulatory programs is influenced by their initial specification at the dorsal neural tube.

HAND1 loss-of-function within the embryonic myocardium reveals survivable congenital cardiac defects and adult heart failure.

AIMS: To examine the role of the basic Helix-loop-Helix (bHLH) transcription factor HAND1 in embryonic and adult myocardium. METHODS AND RESULTS: Hand1 is expressed within the cardiomyocytes of the left ventricle (LV) and myocardial cuff between embryonic days (E) 9.5-13.5. Hand gene dosage plays an important role in ventricular morphology and the contribution of Hand1 to congenital heart defects requires further interrogation. Conditional ablation of Hand1 was carried out using either Nkx2.5 knockin Cre (Nkx2.5Cre) or α-myosin heavy chain Cre (αMhc-Cre) driver. Interrogation of transcriptome data via ingenuity pathway analysis reveals several gene regulatory pathways disrupted including translation and cardiac hypertrophy-related pathways. Embryo and adult hearts were subjected to histological, functional, and molecular analyses. Myocardial deletion of Hand1 results in morphological defects that include cardiac conduction system defects, survivable interventricular septal defects, and abnormal LV papillary muscles (PMs). Resulting Hand1 conditional mutants are born at Mendelian frequencies; but the morphological alterations acquired during cardiac development result in, the mice developing diastolic heart failure. CONCLUSION: Collectively, these data reveal that HAND1 contributes to the morphogenic patterning and maturation of cardiomyocytes during embryogenesis and although survivable, indicates a role for Hand1 within the developing conduction system and PM development.

Twist1 Inactivation in Dmp1-Expressing Cells Increases Bone Mass but Does Not Affect the Anabolic Response to Sclerostin Neutralization.

Wnt signaling plays a major role in bone metabolism. Advances in our understanding of secreted regulators of Wnt have yielded several therapeutic targets to stimulate osteoanabolism-the most promising of which is the Wnt inhibitor sclerostin. Sclerostin antibody recently gained approval for clinical use to treat osteoporosis, but the biology surrounding sclerostin antagonism is still incompletely understood. Numerous factors regulate the efficacy of sclerostin inhibition on bone formation, a process known as self-regulation. In previous communications we reported that the basic helix-loop-helix transcription factor Twist1-a gene know to regulate skeletal development-is highly upregulated among the osteocyte cell population in mice treated with sclerostin antibody. In this communication, we tested the hypothesis that preventing Twist1 upregulation by deletion of Twist1 from late-stage osteoblasts and osteocytes would increase the efficacy of sclerostin antibody treatment, since Twist1 is known to restrain osteoblast activity in many models. Twist1-floxed loss-of-function mice were crossed to the Dmp1-Cre driver to delete Twist1 in Dmp1-expressing cells. Conditional Twist1 deletion was associated with a mild but significant increase in bone mass, as assessed by dual energy x-ray absorptiometry (DXA) and microCT (µCT) for many endpoints in both male and female mice. Biomechanical properties of the femur were not affected by conditional mutation of Twist1. Sclerostin antibody improved all bone properties significantly, regardless of Twist1 status, sex, or endpoint examined. No interactions were detected when Twist1 status and antibody treatment were examined together, suggesting that Twist1 upregulation in the osteocyte population is not an endogenous mechanism that restrains the osteoanabolic effect of sclerostin antibody treatment. In summary, Twist1 inhibition in the late-stage osteoblast/osteocyte increases bone mass but does not affect the anabolic response to sclerostin neutralization.

Variation in a Left Ventricle-Specific Hand1 Enhancer Impairs GATA Transcription Factor Binding and Disrupts Conduction System Development and Function.

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 (r2>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.

Partially Penetrant Cardiac Neural Crest Defects in Hand1 Phosphomutant Mice: Dimer Choice That Is Not So Critical.

Hand1 is a basic Helix-loop-Helix transcription factor that exhibits post-translationally regulated dimer partner choice that allows for a diverse set of Hand1 transcriptional complexes. Indeed, when Hand1 phosphoregulation is altered, conditionally activated hypophorylation (Hand1PO4-) and phosphorylation mimic (Hand1PO4+) Hand1 alleles disrupt both craniofacial and limb morphogenesis with 100% penetrance. Interestingly, activation of conditional Hand1 Phosphomutant alleles within post-migratory neural crest cells produce heart defects that include ventricular septal defects, double-outlet right ventricle, persistent truncus arteriosus with partial penetrance. Single versus double-lobed thymus is a distinguishing feature between Wnt1-Cre;Hand1PO4-/+ and Wnt1-Cre;Hand1PO4+/+ mice. These data show that although Hand1 dimer regulation plays critical and consistent roles in disrupting craniofacial and limb morphogenesis, Hand1 dimer regulation during cardiac outflow track formation is less critical for normal morphogenesis. This review will present the OFT phenotypes observed in Hand1 Phosphomutant mice, and discuss possible mechanisms of how penetrance differences within the same tissues within the same embryos could be variable.

Hand Factors in Cardiac Development.

Congenital heart defects account for 1% of infant mortality and 10% of in utero deaths. As the vertebrate embryo develops, multiple tissue types develop in tandem to morphologically pattern the functional heart. Underlying cardiac development is a network of transcription factors known to tightly control these morphological events. Members of the Twist family of basic helix-loop-helix transcription factors, Hand1 and Hand2, are essential to this process. The expression patterns and functional role of Hand factors in neural crest cells, endocardium, myocardium, and epicardium is indicative of their importance during cardiogenesis; however, to date, an extensive understanding of the transcriptional targets of Hand proteins and their overall mechanism of action remain unclear. In this review, we summarize the recent findings that further outline the crucial functions of Hand factors during heart development and in post-natal heart function. Anat Rec, 302:101-107, 2019. © 2018 Wiley Periodicals, Inc.

Notch signaling regulates Hey2 expression in a spatiotemporal dependent manner during cardiac morphogenesis and trabecular specification.

Hey2 gene mutations in both humans and mice have been associated with multiple cardiac defects. However, the currently reported localization of Hey2 in the ventricular compact zone cannot explain the wide variety of cardiac defects. Furthermore, it was reported that, in contrast to other organs, Notch doesn't regulate Hey2 in the heart. To determine the expression pattern and the regulation of Hey2, we used novel methods including RNAscope and a Hey2 CreERT2 knockin line to precisely determine the spatiotemporal expression pattern and level of Hey2 during cardiac development. We found that Hey2 is expressed in the endocardial cells of the atrioventricular canal and the outflow tract, as well as at the base of trabeculae, in addition to the reported expression in the ventricular compact myocardium. By disrupting several signaling pathways that regulate trabeculation and/or compaction, we found that, in contrast to previous reports, Notch signaling and Nrg1/ErbB2 regulate Hey2 expression level in myocardium and/or endocardium, but not its expression pattern: weak expression in trabecular myocardium and strong expression in compact myocardium. Instead, we found that FGF signaling regulates the expression pattern of Hey2 in the early myocardium, and regulates the expression level of Hey2 in a Notch1 dependent manner.

The HAND1 frameshift A126FS mutation does not cause hypoplastic left heart syndrome in mice.

AIMS: To test if a human Hand1 frame shift mutation identified in human samples is causative of hypoplastic left heart syndrome (HLHS). METHODS AND RESULTS: HLHS is a poorly understood single ventricle congenital heart defect that affects two to three infants in every 10 000 live births. The aetiologies of HLHS are largely unknown. The basic helix-loop-helix transcription factor HAND1 is required for normal heart development. Interrogation of HAND1 sequence from fixed HLHS tissues identified a somatic frame-shift mutation at Alanine 126 (NP_004812.1 p.Ala126Profs13X defined as Hand1A126fs). Hand1A126fs creates a truncated HAND1 protein that predictively functions as dominant negative. To determine if this mutation is causative of HLHS, we engineered a conditional Hand1A126fs mouse allele. Activation of this allele with Nkx2.5Cre results in E14.5 lethality accompanied by cardiac outflow tract and intraventricular septum abnormalities. Using αMHC-Cre or Mef2CAHF-Cre to activate Hand1A126fs results in reduced phenotype and limited viability. Left ventricles of Hand1A126FS mutant mice are not hypoplastic. CONCLUSIONS: Somatically acquired Hand1A126FS mutation is not causative of HLHS. Hand1A126FS mutation does exhibit embryonic lethal cardiac defects that reflect a dominant negative function supporting the critical role of Hand1 in cardiogenesis.

Hand factor ablation causes defective left ventricular chamber development and compromised adult cardiac function.

Coordinated cardiomyocyte growth, differentiation, and morphogenesis are essential for heart formation. We demonstrate that the bHLH transcription factors Hand1 and Hand2 play critical regulatory roles for left ventricle (LV) cardiomyocyte proliferation and morphogenesis. Using an LV-specific Cre allele (Hand1LV-Cre), we ablate Hand1-lineage cardiomyocytes, revealing that DTA-mediated cardiomyocyte death results in a hypoplastic LV by E10.5. Once Hand1-linage cells are removed from the LV, and Hand1 expression is switched off, embryonic hearts recover by E16.5. In contrast, conditional LV loss-of-function of both Hand1 and Hand2 results in aberrant trabeculation and thickened compact zone myocardium resulting from enhanced proliferation and a breakdown of compact zone/trabecular/ventricular septal identity. Surviving Hand1;Hand2 mutants display diminished cardiac function that is rescued by concurrent ablation of Hand-null cardiomyocytes. Collectively, we conclude that, within a mixed cardiomyocyte population, removal of defective myocardium and replacement with healthy endogenous cardiomyocytes may provide an effective strategy for cardiac repair.