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The heart is lined by a single layer of mesothelial cells called the epicardium that provides important cellular contributions for embryonic heart formation. The epicardium harbors a population of progenitor cells that undergo epithelial-to-mesenchymal transition displaying characteristic conversion of planar epithelial cells into multipolar and invasive mesenchymal cells before differentiating into nonmyocyte cardiac lineages, such as vascular smooth muscle cells, pericytes, and fibroblasts. The epicardium is also a source of paracrine cues that are essential for fetal cardiac growth, coronary vessel patterning, and regenerative heart repair. Although the epicardium becomes dormant after birth, cardiac injury reactivates developmental gene programs that stimulate epithelial-to-mesenchymal transition; however, it is not clear how the epicardium contributes to disease progression or repair in the adult. In this review, we will summarize the molecular mechanisms that control epicardium-derived progenitor cell migration, and the functional contributions of the epicardium to heart formation and cardiomyopathy. Future perspectives will be presented to highlight emerging therapeutic strategies aimed at harnessing the regenerative potential of the fetal epicardium for cardiac repair.

Sex disparities in cardiac homeostasis and heart disease are well documented, with differences attributed to actions of sex hormones. However, studies have indicated sex chromosomes act outside of the gonads to function without mediation by gonadal hormones. Here, we performed transcriptional and proteomics profiling to define differences between male and female mouse hearts. We demonstrate, contrary to current dogma, cardiac sex disparities are controlled not only by sex hormones but also through a sex-chromosome mechanism. Using Turner syndrome (XO) and Klinefelter (XXY) models, we find the sex-chromosome pathway is established by X-linked gene dosage. We demonstrate cardiac sex disparities occur at the earliest stages of heart formation, a period before gonad formation. Using these datasets, we identify and define a role for alpha-1B-glycoprotein (A1BG), showing loss of A1BG leads to cardiac defects in females, but not males. These studies provide resources for studying sex-biased cardiac disease states.

MESP1 and MESP2 are transcriptional factors involved in mesoderm specification, somite boundary formation and somite polarity regulation. However, Mesp quadruple mutant zebrafish displayed only abnormal somite polarity without mesoderm specification defects. In order to re-evaluate Mesp1/Mesp2 mutants in mice, Mesp1 and Mesp2 single knockouts (KOs), and a Mesp1/Mesp2 double KO were established using genome-editing techniques without introducing selection markers commonly used before. The Mesp1/Mesp2 double KO embryos exhibited markedly severe mesoderm formation defects that were similar to the previously reported Mesp1/Mesp2 double KO embryos, indicating species differences in the function of MESP family proteins. However, the Mesp1 KO did not display any phenotype, including heart formation defects, which have been reported previously. We noted upregulation of Mesp2 in the Mesp1 KO embryos, suggesting that MESP2 rescues the loss of MESP1 in mesoderm specification. We also found that Mesp1 and Mesp2 expression in the early mesoderm is regulated by the cooperation of two independent enhancers containing T-box- and TCF/Lef-binding sites. Deletion of both enhancers caused the downregulation of both genes, resulting in heart formation defects. This study suggests dose-dependent roles of MESP1 and MESP2 in early mesoderm formation.

Cells display distinct metabolic characteristics depending on its differentiation stage. The fuel type of the cells serves not only as a source of energy but also as a driver of differentiation. Glucose, the primary nutrient to the cells, is a critical regulator of rapidly growing embryos. This metabolic change is a consequence as well as a cause of changes in genetic program. Disturbance of fetal glucose metabolism such as in diabetic pregnancy is associated with congenital heart disease. In utero hyperglycemia impacts the left-right axis establishment, migration of cardiac neural crest cells, conotruncal formation and mesenchymal formation of the cardiac cushion during early embryogenesis and causes cardiac hypertrophy in late fetal stages. In this review, we focus on the role of glucose in cardiogenesis and the molecular mechanisms underlying heart diseases associated with hyperglycemia.

Heart formation in the zebrafish involves a rapid, complex series of morphogenetic events in three-dimensional space that spans cardiac lineage specification through to chamber formation and maturation. This process is tightly orchestrated by a cardiac gene regulatory network (GRN), which ensures the precise spatio-temporal deployment of genes critical for heart formation. Alterations of the timing or spatial localisation of gene expression can have a significant impact in cardiac ontogeny and may lead to heart malformations. Hence, a better understanding of the cellular and molecular basis of congenital heart disease relies on understanding the behaviour of cardiac GRNs with precise spatiotemporal resolution. Here, we review the recent technical advances that have expanded our capacity to interrogate the cardiac GRN in zebrafish. In particular, we focus on studies utilising high-throughput technologies to systematically dissect gene expression patterns, both temporally and spatially during heart development.

The use of alternative biocides has increased due to their economic and ecological relevance. Although data regarding the toxicity of commercial alternative biocides in marine organisms are accumulating, little is known about their toxic pathways or mechanisms. To compare the toxic effects of commercial alternative biocides on non-target pelagic fish (flounder) embryos, we investigated the adverse effects of developmental malformation and transcriptional changes. Three biocides including Diuron, Irgarol 1051® and Sea-Nine 211® produced a largely overlapping suite of developmental malformations, including tail-fin fold defects and dorsal body axis curvature. In our test, the potencies of these biocides were ranked in the following order with respect to malformation and mortalities: Sea-Nine 211® > Irgarol 1051® > Diuron. Consistent with the toxicity rankings, the expression of genes related to heart formation was greater in embryonic flounder exposed to Sea-Nine 211® than in those exposed to Irgarol 1051® or Diuron, while expression of genes related to fin malformation was greater in the Irgarol 1051® exposure group. In analyses of differential gene expression (DEG) profiles (fold change of genes with a cutoff P

Proper heart formation requires coordinated development of two anatomically distinct groups of cells - the first and second heart fields (FHF and SHF). Given that congenital heart defects are often restricted to derivatives of the FHF or SHF, it is crucial to understand the mechanisms controlling their development. Wnt signaling has previously been implicated in SHF proliferation; however, the source of Wnts remains unknown. Through comparative gene analysis, we found upregulation of Wnts and Wnt receptor/target genes in the FHF and SHF, respectively, raising the possibility that early cardiac progenitors may secrete Wnts to influence SHF cell fate. To probe this further, we deletedWntless (Wls), a gene required for Wnt ligand secretion, in various populations of precardiac cells. Deletion of Wls in Mesp1+cells resulted in formation of a single chamber heart with left ventricle identity, implying compromised SHF development. This phenotype was recapitulated by deleting Wls in cells expressing Islet1, a pan-cardiac marker. Similarly, Wls deletion in cells expressing Nkx2.5, a later-expressed pan-cardiac marker, resulted in hypoplastic right ventricle, a structure derived from the SHF. However, no developmental defects were observed when deleting Wls in SHF progenitors. To gain mechanistic insights, we isolated Mesp1-lineage cells from developing embryos and performed single-cell RNA-sequencing. Our comprehensive single cell transcriptome analysis revealed that Wls deletion dysregulates developmental trajectories of both anterior and posterior SHF cells, marked by impaired proliferation and premature differentiation. Together, these results demonstrate a critical role of local precardiac mesodermal Wnts in SHF fate decision, providing fundamental insights into understanding heart field development and chamber formation.Significance StatementThere is significant interest in understanding the mechanisms underlying heart formation to develop treatments and cures for patients suffering from congenital heart disease. In particular, we were interested in the intricacies of first (FHF) and second heart field (SHF) development, as many congenital heart defects present with heart field-specific etiologies. Here, we uncovered a novel relationship between specified cardiac progenitor cells and second heart field progenitors. Through genetic manipulation of Wnt secretion in developing mouse embryos, we identified a population of cardiac progenitor cells that acts as a local source of Wnts which are necessary for proper SHF development. Our single cell transcriptomic analysis of developing anterior mesoderm showed cardiac progenitor-secreted Wnts function through regulation of differentiation and proliferation among SHF progenitors. Thus, this study provides insight into the source and timing of Wnts required for SHF development, and points to the crucial role of co-developing cell populations in heart development.

The origins of congenital heart diseases, the most common congenital diseases are still largely unknown. Environmental factors are now emerging as major causes of these diseases. Vitamin D deficiency has become a public health burden, notably for childbearing age, pregnant and breastfeeding women. Since maternal 25-hydroxyvitamin D (25(OH)D) determined fetal and neonatal 25(OH)D status, foetuses exposed to insufficient levels of vitamin D, may feature developmental defects.Herein, we investigated the effects of maternal vitamin D deficiency on cardiovascular defects in early and later life of offsprings in two generations as well as the molecular mechanisms underlying vitamin D effect.Eight weeks before and during pregnancy, C57BL/6JRj female mice received a sufficient or vitamin D deficient diet ((1.0 IU/g in control vs 0.0 IU/g in Vitamin D Deficient (VDD) group). E16.5 Embryos of maternal VDD diet featured hypertrophic heart revealed by a thicker left ventricular (LV) wall and septum. RNAseq analysis of LV revealed 1555 transcripts differentially expressed in the VDD group and among them cardiac transcription factors and constitutive cardiac genes (tbx5, gata4, myl2). Anti-Vitamin D receptor (VDR) Chip-seq from chromatin of E16.5 LV uncovered different targeting of tbx5 and tbx3 loci by VDR in the VDD vs control embryos. Anti-CTCF ChIP-loop experiments focusing on the Tbx3 and Tbx5 loci uncovered a change in the Topology Associated Domains associated with these loci.Echocardiography of 2-months-old VDD offspring revealed a significantly thicker left ventricle and increased fractional shortening while 6-months-old mice featured cardiac decompensation and in turn failing LV.Maternal vitamin D deficiency severely affects heart formation following a change in chromatin conformation on cardiac gene loci and impacts function of adult hearts in two generations. These defects are likely to be at the origin of cardiovascular diseases in the adulthood.

Cardiovascular disease is a serious threat to human health and a leading cause of mortality worldwide. Recent years have witnessed exciting progress in the understanding of heart formation and development, enabling cardiac biologists to make significant advance in the field of therapeutic heart regeneration. Most of our understanding of heart development and regeneration, including the genes and signaling pathways, are driven by pioneering works in non-mammalian model organisms, such as fruit fly, fish, frog, and chicken. Compared to mammalian animal models, non-mammalian model organisms have special advantages in high-throughput applications such as disease modeling, drug discovery, and cardiotoxicity screening. Genetically engineered animals of cardiovascular diseases provide valuable tools to investigate the molecular and cellular mechanisms of pathogenesis and to evaluate therapeutic strategies. A large number of congenital heart diseases (CHDs) non-mammalian models have been established and tested for the genes and signaling pathways involved in the diseases. Here, we reviewed the mechanisms of heart development and regeneration revealed by these models, highlighting the advantages of non-mammalian models as tools for cardiac research. The knowledge from these animal models will facilitate therapeutic discoveries and ultimately serve to accelerate translational medicine.

During embryonic heart development, epicardial cells residing within the outer layer of the heart undergo epithelial-mesenchymal transition (EMT) and migrate into the myocardium to support and stimulate organ growth and morphogenesis. Disruption of epicardial EMT results in aberrant heart formation and embryonic lethality. Despite being an essential process during development, the regulation of epicardial EMT is poorly understood. Here we report EMT on the epicardial surface of the embryonic heart at subcellular resolution using scanning electron microscopy (SEM). We identified high- and low-EMT regions within the mesothelial layer of the epicardium and an association with key components of the extracellular matrix (ECM). The ECM basement membrane-associated proteoglycan agrin was found to localize in the epicardium in regions actively undergoing EMT. Deletion of agrin resulted in impaired EMT and compromised development of the epicardium, accompanied by down-regulation of the epicardial EMT regulator WT1. Agrin enhanced EMT in human embryonic stem cell-derived epicardial-like cells by decreasing β-catenin and promoting pFAK localization at focal adhesions. In addition, agrin promoted the aggregation of its receptor dystroglycan to the Golgi apparatus in murine epicardial cells and loss of agrin resulted in dispersal of dystroglycan throughout the epicardial cells in embryos, disrupting basement membrane integrity and impairing EMT. Our results provide new insights into the role of the ECM in heart development, and implicate agrin as a critical regulator of EMT, functioning to ensure dystroglycan connects signals between the ECM and activated epicardial cells.Summary statementThe basement membrane-associated proteoglycan agrin regulates epicardial epithelia-to-mesenchyme transition (EMT) through dystroglycan localizing on the Golgi apparatus. This ensures ECM and cytoskeletal connectivity and mechanical integrity of the transitioning epicardium and has important implications for the role of the extracellular matrix (ECM) in heart development.

Heart development in the chicken embryo is regulated by a concert of cardiogenic morphogens and signaling molecules, but the physiological signal molecule nitric oxide(NO) has not been studied in the context of heart formation. A dynamic investigation of endoderm NO formation demonstrates for the first time a correlation with the established development events of the cardiac heart fields and heart tube. Manipulation of endoderm NO signaling demonstrate a role of NO signaling in the differentiation and proliferation of cardiac progenitors for heart tube formation and cardiac heart field development. To investigate NO in the proliferation of myocardial cells in the heart tube embryos, a computer vision based artificial intelligence approach is followed to automate the long and tedious job of counting cells in a large image dataset. We document NO as an important signaling molecule in the regulation of nascent embryonic cardiogenesis whose effects on other early cardiogenic morphogens is unknown.

The role mechanical forces play in shaping the structure and function of the heart is critical to understanding heart formation and the etiology of disease but is challenging to study in patients. Engineered heart tissues (EHTs) incorporating human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes have the potential to provide insight into these adaptive and maladaptive changes in the heart. However, most EHT systems are unable to model both preload (stretch during chamber filling) and afterload (pressure the heart must work against to eject blood). Here, we have developed a new dynamic EHT (dyn-EHT) model that enables us to tune preload and have unconstrained fractional shortening of >10%. To do this, 3D EHTs are integrated with an elastic polydimethylsiloxane (PDMS) strip that provides mechanical pre- and afterload to the tissue in addition to enabling contractile force measurements based on strip bending. Our results demonstrate in wild-type EHTs that dynamic loading is beneficial based on the magnitude of the forces, leading to improved alignment, conduction velocity, and contractility. For disease modeling, we use hiPSC–derived cardiomyocytes from a patient with arrhythmogenic cardiomyopathy (ACM) due to mutations in desmoplakin. We demonstrate that manifestation of this desmosome-linked disease state requires the dyn-EHT conditioning and that it cannot be induced using 2D or standard 3D EHT approaches. Thus, dynamic loading strategy is necessary to provoke a disease phenotype (diastolic lengthening, reduction of desmosome counts, and reduced contractility), which are akin to primary endpoints of clinical disease, such as chamber thinning and reduced cardiac output.Single Sentence SummaryDevelopment of a dynamic mechanical loading platform to improve contractile function of engineered heart tissues and study cardiac disease progression.

ADP-ribosylhydrolase-like 1 (Adprhl1) is a pseudoenzyme expressed in the developing heart myocardium of all vertebrates. In the amphibianXenopus laevis, knockdown of the two cardiac Adprhl1 protein species (40 and 23 kDa) causes failure of chamber outgrowth but this has only been demonstrated using antisense morpholinos that interfere with RNA-splicing. Transgenic production of 40 kDa Adprhl1 provides only part rescue of these defects. CRISPR/Cas9 technology now enables targeted mutation of theadprhl1gene in G0-generation embryos with routine cleavage of all alleles. Testing multiple gRNAs distributed across the locus reveals exonic locations that encode critical amino acids for Adprhl1 function. The gRNA recording the highest frequency of a specific ventricle outgrowth phenotype directs Cas9 cleavage of an exon 6 sequence, where microhomology mediated end-joining biases subsequent DNA repairs towards three small in-frame deletions. Mutant alleles encode discrete loss of 1, 3 or 4 amino acids from a di-arginine (Arg271-Arg272) containing peptide loop at the centre of the ancestral ADP-ribosylhydrolase site. Thus despite lacking catalytic activity, it is the modified (adenosine-ribose) substrate binding cleft of Adprhl1 that fulfils an essential role during heart formation. Mutation results in striking loss of myofibril assembly in ventricle cardiomyocytes. The defects suggest Adprhl1 participation from the earliest stage of cardiac myofibrillogenesis and are consistent with previous MO results and Adprhl1 protein localization to actin filament Z-disc boundaries. A single nucleotide change to the gRNA sequence renders it inactive. Mice lackingAdprhl1exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.HighlightsComparison ofadprhl1morpholinos. Knockdown of the twoXenopuscardiac Adprhl1 protein species (40 and 23 kDa) causes failure of ventricle outgrowth.CRISPR/Cas9 targeted gene mutation ofadprhl1with multiple gRNAs reveals exonic locations that encode critical amino acids for Adprhl1 function.Repair of DSBs at exon 6 yields small in-frame deletions that cause specific ventricle myofibril assembly defects.The deletions disturb a conserved di-arginine containing peptide loop at the centre of the ancestral substrate binding cleft/ADP-ribosylhydrolase site of this pseudoenzyme.Mice lackingAdprhl1exons 3-4 are normal but production of the smaller ADPRHL1 species is unaffected, providing further evidence that cardiac activity is concentrated at the C-terminal protein portion.

During heart formation, the heart grows and undergoes dramatic morphogenesis to achieve efficient embryonic function. Both in fish and amniotes, much of the growth occurring after initial heart tube formation arises from second heart field (SHF)-derived progenitor cell addition to the arterial pole, allowing chamber formation. In zebrafish, this process has been extensively studied during embryonic life, but it is unclear how larval cardiac growth occurs beyond 3 days post-fertilisation (dpf). By quantifying zebrafish myocardial growth using live imaging of GFP-labelled myocardium we show that the heart grows extensively between 3 and 5 dpf. Using methods to assess cell division, cellular development timing assay and Kaede photoconversion, we demonstrate that proliferation, CM addition, and hypertrophy contribute to ventricle growth. Mechanistically, we show that reduction in Mef2c activity (mef2ca+/−;mef2cb−/−), downstream or in parallel with Nkx2.5 and upstream of Ltbp3, prevents some CM addition and differentiation, resulting in a significantly smaller ventricle by 3 dpf. After 3 dpf, however, CM addition in mef2ca+/−;mef2cb−/− mutants recovers to a normal pace, and the heart size gap between mutants and their siblings diminishes into adulthood. Thus, as in mice, there is an early time window when SHF contribution to the myocardium is particularly sensitive to loss of Mef2c activity., Highlights • Live image analysis showed extensive growth in zebrafish heart between 3 and 5 dpf. • Proliferation, hypertrophy and addition of external CM progenitors contribute to heart growth. • Reduced Mef2c activity, downstream or in parallel with Nkx2.5 and upstream of Ltbp3, diminishes growth resulting in a smaller ventricle by 3 dpf. • SHF CM contribution to growth is particularly sensitive to loss of Mef2c at an early heart developmental time window.

Avian embryos have been used for centuries to study development due to the ease of access. Because the embryos are sheltered inside the eggshell, a small window in the shell is ideal for visualizing the embryos and performing different interventions. The window can then be covered, and the embryo returned to the incubator for the desired amount of time, and observed during further development. Up to about 4 days of chicken development (out of 21 days of incubation), when the egg is opened the embryo is on top of the yolk, and its heart is on top of its body. This allows easy imaging of heart formation and heart development using non-invasive techniques, including regular optical microscopy. After day 4, the embryo starts sinking into the yolk, but still imaging technologies, such as ultrasound, can tomographically image the embryo and its heart in vivo. Importantly, because like the human heart the avian heart develops into a four-chambered heart with valves, heart malformations and pathologies that human babies suffer can be replicated in avian embryos, allowing a unique developmental window into human congenital heart disease. Here, we review avian heart formation and provide comparisons to the mammalian heart.

Summary Actomyosin networks provide the major contractile machinery for regulating cell and tissue morphogenesis during development. These networks undergo dynamic rearrangements, enabling cells to have a broad range of mechanical actions. How cells integrate different mechanical stimuli to accomplish complicated tasks in vivo remains unclear. Here, we explore this problem in the context of cell matching, where individual cells form precise inter-cellular connections between partner cells. To study the dynamic roles of actomyosin networks in regulating precise cell matching, we focused on the process of heart formation during Drosophila embryogenesis, where selective filopodia-binding adhesions ensure precise cell alignment. We find that non-muscle Myosin II clusters periodically oscillate within cardioblasts with ~4-min intervals. We observe that filopodia dynamics—including protrusions, retraction, binding stabilization, and binding separation—are correlated with the periodic localization of Myosin II clusters at the cell leading edge. Perturbing the Myosin II activity and oscillatory pattern alters the filopodia properties and binding dynamics and results in mismatched cardioblasts. By simultaneously changing the activity of Myosin II and filopodia adhesion levels, we further demonstrate that levels of Myosin II and adhesion are balanced to ensure precise connectivity between cardioblasts. Combined, we propose a mechanical proofreading machinery of robust cell matching, whereby oscillations of Myosin II within cardioblasts periodically probe filopodia adhesion strength and ensure correct cell-cell connection formation.

During embryonic development, the heart undergoes complex morphogenesis from a liner tube into the four chambers consisting of ventricles, atria and valves. At the same time, the cardiomyocytes compact into a dense, aligned, and highly vascularized myocardium. The extracellular matrix (ECM) is known to play an important role in this process but understanding of the expression and organization remains incomplete. Here, we performed 3D confocal imaging of ECM in the left ventricle and whole heart of embryonic chick from stages Hamburger-Hamilton 28&ndash, 35 (days 5&ndash, 9) as an accessible model of heart formation. First, we observed the formation of a fibronectin-rich, capillary-like networks in the myocardium between day 5 and day 9 of development. Then, we focused on day 5 prior to vascularization to determine the relative expression of fibronectin, laminin, and collagen type IV. Cardiomyocytes were found to uniaxially align prior to vascularization and, while the epicardium contained all ECM components, laminin was reduced, and collagen type IV was largely absent. Quantification of fibronectin revealed highly aligned fibers with a mean diameter of ~500 nm and interfiber spacing of ~3 &micro, m. These structural parameters (volume, spacing, fiber diameter, length, and orientation) provide a quantitative framework to describe the organization of the embryonic ECM.

Background Sugammadex is a new neuromuscular blockade reversal agent. Recently, it has been used in patients under general anesthesia. However, sugammadex could be toxic to fetuses and pediatric patients under 3 years of age. In this study, we demonstrated the safety of sugammadex in fetuses, using zebrafish larvae. Furthermore, its neurotoxicity was evaluated using neuronal cell lines. Methods We used SH-SY5Y cells to determine the viability of neuronal cells treated with sugammadex. Zebrafish larvae were used to determine the teratogenic effects of sugammadex. Results Sugammadex showed no adverse effects on neuronal cells and zebrafish larvae. The survival rates of neuronal cells were not different in all concentrations. In addition, the heart formation of zebrafish embryos, which were exposed to various concentrations of sugammadex, were not different. Conclusion This study demonstrated the feasibility of using sugammadex during pregnancy. However, further clinical studies will be required to extrapolate these results to humans., Graphical Abstract

Physical forces are important participants in the cellular dynamics that shape developing organs. During heart formation, for example, contractility and blood flow generate biomechanical cues that influence patterns of cell behavior. Here, we address the interplay between function and form during the assembly of the cardiac outflow tract (OFT), a crucial connection between the heart and vasculature that develops while circulation is underway. In zebrafish, we find that the OFT expands via accrual of both endocardial and myocardial cells. However, when cardiac function is disrupted, OFT endocardial growth ceases, accompanied by reduced proliferation and reduced addition of cells from adjacent vessels. The flow-responsive TGFβ receptor Acvrl1 is required for addition of endocardial cells, but not for their proliferation, indicating distinct modes of function-dependent regulation for each of these essential cell behaviors. Together, our results indicate that cardiac function modulates OFT morphogenesis by triggering endocardial cell accumulation that induces OFT lumen expansion and shapes OFT dimensions; moreover, these morphogenetic mechanisms provide new perspectives regarding the potential causes of cardiac birth defects.

The role of hemodynamics in cardiovascular development is not well understood. Indeed, it would be remarkable if it were, given the dauntingly complex array of intricately synchronized genetic, molecular, mechanical, and environmental factors at play. However, with congenital heart defects affecting around 1 in 100 human births, and numerous studies pointing to hemodynamics as a factor in cardiovascular morphogenesis, this is not an area in which we can afford to remain in the dark. This review seeks to present the case for the importance of research into the biomechanics of the developing cardiovascular system. This is accomplished by i) illustrating the basics of some of the highly complex processes involved in heart development, and discussing the known influence of hemodynamics on those processes; ii) demonstrating how altered hemodynamic environments have the potential to bring about morphological anomalies, citing studies in multiple animal models with a variety of perturbation methods; iii) providing examples of widely used technological innovations which allow for accurate measurement of hemodynamic parameters in embryos; iv) detailing the results of studies in avian embryos which point to exciting correlations between various hemodynamic manipulations in early development and phenotypic defect incidence in mature hearts; and finally, v) stressing the relevance of uncovering specific biomechanical pathways involved in cardiovascular formation and remodeling under adverse conditions, to the potential treatment of human patients. The time is ripe to unravel the contributions of hemodynamics to cardiac development, and to recognize their frequently neglected role in the occurrence of heart malformation phenotypes.

Summary How individual cells form precise connections with partners in a complicated environment has been a longstanding question. However, most cell matching studies have used qualitative approaches, which may miss subtle but significant morphological changes. Here, we describe the use of embryonic Drosophila heart formation as a simplified system to quantitatively study cell matching. We provide a step-by-step protocol for large-scale embryo preparation and immunostaining and imaging details. We also describe steps for quantifying cellular mismatch from the batch images. For complete details on the use and execution of this protocol, please refer to Zhang et al. (2018 and 2020 ).

Pyrethroid and organochlorine insecticides are enormously used to control agricultural and indoor insect pests. The metabolites of pyrethroid and endosulfan were used to evaluate environmental toxicities using a representative animal model, zebrafish (Danio rerio) embryos in this study. The LC50 values in 3-phenoxy benzoic acid (3-PBA) and endosulfan sulfate (ES) were 1461 μg/L and 1459 μg/L, respectively. At the concentration of 2000 μg/L, spine curvature was observed in the ES-treated embryos. ES showed seizure-like events with an EC50 value of 354 μg/L. At the concentration of 1000 μg/L, the pericardial edema was observed in 3-PBA-treated embryos. The inhibition of heart development and the reduction of beating rates were observed in Tg(cmlc2:EGFP) embryos after the exposure to 3-PBA. Down-regulation of the vmhc gene coding ventricular myosin during heart development was significantly found in 3-PBA-treated embryos at 48 hpf, but recovered afterward. It indicates that ventricular malformation occurred at the initial stage of 3-PBA exposure. Considered together, both 3-PBA and ES need public concerns with periodic monitoring of these metabolites in households and agricultural areas to prevent humans and environmental organisms from their unexpected attacks.

Pulmonary hypertension and pulmonary heart are severe complications of broncho-pulmonary dysplasia, accompanied by poor prognosis and high mortality. Based on Doppler echocardio-graphy in 100 children with bronchopulmonary dysplasia, the frequency of pulmonary hypertension de-velopment and pulmonary heart formation was determined. The risk factors for these complications and mortality outcomes have been defined. The quantitative evaluation of the informative value of electro-cardiographic, radiographic, computer tomographic signs of pulmonary hypertension in children with bronchopulmonary dysplasia was performed. The correspondence of the functional classes of pulmonary hypertension in the Panamanian classification of hypertensive vascular lung disease in children (2011) to the degree of increase in systolic pressure in the pulmonary artery and the severity of hypoxemia was defined.

Aquatic sediments act as a storage for diverse mixtures of organic and inorganic contaminants. Nevertheless, most evaluations of contaminated sediments have been limited to the assessment of concentrations of target compounds and lethal effects on some test species. To identify the organic contaminants causing sub-lethal effects of contaminated sediment, this study combined chemical and biological (non)target analysis involving comprehensive two-dimensional gas chromatography coupled with a time-of-flight Mass Spectrometer (GCxGC/ToF-MS) analysis, embryonic malformation and high-throughput sequencing (RNA-seq) analysis on developing flounder. Polycyclic aromatic hydrocarbons were more abundant in the sediment extract of Yeongil Bay (SEY), while Jinhae Bay (SEJ) was contaminated with a large amount of unidentified chemicals. The unidentified chemicals of SEJ included branched alkanes, oxygenated cycloalkanes, heterogeneous hydrocarbons, and other unknown compounds. Percentage of pericardial edema was the highest in embryonic flounder exposed to SEY. Consistent with the morphogenesis results, the expression level of genes related to heart formation including the nkx2.5 and robo1 was greater in embryonic flounder exposed to SEY. In the analyses of differential gene expression profiles (cutoff P < 0.05), by RNA-seq, embryos exposed to SEJ showed changes related to cell differentiation, cell part morphogenesis, neurogenesis, and neuron development. Genes related to neurogenesis and positive regulation of molecular functions variated significantly in embryos exposed to SEY. These results demonstrated the advantages of combining target and non-target analysis to accurately evaluate the major chemical groups causing sediment toxicity. Therefore, this work provided a useful approach to tracking and revealing the causes of toxic effects and identifying potential toxic mechanisms.

To date, transcript variants of the nuclear envelope protein Nurim and their expression profiles in mice have never been elucidated. In this study, we determined that the primary Nurim variant a was abundantly expressed in mouse heart, liver, spleen and kidney. The protein level of isoform a is initiated at an early stage of heart formation and demonstrated a significant increase in expression throughout embryonic heart development. Interestingly, Nurim b is also up-regulated from E12.5 to E18.5 in different individuals. Our research represents the first report on alternative splicing variants of mouse Nurim and their differential expression profile during embryonic development. These studies suggest a potential role for Nurim in early heart morphogenesis and should help further elucidate the function of Nurim.

During early cardiogenesis, bilateral fields of mesenchymal heart progenitor cells (HPCs) move from the anterior lateral plate mesoderm to the ventral midline, undergoing a mesenchymal-to-epithelial transition (MET) en route to forming a single epithelial sheet. Through tracking of tissue-level deformations in the heart-forming region (HFR) as well as movement trajectories and traction generation of individual HPCs, we find that the onset of MET correlates with a peak in mechanical stress within the HFR and changes in HPC migratory behaviors. Small-molecule inhibitors targeting actomyosin contractility reveal a temporally specific requirement of bulk tissue compliance to regulate heart development and MET. Targeting mutant constructs to modulate contractility and compliance in the underlying endoderm, we find that MET in HPCs can be accelerated in response to microenvironmental stiffening and can be inhibited by softening. To test whether MET in HPCs was responsive to purely physical mechanical cues, we mimicked a high-stress state by injecting an inert oil droplet to generate high strain in the HFR, demonstrating that exogenously applied stress was sufficient to drive MET. MET-induced defects in anatomy result in defined functional lesions in the larval heart, implicating mechanical signaling and MET in the etiology of congenital heart defects. From this integrated analysis of HPC polarity and mechanics, we propose that normal heart development requires bilateral HPCs to undergo a critical behavioral and phenotypic transition on their way to the ventral midline, and that this transition is driven in response to the changing mechanical properties of their endoderm substrate.

Genetic variation in specific transcription factors during heart formation may lead to congenital heart disease (CHD) or even miscarriage. The aim of the present study was to identify CHD‑associated genes using next generation sequencing (NGS). The whole exome DNA sequence was obtained from a stillborn fetus diagnosed with tricuspid atresia and complete transposition of the great arteries using high‑throughput sequencing methods. Subsequently, genetic variants of CHD‑associated genes were selected and verified in 215 non‑syndromic CHD patients and 249 healthy control subjects using polymerase chain reaction combined with Sanger sequencing. Genetic variants of previously reported CHD‑inducing genes, such as cysteine rich with EGF like domains 1 and cbp/p300‑interacting transactivator with Glu/Asp rich carboxy‑terminal domain 2, were discovered through the NGS analysis. In addition, a novel non‑synonymous mutation of the iroquois homeobox 1 (IRX1) gene (p.Gln240Glu) was identified. A total of three non‑synonymous mutations (p.Gln240Glu, p.Ser298Asn and p.Ala381Glu) of the IRX1 gene were verified in 215 non‑syndromic CHD patients, but not in 249 healthy volunteers. The results demonstrated that NGS is a powerful tool to study the etiology of CHD. In addition, the results suggest that genetic variants of the IRX1 gene may contribute to the pathogenesis of CHD.

Physical forces are important participants in the cellular dynamics that shape developing organs. During heart formation, for example, contractility and blood flow generate biomechanical cues that influence patterns of cell behavior. Here, we address the interplay between function and form during the assembly of the cardiac outflow tract (OFT), a crucial connection between the heart and vasculature that develops while circulation is underway. In zebrafish, we find that the OFT expands via accrual of both endocardial and myocardial cells. However, when cardiac function is disrupted, OFT endocardial growth ceases, accompanied by reduced proliferation and reduced addition of cells from adjacent vessels. The TGFβ receptor Acvrl1 is required for addition of endocardial cells, but not for their proliferation, indicating distinct regulation of these essential cell behaviors. Together, our results suggest that cardiac function modulates OFT morphogenesis by triggering endocardial cell accumulation that induces OFT lumen expansion and shapes OFT dimensions.

Congenital Heart Diseases (CHDs) are major cause of prenatal and neonatal mortality. These defects occur due to abnormalities during heart formation in utero. The treatments of CHDs pose a major challenge due to lack of in depth understanding of underlying factors. There are certain evidences that suggest abnormal mitochondrial function in these cardiomyopathies. A large percentage of prenatal dilated cardiomyopathies reportedly exhibit defects in mitochondrial oxidative phosphorylation. However, we still have a very little understanding of causal role played by mitochondria in CHDs. Considering the fact that heart has highest density of mitochondria followed by skeletal muscles and central nervous system, investigation of mitochondrial involvement in CHDs is a very intriguing question. The Drosophila embryonic heart is termed as dorsal vessel. It is a simple contractile tube which is similar to the tubular heart of early vertebrate embryo. Despite the evolutionary distance between Drosophila and vertebrates, cardio genesis is governed by similar fundamental mechanisms. The transcription factors as well as signalling pathways involved in cardio genesis are highly conserved among Drosophila and vertebrates. Dorsal vessel is comprised of merely104 cardio blasts; an extremely simple organ; hence enables pinpointing any minor cardiac deformity. The anterior portion is termed aorta whereas posterior portion is termed Heart proper. Hemolymph flows from posterior to anterior of the dorsal vessel. The posterior portion has specific subset of cardio blasts that form the inflow tracts termed as Ostia. Dorsal vessel has segmental repeated pattern of cell types which is a hallmark of insects. The cardio blasts are flanked by pericardial cells which function as nephrocytes. Drosophila can prove to be a good model system to understand cardiac development and function, which can open new doors to understand cardiomyopathies and improve their treatment. In order to perturb mitochondrial complex-I activity during embryonic development, two components of complex-I of ETC; ND75 which is the largest core subunit of complex-I and ND42 , an accessory subunit required for the assembly of complex-I of ETC were knocked down at different temporal and spatial windows and embryos were observed for lethality in terms of hatching rate. Severe embryonic lethality was observed in F1 progeny embryos by knocking down ND42 and ND75 with Twist Gal4. Twist Gal4 is the early mesoderm Gal4 driver. Its expression starts at stage 4 when mesoderm begins to form. Live imaging of these embryos indicate cardiac malfunction at stage 17 of embryonic development. Quantitative analysis of cardiac parameters using SOHA (Semi-automated Heartbeat Analysis) confirmed the cardiac defects with respect to different parameters for example Heart rate, Heart period, systole-diastole diameters and systole -diastole intervals. Further investigation of the cardiac defect in knock down embryos at cellular level revealed an intriguing cell specification defect at stage 16 of embryonic development. In wild type stage 16 embryo, cardiac tube is formed of 4 Tinman positive and 2 Seven up positive cardio blasts in each hemi segment. However, in complex-I mutant embryos, we found that this 4+2 pattern of cardio blasts is altered to 2+4 pattern. By tracking this phenotype to the earlier stages, we found that this change in cell identity initiates at stage 13 where some of Tinman positive cells begin to lose Tinman expression and ectopically begins to express Seven up . Therefore, identity of cells is changed due to metabolic disturbances . In the complex-I knockdown embryos, ROS levels were found to be high as indicated by mitoSOX dye and gstD GFP reporter line. Co-immunostaining of Twist protein and gst D GFP showed that ROS levels were only increased in the Twist expressing mesoderm cells at stage 10 of embryonic development, suggesting that knockdown of ND42/ND75 using Twist Gal4 has resulted in elevated ROS levels in mesodermal population. We were able to establish ROS as the key mediator to elicit retrograde response from mitochondria to regulate cell fate specification since over-expression of SOD2 in the background of ND42/ND75 knockdown was able to significantly rescue embryonic lethality, cardiac malfunctioning and cell fate specification defects.

Cardiac development is governed by a complex network of transcription factors (TFs) that regulate cell fates in a spatiotemporal manner. Among these, the GATA family of zinc finger TFs plays prominent roles in regulating the development of the myocardium, endocardium, and outflow tract. This family comprises six members three of which, GATA4, 5, and 6, are predominantly expressed in cardiac cells where they activate specific downstream gene targets via interactions with one another and with other TFs and signaling molecules. Their critical function in heart formation is evidenced by the phenotypes of animal models lacking these factors and by the broad spectrum of human congenital heart diseases associated with mutations in their genes. Similarly, in the postnatal heart, these proteins play significant and nonredundant roles in cardiac function, regulating adaptive stress responses including cardiomyocyte hypertrophy and survival, as well as endothelial homeostasis and angiogenesis. As such, decreased expression of either GATA4, 5, or 6 results in impaired cardiovascular homeostasis and increased risk of premature and serious cardiovascular events such as hypertension, arrhythmia, aortopathy, and heart failure. Although a great deal of progress has been made in understanding GATA-dependent regulatory processes in the heart, the molecular mechanisms underlying the specificity of GATA factors and their upstream regulation remain incompletely understood. The knowledge and tools developed since their discovery 25 years ago should accelerate progress toward further elucidation of their mechanisms of action in health and disease. This in turn will greatly improve diagnosis and care for the millions of individuals affected by congenital and acquired cardiac disease worldwide.

The second heart field (SHF) harbors progenitors that are important for heart formation, but little is known about its morphogenesis. We show that SHF population in the mouse splanchnic mesoderm (SpM-SHF) undergoes polarized morphogenesis to preferentially elongate anteroposteriorly. Loss of Wnt5, a putative ligand of the planar cell polarity (PCP) pathway, causes the SpM-SHF to expand isotropically. Temporal tracking reveals that the Wnt5a lineage is a unique subpopulation specified as early as E7.5, and undergoes bi-directional deployment to form specifically the pulmonary trunk and the dorsal mesenchymal protrusion (DMP). In Wnt5a(−/−) mutants, Wnt5a lineage fails to extend into the arterial and venous poles, leading to both outflow tract and atrial septation defects that can be rescued by an activated form of PCP effector Daam1. We identify oriented actomyosin cables in the medial SpM-SHF as a potential Wnt5a-mediated mechanism that promotes SpM-SHF lengthening and restricts its widening. Finally, the Wnt5a lineage also contributes to the pulmonary mesenchyme, suggesting that Wnt5a/PCP is a molecular circuit recruited by the recently identified cardiopulmonary progenitors to coordinate morphogenesis of the pulmonary airways and the cardiac septations necessary for pulmonary circulation. This article has an associated ‘The people behind the papers’ interview.

Heart formation involves a complex series of tissue rearrangements, during which regions of the developing organ expand, bend, converge, and protrude in order to create the specific shapes of important cardiac components. Much of this morphogenesis takes place while cardiac function is underway, with blood flowing through the rapidly contracting chambers. Fluid forces are therefore likely to influence the regulation of cardiac morphogenesis, but it is not yet clear how these biomechanical cues direct specific cellular behaviors. In recent years, the optical accessibility and genetic amenability of zebrafish embryos have facilitated unique opportunities to integrate the analysis of flow parameters with the molecular and cellular dynamics underlying cardiogenesis. Consequently, we are making progress toward a comprehensive view of the biomechanical regulation of cardiac chamber emergence, atrioventricular canal differentiation, and ventricular trabeculation. In this review, we highlight a series of studies in zebrafish that have provided new insight into how cardiac function can shape cardiac morphology, with a particular focus on how hemodynamics can impact cardiac cell behavior. Over the long-term, this knowledge will undoubtedly guide our consideration of the potential causes of congenital heart disease.

3′-5′ cyclic nucleotide phosphodiesterases (PDEs) are a large family of enzymes playing a fundamental role in the control of intracellular levels of cAMP and cGMP. Emerging evidence suggested an important role of phosphodiesterases in heart formation, but little is known about the expression of phosphodiesterases during cardiac development. In the present study, the pattern of expression and enzymatic activity of phosphodiesterases was investigated at different stages of heart formation. C57BL/6 mice were mated and embryos were collected from 14.5 to 18.5 days of development. Data obtained by qRT-PCR and Western blot analysis showed that seven different isoforms are expressed during heart development, and PDE1C, PDE2A, PDE4D, PDE5A and PDE8A are modulated from E14.5 to E18.5. In heart homogenates, the total cAMP and cGMP hydrolytic activity is constant at the evaluated times, and PDE4 accounts for the majority of the cAMP hydrolyzing ability and PDE2A accounts for cGMP hydrolysis. This study showed that a subset of PDEs is expressed in developing mice heart and some of them are modulated to maintain constant nucleotide phosphodiesterase activity in embryonic and fetal heart.

Asb2, ankyrin repeat, and SOCS box protein 2 form an E3 ubiquitin ligase complex. Asb2 ubiquitin ligase activity drives the degradation of filamins, which have essential functions in humans. The placenta is a temporary organ that forms during pregnancy, and normal placentation is important for survival and growth of the fetus. Recent studies have shown that approximately 25-30% of knockout (KO) mice have non-viable offspring, and 68% of knockout lines exhibit placental dysmorphologies. There are very few studies on Asb2, with insufficient research on its role in placental development. Therefore, we generated Asb2 knockout mice and undertook to investigate Asb2 expression during organogenesis, and to identify its role in early embryonic and placental development. The external morphology of KO embryos revealed abnormal phenotypes including growth retardation, pericardial effusion, pale color, and especially heart beat defect from E 9.5. Furthermore, Asb2 expression was observed in the heart from E 9.5, indicating that it is specifically expressed during early heart formation, resulting in embryonic lethality. Histological analysis of E 10.5 KO heart showed malformations such as failure of chamber formation, reduction in trabeculated myocardium length, absence of mesenchymal cells, and destruction of myocardium wall. Moreover, the histological results of Asb2-deficient placenta showed abnormal phenotypes including small labyrinth and reduced vascular complexity, indicating that failure to establish mature circulatory pattern affects the embryonic development and results in early mortality. Collectively, our results demonstrate that Asb2 knockout mice have placental defects, that subsequently result in failure to form a normal cardiac septum, and thereby result in embryo mortality in utero at around E 9.5.

Historic industrial pollution of the Elizabeth River, Virginia resulted in polycyclic aromatic hydrocarbon (PAH) contamination in sediments. Atlantic killifish (Fundulus heteroclitus) inhabiting the Atlantic Wood (AW) industrial site adapted to complex PAH mixture at this Superfund site. Their embryos have proved highly resistant to cardiac abnormalities indicative of PAH toxicity. In this study, embryos spawned from adults collected at AW and King’s Creek (KC), a reference site, were exposed at 24 h post fertilization (hpf) to Elizabeth River Sediment Extract (ERSE), a complex PAH mixture, in a range of concentrations (0, 5.04, 50.45, 100.90, 151.35, or 252.25 µg/L total PAHs). Embryos were processed for histology at 144 hpf to enable evaluations of hearts at tissue and cellular levels. Morphometry and severity scoring were used to evaluate the extent of alterations. Unexposed embryos were similar in both populations. ERSE exposure resulted in multiple changes to hearts of KC embryos but not AW. Alterations were particularly evident in KC embryos exposed to concentrations above 1% ERSE (50.45 µg/L), which had thinner ventricular walls and larger pericardial edema. Individuals with moderate pericardial edema maintained arrangement and proximity of heart chambers, but changes were seen in ventricular myocytes. Severe pericardial edema was prevalent in exposed KC embryos and typically resulted in tube heart formation. Ventricles of tube hearts had very thin walls composed of small, basophilic cells and lacked trabeculae. Edematous pericardial fluid contained small amounts of proteinaceous material, as did controls, and was free of cells. This fluid was primarily unstained, suggesting water influx due to increased permeability. The use of histological approaches provided more specific detail for tissue and cellular effects in hearts of embryos exposed to PAHs and enabled understanding of potential links to later life effects of early life exposure.

Down syndrome (DS) is a significant risk factor for congenital heart disease (CHD), increasing the incidence 50 times over the general population. However, half of people with DS have a normal heart and thus trisomy 21 is not sufficient to cause CHD by itself. Ts65Dn mice are trisomic for orthologs of >100 Hsa21 genes, and their heart defect frequency is significantly higher than their euploid littermates. Introduction of a null allele of Creld1 into Ts65Dn increases the penetrance of heart defects significantly. However, this increase was not seen when the Creld1 null allele was introduced into Ts1Cje, a mouse that is trisomic for about two thirds of the Hsa21 orthologs that are triplicated in Ts65Dn. Among the 23 genes present in three copies in Ts65Dn but not Ts1Cje, we identified Jam2 as necessary for the increased penetrance of Creld1-mediated septal defects in Ts65Dn. Thus, overexpression of the trisomic gene, Jam2, is a necessary potentiator of the disomic genetic modifier, Creld1. No direct physical interaction between Jam2 and Creld1 was identified by several methods. Regions of Hsa21 containing genes that are risk factors of CHD have been identified, but Jam2 (and its environs) has not been linked to heart formation previously. The complexity of this interaction may be more representative of the clinical situation in people than consideration of simple single-gene models.

GATA binding protein 6 (GATA6) is a transcription factor that is expressed in the early blastocyst stage and controls the expression of important genes in the differentiation and development of the heart, pancreas, and intestine. This study confirmed the role of GATA6 in cell differentiation and or- gan development using mouse embryonic stem cells and zebrafish, respectively. First, the mouse em- bryonic stem cells were differentiated into pacemaker cardiomyocytes. An RT-PCR analysis revealed that the expression of the GATA6 gene was greatly increased from day 4 of differentiation. The ex- pression of GATA6 was upregulated prior to increased expression of NK2 homeobox 5 (Nkx2.5) and myocyte enhancer factor 2C (MEF2C), which are critical transcription factors involved in regulating heart formation. To examine the role of GATA6 in development, GATA6 morpholino was micro- injected into zebrafish embryos. Knockdown of GATA6 expression significantly decreased the heart size and heart rate in the zebrafish compared to a control. In addition, the brains were degenerated in the GATA6 morpholino-injected zebrafish. Acridine orange staining showed that knockdown of GATA6 expression increased apoptotic cells in the brain. Interestingly, knockdown of GATA6 ex- pression decreased apoptotic cells in the early bud stage. This study points to the importance of the GATA6 gene in heart and brain development.

The heart is the first organ to be functional in the fetus. Heart formation is a complex morphogenetic process regulated by both genetic and epigenetic mechanisms. Congenital heart diseases (CHD) are the most prominent congenital diseases. Genetics is not sufficient to explain these diseases or the impact of them on patients. Epigenetics is more and more emerging as a basis for cardiac malformations. This review brings the essential knowledge on cardiac biology of development. It further provides a broad background on epigenetics with a focus on three-dimensional conformation of chromatin. Then, we summarize the current knowledge of the impact of epigenetics on cardiac cell fate decision. We further provide an update on the epigenetic anomalies in the genesis of CHD.

Adult cardiac pathologies are associated with misrelated embryonic programs hence understanding cardiac development is essential for both basic and translational research. In this study we investigate the roles of Hand1 in CNC during heart formation. We find that loss of Hand1 in CNC leads to coronary developmental defects. The coronary vasculature develops but is mis-patterned with wide areas of the heart devoid of large vessels. We show that vasculogenesis was unaffected but angiogenesis was defective in mutant embryos due to defective coronary ostia development. Study also confirmed that Ednra loss results in a thin myocardial wall but also made the unique observation that Ednrb null mice had a more severe thinning of myocardial wall compared to Ednra null mice. Ednrb is also expressed in the sympathetic ganglia of the developing embryo and its loss results in hypoplastic sympathetic ganglia. Sympathetic ganglia and their support cells are NCderived structures. By in situ analyses we discovered a loss of Hand2 in the sympathetic ganglia in the DKO. Hand2 is required for the expression of norepinephrine biosynthetic enzymes in the developing sympathetic nervous system (SNS). Norepinephrine activates the heart to contract and previous reports from our group show that a lack of norepinephrine synthesis leads to a thin myocardial wall phenotype. This could explain the thin wall myocardium seen in Ednrb null and DKO embryos.

Vertebrate heart development requires spatiotemporal regulation of gene expression to specify cardiomyocytes, increase the cardiomyocyte population through proliferation, and to establish and maintain atrial and ventricular cardiac chamber identities. The evolutionarily conserved chromatin factor Gon4-like (Gon4l), encoded by the zebrafish ugly duckling (udu) locus, has previously been implicated in cell proliferation, cell survival, and specification of mesoderm-derived tissues including blood and somites, but its role in heart formation has not been studied. Here we report two distinct roles of Gon4l/Udu in heart development: regulation of cell proliferation and maintenance of ventricular identity. We show that zygotic loss of udu expression causes a significant reduction in cardiomyocyte number at one day post fertilization that becomes exacerbated during later development. We present evidence that the cardiomyocyte deficiency in udu mutants results from reduced cell proliferation, unlike hematopoietic deficiencies attributed to TP53-dependent apoptosis. We also demonstrate that expression of the G1/S-phase cell cycle regulator, cyclin E2 (ccne2), is reduced in udu mutant hearts, and that the Gon4l protein associates with regulatory regions of the ccne2 gene during early embryogenesis. Furthermore, udu mutant hearts exhibit a decrease in the proportion of ventricular cardiomyocytes compared to atrial cardiomyocytes, concomitant with progressive reduction of nkx2.5 expression. We further demonstrate that udu and nkx2.5 interact to maintain the proportion of ventricular cardiomyocytes during development. However, we find that ectopic expression of nkx2.5 is not sufficient to restore ventricular chamber identity suggesting that Gon4l regulates cardiac chamber patterning via multiple pathways. Together, our findings define a novel role for zygotically-expressed Gon4l in coordinating cardiomyocyte proliferation and chamber identity maintenance during cardiac development.

Versican is a chondroitin sulfate proteoglycan found in the extracellular matrix that is important for changes in cell phenotype associated with development and disease. Versican has been shown to be involved in cardiovascular disorders, as well as lung disease and fibrosis, inflammatory bowel disease, cancer, and several other diseases that have an inflammatory component. Versican was first identified as a fibroblast proteoglycan and forms large multi-molecular complexes with hyaluronan and other components of the provisional matrix during wound healing and inflammation. The biology of versican has been well studied. Versican plays a major role in embryogenesis, particularly heart formation, where versican deletion proves lethal. The ability to purify versican to characterize and to use in experimental systems is vital to defining its role in development and disease. Protein expression systems have proven challenging to obtain milligram quantities of full length versican. Here, we describe proteoglycan biochemical purification techniques that have been developed by others, but which we have adapted to use with our source tissues and cells. We also include methods for immunohistochemical localization and quantitation of versican in tissue sections.

The vertebrate heart develops from several progenitor lineages. After early-differentiating first heart field (FHF) progenitors form the linear heart tube, late-differentiating second heart field (SHF) progenitors extend the atrium and ventricle, and form inflow and outflow tracts (IFT/OFT). However, the position and migration of late-differentiating progenitors during heart formation remains unclear. Here, we track zebrafish heart development using transgenics based on the cardiopharyngeal gene tbx1. Live imaging uncovers a tbx1 reporter-expressing cell sheath that continuously disseminates from the lateral plate mesoderm towards the forming heart tube. High-speed imaging and optogenetic lineage tracing corroborates that the zebrafish ventricle forms through continuous addition from the undifferentiated progenitor sheath followed by late-phase accrual of the bulbus arteriosus (BA). FGF inhibition during sheath migration reduces ventricle size and abolishes BA formation, refining the window of FGF action during OFT formation. Our findings consolidate previous end-point analyses and establish zebrafish ventricle formation as a continuous process., Late-differentiating second heart field progenitors contribute to atrium, ventricle, and outflow tract in the zebrafish heart but how remains unclear. Here, the authors image heart formation in transgenics based on the cardiopharyngeal gene tbx1 and show that progenitors are continuously added.

The heart switches its energy substrate from glucose to fatty acids at birth, and maternal hyperglycemia is associated with congenital heart disease. However, little is known about how blood glucose impacts heart formation. Using a chemically defined human pluripotent stem-cell-derived cardiomyocyte differentiation system, we found that high glucose inhibits the maturation of cardiomyocytes at genetic, structural, metabolic, electrophysiological, and biomechanical levels by promoting nucleotide biosynthesis through the pentose phosphate pathway. Blood glucose level in embryos is stable in utero during normal pregnancy, but glucose uptake by fetal cardiac tissue is drastically reduced in late gestational stages. In a murine model of diabetic pregnancy, fetal hearts showed cardiomyopathy with increased mitotic activity and decreased maturity. These data suggest that high glucose suppresses cardiac maturation, providing a possible mechanistic basis for congenital heart disease in diabetic pregnancy., eLife digest Congenital heart disease is the most common type of birth defect, affecting nearly 1 in 100 children born. It can involve a weak heart, narrowed arteries, narrowed heart valves, or the main arteries of the heart switching places. These conditions can be fatal if untreated and often need surgery to correct. The mother’s blood sugar levels during pregnancy can have a large effect on how likely the baby is to have congenital heart disease. If a pregnant woman has poorly controlled diabetes with rapidly fluctuating sugar levels, she may be at a higher risk of having a child with the condition. High sugar levels in the mother’s blood make the baby up to five times more likely to have congenital heart disease. It has been difficult to find out exactly how sugar levels interfere with heart development because diabetes can affect the fetus in many ways. Nakano et al. used stem cells and experiments in pregnant mice with diabetes to hone in on how high sugar levels affect the fetus’s heart development. First, heart cells were grown from human stem cells, and exposed to high levels of glucose in a dish. This revealed a new mechanism for how high sugar levels affect heart formation: the cells created too many nucleotides, the building blocks of molecules such as DNA. It turns out that high glucose levels boosted a chemical process in the cell known as the pentose phosphate pathway. Some of the products of this pathway are nucleotides. This made the cells divide rapidly, but did not allow them to mature well compared with cells exposed to normal levels of sugar. In another experiment, Nakano et al. found similar results in pregnant diabetic mice. The heart cells in mouse fetuses also divided quickly but matured slowly when exposed to high sugar levels. An estimated 60 million women at an age to have children have diabetes. These new findings help us to understand why and how these women are more likely to have children with congenital heart disease, and further study will hopefully lead to a better way to prevent this condition.

Many of the major discoveries in the fields of genetics and developmental biology have been made using the fruit fly, Drosophila melanogaster. With regard to heart development, the conserved network of core cardiac transcription factors that underlies cardiogenesis has been studied in great detail in the fly, and the importance of several signaling pathways that regulate heart morphogenesis, such as Slit/Robo, was first shown in the fly model. Recent technological advances have led to a large increase in the genomic data available from patients with congenital heart disease (CHD). This has highlighted a number of candidate genes and gene networks that are potentially involved in CHD. To validate genes and genetic interactions among candidate CHD-causing alleles and to better understand heart formation in general are major tasks. The specific limitations of the various cardiac model systems currently employed (mammalian and fish models) provide a niche for the fly model, despite its evolutionary distance to vertebrates and humans. Here, we review recent advances made using the Drosophila embryo that identify factors relevant for heart formation. These underline how this model organism still is invaluable for a better understanding of CHD.

Distal outgrowth, maturation and remodeling of the endocardial cushion mesenchyme in the atrioventricular (AV) canal are the essential morphogenetic events during four-chambered heart formation. Mesenchymalized AV endocardial cushions give rise to the AV valves and the membranous ventricular septum (VS). Failure of these processes results in several human congenital heart defects. Despite this clinical relevance, the mechanisms governing how mesenchymalized AV endocardial cushions mature and remodel into the membranous VS and AV valves have only begun to be elucidated. The role of BMP signaling in the myocardial and secondary heart forming lineage has been well studied; however, little is known about the role of BMP2 expression in the endocardial lineage. To fill this knowledge gap, we generated Bmp2 endocardial lineage-specific conditional knockouts (referred to as Bmp2 cKOEndo) by crossing conditionally-targeted Bmp2flox/flox mice with a Cre-driver line, Nfatc1Cre, wherein Cre-mediated recombination was restricted to the endocardial cells and their mesenchymal progeny. Bmp2 cKOEndo mouse embryos did not exhibit failure or delay in the initial AV endocardial cushion formation at embryonic day (ED) 9.5-11.5; however, significant reductions in AV cushion size were detected in Bmp2 cKOEndo mouse embryos when compared to control embryos at ED13.5 and ED16.5. Moreover, deletion of Bmp2 from the endocardial lineage consistently resulted in membranous ventricular septal defects (VSDs), and mitral valve deficiencies, as evidenced by the absence of stratification of mitral valves at birth. Muscular VSDs were not found in Bmp2 cKOEndo mouse hearts. To understand the underlying morphogenetic mechanisms leading to a decrease in cushion size, cell proliferation and cell death were examined for AV endocardial cushions. Phospho-histone H3 analyses for cell proliferation and TUNEL assays for apoptotic cell death did not reveal significant differences between control and Bmp2 cKOEndo in AV endocardial cushions. However, mRNA expression of the extracellular matrix components, versican, Has2, collagen 9a1, and periostin was significantly reduced in Bmp2 cKOEndo AV cushions. Expression of transcription factors implicated in the cardiac valvulogenesis, Snail2, Twist1 and Sox9, was also significantly reduced in Bmp2 cKOEndo AV cushions. These data provide evidence that BMP2 expression in the endocardial lineage is essential for the distal outgrowth, maturation and remodeling of AV endocardial cushions into the normal membranous VS and the stratified AV valves.

Cardiac precursor cells (CPCs) in the first heart field (FHF) and the second heart field (SHF) present at both arterial and venous poles assemble to form a cardiac tube in zebrafish. Hippo kinase cascade is essential for proper heart formation; however, it remains elusive how Hippo signal contributes to early cardiac fate determination. We here demonstrate that mutants oflarge tumor suppressor kinase 1/2(lats1/2) exhibited an increase in a SHF marker, Islet1 (Isl1)-positive andhand2promoter-activated venous pole atrial cardiomyocytes (CMs) and that those showed expansion of the domain between between the anterior and the posterior lateral plate mesoderm. Consistently, TEAD-8 dependent transcription was activated in caudal region of the left ALPM cells that gave rise to the venous pole atrial CMs. Yap1/Wwtr1-promotedbmp2bexpression was essential for Smad-regulatedhand2expression in the left ALPM, indicating that Hippo signaling restricts the SHF cells originating from the left ALPM that move toward the venous pole.

Congenital heart defects are the most common malformations in humans, affecting approximately 1% of newborn babies. While genetic causes of congenital heart disease have been studied, only less than 20% of human cases are clearly linked to genetic anomalies. The cause for the majority of the cases remains unknown. Heart formation is a finely orchestrated developmental process and slight disruptions of it can lead to severe malformations. Dysregulation of developmental processes leading to heart malformations are caused by genetic anomalies but also environmental factors including blood flow. Intra-cardiac blood flow dynamics plays a significant role regulating heart development and perturbations of blood flow lead to congenital heart defects in animal models. Defects that result from hemodynamic alterations recapitulate those observed in human babies, even those due to genetic anomalies and toxic teratogen exposure. Because important cardiac developmental events, such as valve formation and septation, occur under blood flow conditions while the heart is pumping, blood flow regulation of cardiac formation might be a critical factor determining cardiac phenotype. The contribution of flow to cardiac phenotype, however, is frequently ignored. More research is needed to determine how blood flow influences cardiac development and the extent to which flow may determine cardiac phenotype.

International audience; Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix–loop–helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation—Tcf3 and Foxa2—and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1–4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.