Cardiac Precursor Research Articles

Novel Leptin-Cardiac TRH pathway responsible for the cardiac alterations in the Hyperleptinemic obesity.

The association between hypertension andobesity-induced cardiac damage is usually accepted.However, no studies have been focused on cardiac alterations in obesity, independently of blood pressure increase. It iswell known that Cardiac TRH induces Left Ventricular Hypertrophy (LVH) and fibrosis, and its inhibition prevents the development ofhypertrophy. Also, it has been described that the adiponectin leptin induces TRH expression. Thus, we hypothesized that in obesity, the increase in TRH induced by hyperleptinemia is responsible for LVH, until now mostly attributed to pressure load. We studied obese Agouti mice suffering from hypertension with hyperleptinemia and found a significant LVH development with increased TRH gene expression. Consequently, we found higher fibrotic (collagens and TGF-β) and hypertrophic markers (BNP and β-MHC) expressionvs lean black controls. As pressure could explain these results, we treated obese mice with diuretic (hydrochlorothiazide 20mg/kg/day) since weaning. Diuretic treatment was successful as the diuretic group was normotensive in contrast to control obese mice. Nevertheless, both groups showed LVH development, higher cardiac precursor TRH gene and peptide expressions and elevated fibrotic and hypertrophic markers expression,pointing out that obesity-induced LVH is not due to hypertension. In addition, we performed Cardiac TRH inhibition by specific siRNA injection compared to control siRNA treatment and evaluated cardiac damage. As expected, expressions and protein increase in hypertrophic and fibrotic markers observed in the AG mouse with the native cTRH system were not seen in the AG mouse with the cTRH silencing. Indeed, the AG + TRH-siRNA group showed hypertrophicmarkers expression and fibrosis measurements similar to the lean BL mice. On the whole, these results point out that the novel Leptin-Cardiac TRH pathway is responsible for the cardiac alterations present in hyperleptinemic obesity, independent of blood pressure, and cTRH long-term silencing since early stages totally preventLVH development and cardiac fibrosis.

Transcriptome and open chromatin analysis reveals the process of myocardial cell development and key pathogenic target proteins in Long QT syndrome type 7

ObjectiveLong QT syndrome type 7 (Andersen–Tawil syndrome, ATS), which is caused by KCNJ2 gene mutation, often leads to ventricular arrhythmia, periodic paralysis and skeletal malformations. The development, differentiation and electrophysiological maturation of cardiomyocytes (CMs) changes promote the pathophysiology of Long QT syndrome type 7(LQT7). We aimed to specifically reproduce the ATS disease phenotype and study the pathogenic mechanism.Methods and resultsWe established a cardiac cell model derived from human induced pluripotent stem cells (hiPSCs) to the phenotypes and electrophysiological function, and the establishment of a human myocardial cell model that specifically reproduces the symptoms of ATS provides a reliable platform for exploring the mechanism of this disease or potential drugs. The spontaneous pulsation rate of myocardial cells in the mutation group was significantly lower than that in the repair CRISPR group, the action potential duration was prolonged, and the Kir2.1 current of the inward rectifier potassium ion channel was decreased, which is consistent with the clinical symptoms of ATS patients. Only ZNF528, a chromatin-accessible TF related to pathogenicity, was continuously regulated beginning from the cardiac mesodermal precursor cell stage (day 4), and continued to be expressed at low levels, which was identified by WGCNA method and verified with ATAC-seq data in the mutation group. Subsequently, it indicated that seven pathways were downregulated (all p < 0.05) by used single sample Gene Set Enrichment Analysis to evaluate the overall regulation of potassium-related pathways enriched in the transcriptome and proteome of late mature CMs. Among them, the three pathways (GO: 0008076, GO: 1990573 and GO: 0030007) containing the mutated gene KCNJ2 is involved that are related to the whole process by which a potassium ion enters the cell via the inward rectifier potassium channel to exert its effect were inhibited. The other four pathways are related to regulation of the potassium transmembrane pathway and sodium:potassium exchange ATPase (p < 0.05). ZNF528 small interfering (si)-RNA was applied to hiPSC-derived cardiomyocytes for CRISPR group to explore changes in potassium ion currents and growth and development related target protein levels that affect disease phenotype. Three consistently downregulated proteins (KCNJ2, CTTN and ATP1B1) associated with pathogenicity were verificated through correlation and intersection analysis.ConclusionThis study uncovers TFs and target proteins related to electrophysiology and developmental pathogenicity in ATS myocardial cells, obtaining novel targets for potential therapeutic candidate development that does not rely on gene editing.Graphical

Generation of human vascularized and chambered cardiac organoids for cardiac disease modelling and drug evaluation.

Human induced pluripotent stem cell (hiPSC)-derived cardiac organoids (COs) have shown great potential in modelling human heart development and cardiovascular diseases, a leading cause of global death. However, several limitations such as low reproducibility, limited vascularization and difficulty in formation of cardiac chamber were yet to be overcome. We established a new method for robust generation of COs, via combination of methodologies of hiPSC-derived vascular spheres and directly differentiated cardiomyocytes from hiPSCs, and investigated the potential application of human COs in cardiac injury modelling and drug evaluation. The human COs we built displayed a vascularized and chamber-like structure, and hence were named vaschamcardioids (vcCOs). These vcCOs exhibited approximately 90% spontaneous beating ratio. Single-cell transcriptomics identified a total of six cell types in the vcCOs, including cardiomyocytes, cardiac precursor cells, endothelial cells, fibroblasts, etc. We successfully recaptured the processes of cardiac injury and fibrosis in vivo on vcCOs, and showed that the FDA-approved medication captopril significantly attenuated cardiac injury-induced fibrosis and functional disorders. In addition, the human vcCOs exhibited an obvious drug toxicity reaction to doxorubicin in a dose-dependent manner. We developed a three-step method for robust generation of chamber-like and vascularized complex vcCOs, and our data suggested that vcCOs might become a useful model for understanding pathophysiological mechanisms of cardiovascular diseases, developing intervention strategies and screening drugs.

Generation of a ISL1 homozygous knockout stem cell line (WAe009-A-1G) by episomal vector-based CRISPR/Cas9 system

The ISL LIM homeobox 1 (ISL1) gene belongs to the LIM/homeodomain transcription factor family and plays a pivotal role in conveying multipotent and proliferative properties of cardiac precursor cells. Mutations in ISL1 are linked to congenital heart disease. To further explore ISL1′s role in the human heart, we have created a homozygous ISL1 knockout (ISL1-KO) human embryonic stem cell line using the CRISPR/Cas9 system. Notably, this ISL1-KO cell line retains normal morphology, pluripotency, and karyotype. This resource serves as a valuable tool for investigating ISL1′s function in cardiomyocyte differentiation.

Multiple Cell Lineages Give Rise to a Cell Type.

It has long been assumed that a specific cell type arises following stepwise specification of cells corresponding to the branching of cell lineages. However, accumulating evidence indicates that multiple and even remote cell lineages can lead to the development of the same cells. Four examples giving different yet new insights will be discussed: skeletal muscle development from precursors with distinct initial histories of transcriptional regulation, lens cell development from remote lineages yet sharing basic transcription factors, blood cell development under intersectional pathways, and neural tissue development from cardiac precursors through the manipulation of just one component of epigenetic regulation. These examples provide flexible and nondogmatic perspectives on developmental cell regulation, fundamentally revising the old model relying on cell lineages.

Abstract 14625: Association of Eicosanoid Metabolites With Cardiac Structure and Function

Introduction: Eicosanoids are bioactive lipids that govern the upstream initiation of pro- and anti-inflammatory activity. They may serve as important pathways by which obesity and cardiometabolic disease lead to the development of heart failure with preserved ejection fraction (HFpEF). Objective: We sought to examine the association of plasma circulating eicosanoid metabolites with subclinical cardiac remodeling as ascertained by cardiac magnetic resonance imaging (CMR) as important determinants of HFpEF risk. Methods: We studied 3880 MESA study participants (mean age 63±10 years, 53% women) who underwent plasma metabolite profiling using a mass spectrometry-based platform and CMR. We used multivariable linear regression to examine the association of eicosanoid metabolites with left ventricular mass index (LVMI, primary outcome), LV end-diastolic volume index (LVEDV), and left atrial volume (LAV). Results: Of 811 eicosanoids, 158 were associated with LVMI (Figure; FDR q &lt;0.05 for all). Specifically, derivatives of eicosadienoic acid (15 oxoEDE), linoleic acid (9,10 diHOME), and arachidonic acid (12(R) HETE) were negatively associated with LVMI and prostaglandin 19R-hydroxy-PGE1 was positively associated with LVMI. Of 158 metabolites, 65 were are also associated with LVEDV including derivatives of linoleic acid (13(S) HOTrE), oxylipin 11-HETE and 15 oxoEDE. Of 158 metabolites, Adrenic acid is also associated with LAV. Conclusions: We found that specific eicosanoids including 15 oxoEDE, 9,10 diHOME, and 12(R) HETE were associated with LVMI, an important measure of subclinical cardiac remodeling and precursor to HFpEF. As causal drivers of HFpEF are poorly characterized, a better understanding of pro- and anti-inflammatory eicosanoid pathways that contribute to cardiac remodeling may have important implications for the prevention of HFpEF.

Erratum for the Research Article "Matrix rigidity controls endothelial differentiation and morphogenesis of cardiac precursors" by Kshitiz et al.

Erratum for the Research Article "Matrix rigidity controls endothelial differentiation and morphogenesis of cardiac precursors" by Kshitiz et al.

A Mesp1-dependent developmental breakpoint in transcriptional and epigenomic specification of early cardiac precursors.

Transcriptional networks governing cardiac precursor cell (CPC) specification are incompletely understood due in part to limitations in distinguishing CPCs from non-cardiac mesoderm in early gastrulation. We leveraged detection of early cardiac lineage transgenes within a granular single cell transcriptomic time course of mouse embryos to identify emerging CPCs and describe their transcriptional profiles. Mesp1, a transiently-expressed mesodermal transcription factor (TF), is canonically described as an early regulator of cardiac specification. However, we observed perdurance of CPC transgene-expressing cells in Mesp1 mutants, albeit mis-localized, prompting us to investigate the scope of Mesp1's role in CPC emergence and differentiation. Mesp1 mutant CPCs failed to robustly activate markers of cardiomyocyte maturity and critical cardiac TFs, yet they exhibited transcriptional profiles resembling cardiac mesoderm progressing towards cardiomyocyte fates. Single cell chromatin accessibility analysis defined a Mesp1-dependent developmental breakpoint in cardiac lineage progression at a shift from mesendoderm transcriptional networks to those necessary for cardiac patterning and morphogenesis. These results reveal Mesp1-independent aspects of early CPC specification and underscore a Mesp1-dependent regulatory landscape required for progression through cardiogenesis.

Return of the Tbx5; lineage-tracing reveals ventricular cardiomyocyte-like precursors in the injured adult mammalian heart

The single curative measure for heart failure patients is a heart transplantation, which is limited due to a shortage of donors, the need for immunosuppression and economic costs. Therefore, there is an urgent unmet need for identifying cell populations capable of cardiac regeneration that we will be able to trace and monitor. Injury to the adult mammalian cardiac muscle, often leads to a heart attack through the irreversible loss of a large number of cardiomyocytes, due to an idle regenerative capability. Recent reports in zebrafish indicate that Tbx5a is a vital transcription factor for cardiomyocyte regeneration. Preclinical data underscore the cardioprotective role of Tbx5 upon heart failure. Data from our earlier murine developmental studies have identified a prominent unipotent Tbx5-expressing embryonic cardiac precursor cell population able to form cardiomyocytes, in vivo, in vitro and ex vivo. Using a developmental approach to an adult heart injury model and by employing a lineage-tracing mouse model as well as the use of single-cell RNA-seq technology, we identify a Tbx5-expressing ventricular cardiomyocyte-like precursor population, in the injured adult mammalian heart. The transcriptional profile of that precursor cell population is closer to that of neonatal than embryonic cardiomyocyte precursors. Tbx5, a cardinal cardiac development transcription factor, lies in the center of a ventricular adult precursor cell population, which seems to be affected by neurohormonal spatiotemporal cues. The identification of a Tbx5-specific cardiomyocyte precursor-like cell population, which is capable of dedifferentiating and potentially deploying a cardiomyocyte regenerative program, provides a clear target cell population for translationally-relevant heart interventional studies.

A transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification.

The major cardiac cell types composing the adult heart arise from common multipotent precursor cells. Cardiac lineage decisions are guided by extrinsic and cell-autonomous factors, including recently discovered long noncoding RNAs (lncRNAs). The human lncRNA CARMEN, which is known to dictate specification toward the cardiomyocyte (CM) and the smooth muscle cell (SMC) fates, generates a diversity of alternatively spliced isoforms. The CARMEN locus can be manipulated to direct human primary cardiac precursor cells (CPCs) into specific cardiovascular fates. Investigating CARMEN isoform usage in differentiating CPCs represents therefore a unique opportunity to uncover isoform-specific functions in lncRNAs. Here, we identify one CARMEN isoform, CARMEN-201, to be crucial for SMC commitment. CARMEN-201 activity is encoded within an alternatively spliced exon containing a MIRc short interspersed nuclear element. This element binds the transcriptional repressor REST (RE1 Silencing Transcription Factor), targets it to cardiogenic loci, including ISL1, IRX1, IRX5, and SFRP1, and thereby blocks the CM gene program. In turn, genes regulating SMC differentiation are induced. These data show how a critical physiological switch is wired by alternative splicing and functional transposable elements in a long noncoding RNA. They further demonstrated the crucial importance of the lncRNA isoform CARMEN-201 in SMC specification during heart development.

Alternative splicing of a transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification

Alternative splicing of a transposable element into the human long noncoding RNA CARMEN is a switch for cardiac precursor cell specification

Abstract 14259: Novel Long Noncoding RNA, Ppp1r1b-LncRNA , Epigenetically Promotes Myogenic Differentiation Program in Cardiac and Skeletal Myocytes

Introduction: Emerged as critical regulators of the transcriptome, the lncRNAs play important roles in cardiac development and might be targeted to treat human cardiomyocyte dysfunction in congenital heart disease. In our work, we identified a novel lncRNA, Ppp1r1b-lncRNA , as an essential, functionally conserved, positive regulator of myogenic differentiation. Hypothesis: Ppp1r1b-lncRNA promotes myogenic differentiation program of cardiac and skeletal myocytes via epigenic mechanisms. Methods: Antisense oligonucleotides (GapmeR) were used to suppress Ppp1r1b-lncRNA . Chromatin immunoprecipitation (CHIP) and chromatin isolation by RNA purification (CHIRP) were used to analyze gene specific histone modification level and lncRNA: chromatin interaction, respectively. RNA pull-down and RNA immunoprecipitation (RIP) were used to identify Ppp1r1b-lncRNA : protein interactome. Results: By silencing Ppp1r1b-lncRNA, skeletal myoblasts and human-induced pluripotent stem cell derived cardiomyocytes (hiPSCs-CMs) failed to develop fully differentiated myotubes. Analysis of GapmeR injected neonatal mouse heart further confirmed the role of Ppp1r1b-lncRNA in normal myogenesis. Members of muscle specific transcription factors, including MyoD1 and TBX5, were not induced during myogenic differentiation in the Ppp1r1b-lncRNA depleted cardiac and skeletal muscle precursor cells. Histone modification analysis revealed enrichment of H3K27 tri-methylation at MyoD1 and TBX5 promoters in GapmeR treated skeletal myoblasts. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and ChIRP analysis revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic regulatory factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Conclusions: Ppp1r1b-lncRNA promotes myogenic differentiation by inhibiting histone methylation on the promotors of myogenic master regulators via competing for PRC2 binding with chromatin during heart and skeletal muscle development.

Impaired SERCA2a phosphorylation causes diabetic cardiomyopathy through impinging on cardiac contractility and precursor protein processing

Abstract Diabetic cardiomyopathy (DCM) is currently a progressive and nonstoppable complication in type 2 diabetic patients. Metabolic insults and insulin resistance are involved in its pathogenesis; however, the underlying mechanisms are still not clearly understood. Here we show that calcium dysregulation can be both a cause and a consequence of cardiac insulin resistance that leads to DCM. A western diet induces the development of DCM through at least three phases in mice, among which an early phase depends on impaired Thr484-phosphorylation of sarcoplasmic/endoplasmic reticulum calcium ATPase 2a (SERCA2a) elicited by insulin resistance. Mutation of SERCA2a-Thr484 to a nonphosphorylatable alanine delays calcium re-uptake into the sarcoplasmic reticulum in the cardiomyocytes and decreases cardiac function at the baseline. Importantly, this mutation blunts the early phase of DCM, but has no effect on disease progression in the following phases. Interestingly, impairment of sarcoplasmic reticulum calcium re-uptake caused by the SERCA2a-Thr484 mutation inhibited processing of insulin receptor precursor through FURIN convertase, resulting in cardiac insulin resistance. Collectively, these data reveal a bidirectional relationship between insulin resistance and impairment of calcium homeostasis, which may underlie the early pathogenesis of DCM. Our findings have therapeutic implications for early intervention of DCM.

Inhibition of RhoA and Cdc42 by miR-133a Modulates Retinoic Acid Signalling during Early Development of Posterior Cardiac Tube Segment.

It is well known that multiple microRNAs play crucial roles in cardiovascular development, including miR-133a. Additionally, retinoic acid regulates atrial marker expression. In order to analyse the role of miR-133a as a modulator of retinoic acid signalling during the posterior segment of heart tube formation, we performed functional experiments with miR-133a and retinoic acid by means of microinjections into the posterior cardiac precursors of both primitive endocardial tubes in chick embryos. Subsequently, we subjected embryos to whole mount in situ hybridisation, immunohistochemistry and qPCR analysis. Our results demonstrate that miR-133a represses RhoA and Cdc42, as well as Raldh2/Aldh1a2, and the specific atrial markers Tbx5 and AMHC1, which play a key role during differentiation. Furthermore, we observed that miR-133a upregulates p21 and downregulates cyclin A by repressing RhoA and Cdc42, respectively, thus functioning as a cell proliferation inhibitor. Additionally, retinoic acid represses miR-133a, while it increases Raldh2, Tbx5 and AMHC1. Given that RhoA and Cdc42 are involved in Raldh2 expression and that they are modulated by miR-133a, which is influenced by retinoic acid signalling, our results suggest the presence of a negative feedback mechanism between miR-133a and retinoic acid during early development of the posterior cardiac tube segment. Despite additional unexplored factors being possible contributors to this negative feedback mechanism, miR-133a might also be considered as a potential therapeutic tool for the diagnosis, therapy and prognosis of cardiac diseases.

Inhibition of the NOTCH1 Pathway in the Stressed Heart Limits Fibrosis and Promotes Recruitment of Non-Myocyte Cells into the Cardiomyocyte Fate.

Cardiac pathologies lead to an acute or gradual loss of cardiomyocytes. Because of the limited regenerative capacity of the mammalian heart, cardiomyocytes are only replaced by fibrotic tissue. Excessive fibrosis contributes to the deterioration of cardiac function and the transition to heart failure, which is the leading cause of morbidity and mortality worldwide. Currently, no treatments can promote replenishment of the injured heart with newly formed cardiomyocytes. In this context, regenerative strategies explore the possibility to promote recovery through induction of cardiomyocyte production from pre-existing cardiomyocytes. On the other hand, cardiac non-myocyte cells can be directly reprogrammed into induced cardiac precursor cells and cardiomyocytes, suggesting that these cells could be exploited to produce cardiomyocytes in vivo. Here, we provide evidence that the sequential activation and inhibition of the NOTCH1 signaling pathway in the stressed heart decreases fibrosis and improves cardiac function in the stressed heart. This is accompanied by the emergence of new cardiomyocytes from non-myocyte origin. Overall, our data show how a developmental pathway such as the NOTCH pathway can be manipulated to provide therapeutic benefit in the damaged heart.

CCBE1 in Cardiac Development and Disease.

The collagen- and calcium-binding EGF-like domains 1 (CCBE1) is a secreted protein extensively described as indispensable for lymphangiogenesis during development enhancing VEGF-C signaling. In human patients, mutations in CCBE1 have been found to cause Hennekam syndrome, an inherited disease characterized by malformation of the lymphatic system that presents a wide variety of symptoms such as primary lymphedema, lymphangiectasia, and heart defects. Importantly, over the last decade, an essential role for CCBE1 during heart development is being uncovered. In mice, Ccbe1 expression was initially detected in distinct cardiac progenitors such as first and second heart field, and the proepicardium. More recently, Ccbe1 expression was identified in the epicardium and sinus venosus (SV) myocardium at E11.5–E13.5, the stage when SV endocardium–derived (VEGF-C dependent) coronary vessels start to form. Concordantly, CCBE1 is required for the correct formation of the coronary vessels and the coronary artery stem in the mouse. Additionally, Ccbe1 was found to be enriched in mouse embryonic stem cells (ESC) and revealed as a new essential gene for the differentiation of ESC-derived early cardiac precursor cell lineages. Here, we bring an up-to-date review on the role of CCBE1 in cardiac development, function, and human disease implications. Finally, we envisage the potential of this molecule’s functions from a regenerative medicine perspective, particularly novel therapeutic strategies for heart disease.

Similarity Score for the Identification of Active Sites in Patients With Atrial Fibrillation.

BackgroundAtrial fibrillation (AF) is the most common cardiac arrhythmia and precursor to other cardiac diseases. Catheter ablation is associated with limited success rates in patients with persistent AF. Currently, existing mapping systems fail to identify critical target sites for ablation. Recently, we proposed and validated several individual techniques, such as dominant frequency (DF), multiscale frequency (MSF), kurtosis (Kt), and multiscale entropy (MSE), to identify active sites of arrhythmias using simulated intracardiac electrograms (iEGMs). However, the individual performances of these techniques to identify arrhythmogenic substrates are not reliable.ObjectiveThis study aimed to develop a similarity score using various iEGM analysis techniques to more accurately identify the spatial location of active sites of arrhythmia in patients with AF.MethodsClinical bipolar iEGMs were obtained from patients with AF who underwent either successful (m = 4) or unsuccessful (m = 4) catheter ablation. A similarity score (0–3) was developed via the earth mover’s distance (EMD) approach based on a combination of DF, MSF, MSE, and Kt techniques.ResultsIndividual techniques successfully discriminated between successful and unsuccessful AF ablation patients but were not reliable in identifying active spatial sites of AF. However, the proposed similarity score was able to pinpoint the spatial sites with high values (active AF sites) that were observed only in patients with unsuccessful AF termination, suggesting that these active sites were missed during the ablation procedure.ConclusionArrhythmogenic substrates with abnormal electrical activity are identified in patients with unsuccessful AF termination after catheter ablation, suggesting clinical efficacy of similarity score.

Dynamic MicroRNA Expression Profiles During Embryonic Development Provide Novel Insights Into Cardiac Sinus Venosus/Inflow Tract Differentiation.

MicroRNAs have been explored in different organisms and are involved as molecular switches modulating cellular specification and differentiation during the embryonic development, including the cardiovascular system. In this study, we analyze the expression profiles of different microRNAs during early cardiac development. By using whole mount in situ hybridization in developing chick embryos, with microRNA-specific LNA probes, we carried out a detailed study of miR-23b, miR-130a, miR-106a, and miR-100 expression during early stages of embryogenesis (HH3 to HH17). We also correlated those findings with putative microRNA target genes by means of mirWalk and TargetScan analyses. Our results demonstrate a dynamic expression pattern in cardiac precursor cells from the primitive streak to the cardiac looping stages for miR-23b, miR-130a, and miR-106a. Additionally, miR-100 is later detectable during cardiac looping stages (HH15-17). Interestingly, the sinus venosus/inflow tract was shown to be the most representative cardiac area for the convergent expression of the four microRNAs. Through in silico analysis we revealed that distinct Hox family members are predicted to be targeted by the above microRNAs. We also identified expression of several Hox genes in the sinus venosus at stages HH11 and HH15. In addition, by means of gain-of-function experiments both in cardiomyoblasts and sinus venosus explants, we demonstrated the modulation of the different Hox clusters, Hoxa, Hoxb, Hoxc, and Hoxd genes, by these microRNAs. Furthermore, we correlated the negative modulation of several Hox genes, such as Hoxa3, Hoxa4, Hoxa5, Hoxc6, or Hoxd4. Finally, we demonstrated through a dual luciferase assay that Hoxa1 is targeted by miR-130a and Hoxa4 is targeted by both miR-23b and miR-106a, supporting a possible role of these microRNAs in Hox gene modulation during differentiation and compartmentalization of the posterior structures of the developing venous pole of the heart.

DNA‐Binding Specificity of Cardiac Transcription Factor Complex Formed by NKX2‐5 and TBX5

Transcription factors (TFs) are sequence-specific DNA-binding proteins responsible for cellular differentiation and development. Eukaryotic TFs are known for binding DNA as multimeric complexes to regulate gene expression. Transcription factors NKX2-5 and TBX5 are fundamental elements of the gene regulatory networks that control mammalian heart development. More importantly, NKX2-5 and TBX5 can bind to DNA as a cooperative complex. Our aim was to identify the intrinsic DNA-binding preferences and the motif spacing and orientation pattern of the NKX2-5:TBX5 heterodimer complex. In this study, we expressed full-length GST-tagged NKX2-5 and TBX5 using a wheat germ cell-free system. We successfully determined the in vitro DNA-binding specificity of the NKX2-5:TBX5 complex using Systematic Evolution of Ligands by Exponential Enrichment (SELEX-seq). Our preliminary results shows NKX2-5:TBX5 binding in a head-to-tail orientation with 0bp and 3bps spacing between core binding sites: GGTGT for TBX5 and CACTT for NKX2-5. The spacing preference is consistent with ChIP-exo data from cardiomyocytes and cardiac precursors. Our results highlight the utility of using in vitro techniques to determined DNA-binding specificity to understand genomic binding. The findings of our study will help understand the spatial and temporal gene regulation rules involved in normal heart development.

Anterior lateral plate mesoderm gives rise to multiple tissues and requires tbx5a function in left-right asymmetry, migration dynamics, and cell specification of late-addition cardiac cells

In this study, we elucidate a single cell resolution fate map in the zebrafish in a sub-section of the anterior Lateral Plate Mesoderm (aLPM) at 18 hpf. Our results show that this tissue is not organized into segregated regions but gives rise to intermingled pericardial sac, peritoneum, pharyngeal arch and cardiac precursors. We further report upon asymmetrical contributions of lateral aLPM-derived heart precursors—specifically that twice as many heart precursors arise from the right side versus the left side of the embryo. Cell tracking analyses and large-scale cell labeling of the lateral aLPM corroborate these differences and show that the observed asymmetries are dependent upon Tbx5a expression. Previously, it was shown that cardiac looping was affected in Tbx5a knock-down and knock-out zebrafish (Garrity et al., 2002; Parrie et al., 2013); our present data also implicate tbx5a function in cell specification, establishment and maintenance of cardiac left-right asymmetry.