Coexistent HCN4 and GATA5 Rare Variants and Atrial Fibrillation in a Large Spanish Family.

BACKGROUND: Familial association of atrial fibrillation (AF) can involve single gene variants related to known arrhythmogenic mechanisms; however, genome-wide association studies often disclose complex genetic variants in familial and nonfamilial AF, making it difficult to relate to known pathogenetic mechanisms. METHODS: The finding of 4 siblings with AF led to studying 47 members of a family. Long-term Holter monitoring (average 298 hours) ruled out silent AF. Whole-exome sequencing was performed, and variants shared by the index cases were filtered and prioritised according to current recommendations. HCN4 currents (IHCN4) were recorded in Chinese hamster ovary cells expressing human p.P1163H or native HCN4 channels with the use of the patch-clamp technique, and topologically associating domain analyses of GATA5 variant were performed. RESULTS: The clinical study diagnosed 2 more AF cases. Five family members carried the heterozygous p.P1163H HCN4 variant, 14 carried the intronic 20,61040536,G,A GATA5 rare variant, and 9 carried both variants (HCN4+GATA5). Five of the 6 AF cases (onset age ranging from 33 to 70 years) carried both variants and 1 carried the GATA5 variant alone. Multivariate analysis showed that the presence of HCN4+GATA5 variants significantly increased AF risk (odds ratio 32.7, 95% confidence interval 1.8-591.4) independently from age, hypertension, and overweight. Functional testing showed that IHCN4 generated by heterozygous p.P1163H were normal. Topologically associating domain analysis suggested that GATA5 could affect the expression of many genes, including those encoding microRNA-1. CONCLUSION: The coincidence of 2 rare gene variants was independently associated with AF, but functional studies do not allow the postulation of the arrhythmogenic mechanisms involved.

Towards Understanding the Gene-Specific Roles of GATA Factors in Heart Development: Does GATA4 Lead the Way?

Transcription factors play crucial roles in the regulation of heart induction, formation, growth and morphogenesis. Zinc finger GATA transcription factors are among the critical regulators of these processes. GATA4, 5 and 6 genes are expressed in a partially overlapping manner in developing hearts, and GATA4 and 6 continue their expression in adult cardiac myocytes. Using different experimental models, GATA4, 5 and 6 were shown to work together not only to ensure specification of cardiac cells but also during subsequent heart development. The complex involvement of these related gene family members in those processes is demonstrated through the redundancy among them and crossregulation of each other. Our recent identification at the genome-wide level of genes specifically regulated by each of the three family members and our earlier discovery that gata4 and gata6 function upstream, while gata5 functions downstream of noncanonical Wnt signalling during cardiac differentiation, clearly demonstrate the functional differences among the cardiogenic GATA factors. Such suspected functional differences are worth exploring more widely. It appears that in the past few years, significant advances have indeed been made in providing a deeper understanding of the mechanisms by which each of these molecules function during heart development. In this review, I will therefore discuss current evidence of the role of individual cardiogenic GATA factors in the process of heart development and emphasize the emerging central role of GATA4.

Identification of non-synonymous variations in ROBO1 and GATA5 genes in a family with bicuspid aortic valve disease.

Bicuspid aortic valve (BAV) is the most common congenital heart defect with a high index of heritability. Patients with BAV have different clinical courses and disease progression. Herein, we report three siblings with BAV and clinical differences. Their clinical presentations include moderate to severe aortic regurgitation, aortic stenosis, and ascending aortic aneurysm. Genetic investigation was carried out using Whole-Exome Sequencing for the three patients. We identified two non-synonymous variants in ROBO1 and GATA5 genes. The ROBO1: p.(Ser327Pro) variant is shared by the three BAV-affected siblings. The GATA5: p.(Gln3Arg) variant is shared only by the two brothers who presented BAV and ascending aortic aneurysm. Their sister, affected by BAV without aneurysm, does not harbor the GATA5: p.(Gln3Arg) variant. Both variants were absent in the patients' fourth brother who is clinically healthy with tricuspid aortic valve. To our knowledge, this is the first association of ROBO1 and GATA5 variants in familial BAV with a potential genotype-phenotype correlation. Our findings are suggestive of the implication of ROBO1 gene in BAV and the GATA5: p.(Gln3Arg) variant in ascending aortic aneurysm. Our family-based study further confirms the intrafamilial incomplete penetrance of BAV and the complex pattern of inheritance of the disease.

GATA4/5/6 family transcription factors are conserved determinants of cardiac versus pharyngeal mesoderm fate.

GATA4/5/6 transcription factors play essential, conserved roles in heart development. To understand how GATA4/5/6 modulates the mesoderm-to-cardiac fate transition, we labeled, isolated, and performed single-cell gene expression analysis on cells that express gata5 at precardiac time points spanning zebrafish gastrulation to somitogenesis. We found that most mesendoderm-derived lineages had dynamic gata5/6 expression. In the absence of Gata5/6, the population structure of mesendoderm-derived cells was substantially altered. In addition to the expected absence of cardiac mesoderm, we confirmed a concomitant expansion of cranial-pharyngeal mesoderm. Moreover, Gata5/6 loss led to extensive changes in chromatin accessibility near cardiac and pharyngeal genes. Functional analyses in zebrafish and the tunicate Ciona, which has a single GATA4/5/6 homolog, revealed that GATA4/5/6 acts upstream of tbx1 to exert essential and cell-autonomous roles in promoting cardiac and inhibiting pharyngeal mesoderm identity. Overall, cardiac and pharyngeal mesoderm fate choices are achieved through an evolutionarily conserved GATA4/5/6 regulatory network.

Molecular genetic study on GATA5 gene promoter in acute myocardial infarction.

BACKGROUND: Acute myocardial infarction (AMI) is a severe type of coronary artery disease, caused by coronary occlusion and followed by cardiac ischaemia. GATA binding protein 5 (GATA5) is an important member of GATA family and plays an important role in vascular inflammation, endothelial function, oxidative stress and cell metabolism. Previous studies have shown that the DNA sequence variants (DSVs) in GATA4 and GATA6 promoter can increase susceptibility to AMI. In this study, we explored the relationship between GATA5 promoter and AMI for the first time, hoping to provide a new genetic basis for understanding the pathogenesis of AMI. METHODS: GATA5 promoter was sequenced in 683 individuals (332 AMI patients and 351 controls). The transcriptional activity of the GATA5 promoter with or without DSVs in HEK-293 cells, H9c2 cells and primary neonatal rat cardiomyocytes were examined by Promega Dual-Luciferase® Reporter Assay system. Electrophoretic mobility shift assay (EMSA) was performed to explore whether the DSVs interfered with the binding of transcription factors (TFs). RESULTS: Nine mutations have been found in GATA5 promoter, eight of them evidently altered the transcriptional activity of the GATA5 promoter, five of them disrupted the binding of TFs (such as farnesoid X receptor). Furthermore, haplotype AT (across rs80197101 and rs77067995) is a dangerous haplotype of AMI. Genotype GA and allele A of rs80197101 and genotype CT and allele T of rs77067995 are the risk factors of AMI. CONCLUSIONS: DSVs in GATA5 promoter can increase susceptibility to AMI. But the mechanism remains to be verified in vivo.

Discovery of rare variants associated with blood pressure regulation through meta-analysis of 1.3 million individuals.

Genetic studies of blood pressure (BP) to date have mainly analyzed common variants (minor allele frequency > 0.05). In a meta-analysis of up to ~1.3 million participants, we discovered 106 new BP-associated genomic regions and 87 rare (minor allele frequency ≤ 0.01) variant BP associations (P < 5 × 10-8), of which 32 were in new BP-associated loci and 55 were independent BP-associated single-nucleotide variants within known BP-associated regions. Average effects of rare variants (44% coding) were ~8 times larger than common variant effects and indicate potential candidate causal genes at new and known loci (for example, GATA5 and PLCB3). BP-associated variants (including rare and common) were enriched in regions of active chromatin in fetal tissues, potentially linking fetal development with BP regulation in later life. Multivariable Mendelian randomization suggested possible inverse effects of elevated systolic and diastolic BP on large artery stroke. Our study demonstrates the utility of rare-variant analyses for identifying candidate genes and the results highlight potential therapeutic targets.

Potential roles of GATA binding protein 5 in cardiovascular diseases.

It is known that functional defects of GATA binding protein 5 (GATA5), an important member of GATA transcription factor family, could cause multiple congenital defects. However, the mechanisms of this transcription factor in cardiovascular diseases are still little known. Finding a genetic approach should help with understanding the possible roles of GATA5 in different cardiovascular diseases and purpose it as a possible therapeutic agent. Hence, this review is divided into three chapters to summarize the roles and main regulatory mechanisms of GATA5 in hypertension, arrhythmia and congenital heart disease, respectively. In each chapter, this review firstly introduces the roles of GATA5 mutations, and then discusses the main regulatory mechanisms of GATA5 in the corresponding diseases (Such as the endothelial dysfunction signaling pathway in the chapter of hypertension, GATA5-NaV1.5 signaling pathway in the chapter of arrhythmia, GATA5-HEY2 and GATA5-Nodal signaling pathway in the chapter of congenital heart disease). Additionally, based on these regulatory networks, it is also speculated that abnormal methylation of the GATA5 gene promoter may lead to cardiovascular diseases such as congenital heart disease. This conjecture is proposed to enrich the regulatory networks of GATA5 and provide a theoretical basis for diagnosis and treatment of cardiovascular diseases.

Specificity, redundancy and dosage thresholds among gata4/5/6 genes during zebrafish cardiogenesis.

The Gata4/5/6 sub-family of zinc finger transcription factors regulate many aspects of cardiogenesis. However, critical roles in extra-embryonic endoderm also challenge comprehensive analysis during early mouse cardiogenesis, while zebrafish models have previously relied on knockdown assays. We generated targeted deletions to disrupt each gata4/5/6 gene in zebrafish and analyzed cardiac phenotypes in single, double and triple mutants. The analysis confirmed that loss of gata5 causes cardia bifida and validated functional redundancies for gata5/6 in cardiac precursor specification. Surprisingly, we discovered that gata4 is dispensable for early zebrafish development, while loss of one gata4 allele can suppress the bifid phenotype of the gata5 mutant. The gata4 mutants eventually develop an age-dependent cardiomyopathy. By combining combinations of mutant alleles, we show that cardiac specification depends primarily on an overall dosage of gata4/5/6 alleles rather than a specific gene. We also identify a specific role for gata6 in controlling ventricle morphogenesis through regulation of both the first and second heart field, while loss of both gata4/6 eliminates the ventricle. Thus, different developmental programs are dependent on total dosage, certain pairs, or specific gata4/5/6 genes during embryonic cardiogenesis.This article has an associated First Person interview with the first author of the paper.

The role of miR-92 in regulating early development and metamorphosis of Japanese flounder Paralichthys olivaceus.

MicroRNAs are a class of short non-coding RNAs that contain approximately 22 nucleotides and play a regulatory role in RNA silencing and translational repression. miR-92 belongs to the miR-17-92 family and has a regulatory effect on cell proliferation, apoptosis, and expression of proto-oncogenes and tumor suppressor genes. However, its function in flatfish is unclear. In this study, we used farmed Japanese flounder, Paralichthys olivaceus, and showed that gata5 is a target gene of miR-92. Experiments on miR-92 overexpression indicated that gata5 and sox17 were downregulated, while the transcription level of ntl increased. By contrast, depletion of miR-92 resulted in increased gata5 and sox17 levels and reduced ntl level. Moreover, thiourea treatment indicated that miR-92 may inhibit the metamorphic development of Japanese flounder. Our study suggests that miR-92 regulates the fate of endoderm and mesoderm by controlling gata5.

Molecular determinants of nephron vascular specialization in the kidney.

Although kidney parenchymal tissue can be generated in vitro, reconstructing the complex vasculature of the kidney remains a daunting task. The molecular pathways that specify and sustain functional, phenotypic and structural heterogeneity of the kidney vasculature are unknown. Here, we employ high-throughput bulk and single-cell RNA sequencing of the non-lymphatic endothelial cells (ECs) of the kidney to identify the molecular pathways that dictate vascular zonation from embryos to adulthood. We show that the kidney manifests vascular-specific signatures expressing defined transcription factors, ion channels, solute transporters, and angiocrine factors choreographing kidney functions. Notably, the ontology of the glomerulus coincides with induction of unique transcription factors, including Tbx3, Gata5, Prdm1, and Pbx1. Deletion of Tbx3 in ECs results in glomerular hypoplasia, microaneurysms and regressed fenestrations leading to fibrosis in subsets of glomeruli. Deciphering the molecular determinants of kidney vascular signatures lays the foundation for rebuilding nephrons and uncovering the pathogenesis of kidney disorders.

Human pre-valvular endocardial cells derived from pluripotent stem cells recapitulate cardiac pathophysiological valvulogenesis.

Genetically modified mice have advanced our understanding of valve development and disease. Yet, human pathophysiological valvulogenesis remains poorly understood. Here we report that, by combining single cell sequencing and in vivo approaches, a population of human pre-valvular endocardial cells (HPVCs) can be derived from pluripotent stem cells. HPVCs express gene patterns conforming to the E9.0 mouse atrio-ventricular canal (AVC) endocardium signature. HPVCs treated with BMP2, cultured on mouse AVC cushions, or transplanted into the AVC of embryonic mouse hearts, undergo endothelial-to-mesenchymal transition and express markers of valve interstitial cells of different valvular layers, demonstrating cell specificity. Extending this model to patient-specific induced pluripotent stem cells recapitulates features of mitral valve prolapse and identified dysregulation of the SHH pathway. Concurrently increased ECM secretion can be rescued by SHH inhibition, thus providing a putative therapeutic target. In summary, we report a human cell model of valvulogenesis that faithfully recapitulates valve disease in a dish.

GATA5 inhibits hepatocellular carcinoma cells malignant behaviours by blocking expression of reprogramming genes.

Evidence indicated that GATA5 may suppress hepatocellular carcinoma (HCC) cell malignant transformation, but the mechanism of how GATA5 affects cancer cell reprogramming to inhibit HCC malignant behaviour is still unclear. In this study, we report that the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM was significantly higher in HCC tissues compared to normal liver tissues. In contrast, the expression of GATA5 was significantly lower in HCC tissues compared to normal liver tissues. Transfection of CDH-GATA5 vectors into HCC cells (HLE, Bel 7402 and PLC/PRF/5 cells) increased the GATA5 expression and decreased the expression of ß-catenin and reprogramming genes p-Oct4, Nanog, Klf4, c-myc and EpCAM. Increased GATA5 expression by transfection with its expression vectors was also able to inhibit the cell growth, colony formation and capability of migration, invasion, while promoting apoptosis in HCC cells. Results revealed that GATA5 co-localization with ß-catenin in the cytoplasm, preventing ß-catenin from entering the nucleus. Treatment with the specific Wnt/ß-catenin pathway inhibitor salinomycin was able to reduce the expression of ß-catenin and reprogramming genes. Salinomycin exerted a similar influence as GATA5, and siRNA-GATA5 restored ß-catenin and reprogramming gene expression. This study demonstrates that an increase in the expression of GATA5 inhibits the expression of ß-catenin and reprogramming genes and suppresses tumour growth, colony formation, metastasis and invasion, while promoting apoptosis in HCC cells. The mechanism of GATA5 inhibiting the malignant behaviours of HCC cells may involve in the disruption of the Wnt/ß-catenin pathway and the reduction of reprogramming gene expression.

Zebrafish microRNA miR-210-5p inhibits primitive myelopoiesis by silencing foxj1b and slc3a2a mRNAs downstream of gata4/5/6 transcription factor genes.

Zebrafish gata4/5/6 genes encode transcription factors that lie on the apex of the regulatory hierarchy in primitive myelopoiesis. However, little is known about the roles of microRNAs in gata4/5/6-regulated processes. Performing RNA-Seq deep sequencing analysis of the expression changes of microRNAs in gata4/5/6-knockdown embryos, we identified miR-210-5p as a regulator of zebrafish primitive myelopoiesis. Knocking down gata4/5/6 (generating gata5/6 morphants) significantly increased miR-210-5p expression, whereas gata4/5/6 overexpression greatly reduced its expression. Consistent with inhibited primitive myelopoiesis in the gata5/6 morphants, miR-210-5p overexpression repressed primitive myelopoiesis, indicated by reduced numbers of granulocytes and macrophages. Moreover, knocking out miR-210 partially rescued the defective primitive myelopoiesis in zebrafish gata4/5/6-knockdown embryos. Furthermore, we show that the restrictive role of miR-210-5p in zebrafish primitive myelopoiesis is due to impaired differentiation of hemangioblast into myeloid progenitor cells. By comparing the set of genes with reduced expression levels in the gata5/6 morphants to the predicted target genes of miR-210-5p, we found that foxj1b and slc3a2a, encoding a forkhead box transcription factor and a solute carrier family 3 protein, respectively, are two direct downstream targets of miR-210-5p that mediate its inhibitory roles in zebrafish primitive myelopoiesis. In summary, our results reveal that miR-210-5p has an important role in the genetic network controlling zebrafish primitive myelopoiesis.

Variants in cardiac GATA genes associated with bicuspid aortic valve.

BACKGROUND: Bicuspid aortic valve (BAV) is a heterogeneous and still not fully understood condition, with diverse genetic aetiology and associated phenotypes ranging from aortic stenosis or regurgitation to aneurysm and dissection. Several genes have been associated with the presence of BAV, notably some members of the GATA family of transcription factors that play important roles in cardiac embryogenesis. METHODS: A case-control study with 122 unrelated and ethnically matched patients with bicuspid and 154 with tricuspid aortic valve was performed. All exons of GATA4, GATA5, and GATA6 genes were sequenced searching for variants. Frequencies were compared and functional effects of rare variants were analysed by informatic prediction tools. RESULTS: Four rare and potentially pathogenic variants were identified in only five sporadic cases: a missense p.Arg202Gln (rs782614097) in GATA5 and three synonymous variants, p.Cys274= (rs55980825) and p.His302= (rs201516339) in GATA4, and p.Asn458= (rs143026087) in GATA6. Minor alleles of p.His302=, p.Arg202Gln and rs3764962 are enriched in BAV patients compared to ExAC database (P = 0.01, 0.03, and 0.01 respectively). In addition, a common polymorphism in GATA5 (p.Asp203=, rs41305803) is associated with BAV showing a protective effect in carriers of the minor allele (OR [95%CI] = 0.45[0.25-0.81]; P = 0.004). CONCLUSION: This study associates additional genetic variants in GATA4 and GATA5 with BAV, supporting the implication of these genes in the development of this valvulopathy. The discovery of all the genetic factors involved will contribute to a better understanding of the process and, therefore, to detect a genetic predisposition and even to the identification of therapeutic targets.

The genetic program of oocytes can be modified in vivo in the zebrafish ovary.

Oocytes, the irreplaceable gametes for generating a new organism, are matured in the ovary of living female animals. It is unknown whether any genetic manipulations can be applied to immature oocytes inside the living ovaries. As a proof-of-concept, we here demonstrate genetic amendments of zebrafish immature oocytes within the ovary. Oocyte microinjection in situ (OMIS) stimulates tissue repair responses, but some of the microinjected immature oocytes are matured, ovulated and fertilizable. By OMIS-mediated Cas9 approach, ntla and gata5 loci of oocytes arrested at prophase I of meiosis are successfully edited before fertilization. Through OMIS, high efficiency of biallelic mutations in single or multiple loci using Cas9/gRNAs allows immediate manifestation of mutant phenotypes in F0 embryos and multiple transgenes can co-express the reporters in F0 embryos with patterns similar to germline transgenic embryos. Furthermore, maternal knockdown of dnmt1 by antisense morpholino via OMIS results in a dramatic decrease of global DNA methylation level at the dome stage and causes embryonic lethality prior to segmentation period. Therefore, OMIS opens a door to efficiently modify the genome and provides a possibility to repair genetically abnormal oocytes in situ.

Prognostic DNA methylation markers for renal cell carcinoma: a systematic review.

AIM: Despite numerous published prognostic methylation markers for renal cell carcinoma (RCC), none of these have yet changed patient management. Our aim is to systematically review and evaluate the literature on prognostic DNA methylation markers for RCC. MATERIALS & METHODS: We conducted an exhaustive search of PubMed, EMBASE and MEDLINE up to April 2017 and identified 49 publications. Studies were reviewed according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement, assessed for their reporting quality using the Reporting Recommendations for Tumor Marker Prognostic Studies (REMARK) criteria, and were graded to determine the level of evidence (LOE) for each biomarker. RESULTS: We identified promoter methylation of BNC1, SCUBE3, GATA5, SFRP1, GREM1, RASSF1A, PCDH8, LAD1 and NEFH as promising prognostic markers. Extensive methodological heterogeneity across the included studies was observed, which hampers comparability and reproducibility of results, providing a possible explanation why these biomarkers do not reach the clinic. CONCLUSION: Potential prognostic methylation markers for RCC have been identified, but they require further validation in prospective studies to determine their true clinical value.

Compound heterozygous GATA5 mutations in a girl with hydrops fetalis, congenital heart defects and genital anomalies.

GATA5 belongs to the GATA family of transcription factors characterized by highly evolutionarily conserved zinc-finger DNA-binding domains. Mouse models have implicated a role of GATA5 during mammalian embryogenesis, including proper heart development and gender-specific regulation of female genitourinary tract formation. Previous studies have found an association of heterozygous missense alterations in GATA5 with a broad variety of heart diseases; however, the clinical relevance of the identified susceptibility variants has remained unclear. Here, we report on a girl with hydrops fetalis, congenital heart defects, clitoromegaly and postnatally increased 17-hydroxyprogesterone levels. By trio whole-exome sequencing, we identified compound heterozygous missense mutations, p.Ser19Trp and p.Arg202Gln, in GATA5 as putative disease-causing alterations. The identified mutations fail to rescue the cardia bifida phenotype in a zebrafish model, mislocalize to subnuclear foci when transiently transfected in HEK293 cells and possess less transcriptional activity. In addition to demonstrating the pathogenicity of identified mutations, our findings show that GATA5 mutations, in addition to heart diseases, can result in congenital abnormalities of the female genitourinary tract in humans.

Essential role for the planarian intestinal GATA transcription factor in stem cells and regeneration.

The cellular turnover of adult tissues and injury-induced repair proceed through an exquisite integration of proliferation, differentiation, and survival signals that involve stem/progenitor cell populations, their progeny, and differentiated tissues. GATA factors are DNA binding proteins that control stem cells and the development of tissues by activating or repressing transcription. Here we examined the role of GATA transcription factors in Schmidtea mediterranea, a freshwater planarian that provides an excellent model to investigate gene function in adult stem cells, regeneration, and differentiation. Smed-gata4/5/6, the homolog of the three mammalian GATA-4,-5,-6 factors is expressed at high levels in differentiated gut cells but also at lower levels in neoblast populations, the planarian stem cells. Smed-gata4/5/6 knock-down results in broad differentiation defects, especially in response to injury. These defects are not restricted to the intestinal lineage. In particular, at late time points during the response to injury, loss of Smed-gata4/5/6 leads to decreased neoblast proliferation and to gene expression changes in several neoblast subpopulations. Thus, Smed-gata4/5/6 plays a key evolutionary conserved role in intestinal differentiation in planarians. These data further support a model in which defects in the intestinal lineage can indirectly affect other differentiation pathways in planarians.

WT1, MSH6, GATA5 and PAX5 as epigenetic oral squamous cell carcinoma biomarkers - a short report.

PURPOSE: Oral squamous cell carcinoma (OSCC) is a frequently occurring aggressive malignancy with a heterogeneous clinical behavior. Based on the paucity of specific early diagnostic and prognostic biomarkers, which hampers the appropriate treatment and, ultimately the development of novel targeted therapies, we aimed at identifying such biomarkers through a genetic and epigenetic analysis of these tumors. METHODS: 93 primary OSCCs were subjected to DNA copy number alteration (CNA) and methylation status analyses using methylation-specific multiplex ligation-dependent probe amplification (MS-MPLA). The genetic and epigenetic OSCC profiles obtained were associated with the patients' clinic-pathological features. RESULTS: We found that WT1 gene promoter methylation is a predictor of a better prognosis and that MSH6 and GATA5 gene promoter methylation serve as predictors of a worse prognosis. GATA5 gene promoter methylation was found to be significantly associated with a shorter survival rate. In addition, we found that PAX5 gene promoter methylation was significantly associated with tongue tumors. To the best of our knowledge, this is the first study that highlights this specific set of genes as epigenetic diagnostic and prognostic biomarkers in OSCC. CONCLUSIONS: Our data highlight the importance of epigenetically assessing OSCCs to identify key genes that may serve as diagnostic and prognostic biomarkers and, potentially, as candidate therapeutic targets.

Mespaa can potently induce cardiac fates in zebrafish.

The Mesp family of transcription factors have been implicated in the early formation and migration of the cardiac lineage, although the precise molecular mechanisms underlying this process remain unknown. In this study we examine the function of Mesp family members in zebrafish cardiac development and find that Mespaa is remarkably efficient at promoting cardiac fates in normally non-cardiogenic cells. However, Mespaa is dispensable for normal cardiac formation. Despite no overt defects in cardiovascular specification, we find a consistent defect in cardiac laterality in mespaa null embryos. This is further exacerbated by the depletion of other mesp paralogues, highlighting a conserved role for the mesp family in left-right asymmetry, distinct from a function in cardiac specification. Despite an early requirement for mespaa to promote cardiogenesis, cells over-expressing mespaa are found to both exhibit unique cellular behaviors and activate the transcription of gata5 only after the completion of gastrulation. We propose that while mespaa remains capable of driving cardiac progenitor formation in zebrafish, it may not play an essential role in the cardiac regulatory network. Furthermore, the late activation of migration and cardiac gene transcription in mespaa over-expressing cells challenges previous studies on the timing of these events and provides intriguing questions for future study.