Zebrafish Cardiogenesis Research Articles

Sin3a associated protein 130kDa, sap130, plays an evolutionary conserved role in zebrafish heart development.

Hypoplastic left heart syndrome (HLHS) is a congenital heart disease where the left ventricle is reduced in size. A forward genetic screen in mice identified SIN3A associated protein 130kDa (Sap130), part of the chromatin modifying SIN3A/HDAC complex, as a gene contributing to the etiology of HLHS. Here, we report the role of zebrafish sap130 genes in heart development. Loss of sap130a, one of two Sap130 orthologs, resulted in smaller ventricle size, a phenotype reminiscent to the hypoplastic left ventricle in mice. While cardiac progenitors were normal during somitogenesis, diminution of the ventricle size suggest the Second Heart Field (SHF) was the source of the defect. To explore the role of sap130a in gene regulation, transcriptome profiling was performed after the heart tube formation to identify candidate pathways and genes responsible for the small ventricle phenotype. Genes involved in cardiac differentiation and cardiac function were dysregulated in sap130a, but not in sap130b mutants. Confocal light sheet analysis measured deficits in cardiac output in MZsap130a supporting the notion that cardiomyocyte maturation was disrupted. Lineage tracing experiments revealed a significant reduction of SHF cells in the ventricle that resulted in increased outflow tract size. These data suggest that sap130a is involved in cardiogenesis via regulating the accretion of SHF cells to the growing ventricle and in their subsequent maturation for cardiac function. Further, genetic studies revealed an interaction between hdac1 and sap130a, in the incidence of small ventricles. These studies highlight the conserved role of Sap130a and Hdac1 in zebrafish cardiogenesis.

Atrial and Sinoatrial Node Development in the Zebrafish Heart.

Proper development and function of the vertebrate heart is vital for embryonic and postnatal life. Many congenital heart defects in humans are associated with disruption of genes that direct the formation or maintenance of atrial and pacemaker cardiomyocytes at the venous pole of the heart. Zebrafish are an outstanding model for studying vertebrate cardiogenesis, due to the conservation of molecular mechanisms underlying early heart development, external development, and ease of genetic manipulation. Here, we discuss early developmental mechanisms that instruct appropriate formation of the venous pole in zebrafish embryos. We primarily focus on signals that determine atrial chamber size and the specialized pacemaker cells of the sinoatrial node through directing proper specification and differentiation, as well as contemporary insights into the plasticity and maintenance of cardiomyocyte identity in embryonic zebrafish hearts. Finally, we integrate how these insights into zebrafish cardiogenesis can serve as models for human atrial defects and arrhythmias.

First person – Jessica Sam

ABSTRACTFirst Person is a series of interviews with the first authors of a selection of papers published in Biology Open, helping early-career researchers promote themselves alongside their papers. Jessica Sam is first author on ‘Specificity, redundancy and dosage thresholds among gata4/5/6 genes during zebrafish cardiogenesis’, published in BiO. Jessica is a PhD student in the lab of Todd Evans at the Department of Surgery, Weill Cornell Medicine, New York, USA, investigating the genetic mechanisms underlying cardiac development and congenital heart disease.

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

ABSTRACTThe 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.

TETs Regulate Proepicardial Cell Migration through Extracellular Matrix Organization during Zebrafish Cardiogenesis

SUMMARYTen-eleven translocation (Tet) enzymes (Tet1/2/3) mediate 5-methylcytosine (5mC) hydroxylation, which can facilitate DNA demethylation and thereby impact gene expression. Studied mostly for how mutant isoforms impact cancer, the normal roles for Tet enzymes during organogenesis are largely unknown. By analyzing compound mutant zebrafish, we discovered a requirement for Tet2/3 activity in the embryonic heart for recruitment of epicardial progenitors, associated with development of the atrial-ventricular canal (AVC). Through a combination of methylation, hydroxymethylation, and transcript profiling, the genes encoding the activin A subunit Inhbaa (in endocardium) and Sox9b (in myocardium) were implicated as demethylation targets of Tet2/3 and critical for organization of AVC-localized extracellular matrix (ECM), facilitating migration of epicardial progenitors onto the developing heart tube. This study elucidates essential DNA demethylation modifications that govern gene expression changes during cardiac development with striking temporal and lineage specificities, highlighting complex interactions in multiple cell populations during development of the vertebrate heart.

The transcription factor Foxc1a in zebrafish directly regulates expression of nkx2.5, encoding a transcriptional regulator of cardiac progenitor cells

Cardiogenesis is a tightly controlled biological process required for formation of a functional heart. The transcription factor Foxc1 not only plays a crucial role in outflow tract development in mice, but is also involved in cardiac structure formation and normal function in humans. However, the molecular mechanisms by which Foxc1 controls cardiac development remain poorly understood. Previously, we reported that zebrafish embryos deficient in foxc1a, an ortholog of mammalian Foxc1, display pericardial edemas and die 9-10 days postfertilization. To further investigate Foxc1a's role in zebrafish cardiogenesis and identify its downstream target genes during early heart development, we comprehensively analyzed the cardiovascular phenotype of foxc1a-null zebrafish embryos. Our results confirmed that foxc1a-null mutants exhibit disrupted cardiac morphology, structure, and function. Performing transcriptome analysis on the foxc1a mutants, we found that the expression of the cardiac progenitor marker gene nkx2.5 was significantly decreased, but the expression of germ layer-patterning genes was unaffected. Dual-fluorescence in situ hybridization assays revealed that foxc1a and nkx2.5 are co-expressed in the anterior lateral plate mesoderm at the somite stage. Chromatin immunoprecipitation and promoter truncation assays disclosed that Foxc1a regulates nkx2.5 expression via direct binding to two noncanonical binding sites in the proximal nkx2.5 promoter. Moreover, functional rescue experiments revealed that developmental stage-specific nkx2.5 overexpression partially rescues the cardiac defects of the foxc1a-null embryos. Taken together, our results indicate that during zebrafish cardiogenesis, Foxc1a is active directly upstream of nkx2.5.

Gene locations may contribute to predicting gene regulatory relationships.

We propose that locations of genes on chromosomes can contribute to the prediction of gene regulatory relationships. We constructed a time-based gene regulatory network of zebrafish cardiogenesis on the basis of a spatio-temporal neighborhood method. Through the network, specific regulatory pathways and order of gene expression during zebrafish cardiogenesis were obtained. By comparing the order with locations of these genes on chromosomes, we discovered that there exists a reversal phenomenon between the order and order of gene locations. The discovery provides an inherent rule to instruct exploration of gene regulatory relationships. Specifically, the discovery can help to predict if regulatory relationships between genes exist and contribute to evaluating the correctness of discovered gene regulatory relationships.

Maternal or zygotic sphingosine kinase is required to regulate zebrafish cardiogenesis.

The sphingosine 1-phosphate (S1P) signaling pathway regulates zebrafish cardiogenesis, and provides a paradigm for how signaling gradients coordinate collective cell migration across tissue layers. It is known that the S1P transporter (Spns2) functions in extra-embryonic YSL to activate G protein-coupled receptor (S1pr2) signaling in endoderm for deposition of positional cues (integrin, fibronectin, etc.). Such cues are recognized by overlying lateral precardiac mesoderm that migrates to the midline and fuses to form the primordial heart tube. However, the source of bio-active S1P is not known. There are multiple receptors and it is not known if there are earlier or even receptor-independent functions for S1P. Because S1P can only be generated by sphingosine kinases, we targeted a mutation to the single kinase gene expressed during early embryogenesis (sphk2). Zygotic mutants survive to adulthood and appear normal, but maternal-zygotic mutant embryos phenocopy null zygotic mutants of spns2 or s1pr2. The data show that maternally derived sphk2 RNA is fully sufficient to generate an S1P signaling gradient in the YSL that ultimately controls precardiac mesoderm migration during embryogenesis. Furthermore, despite maternal expression of sphk2, there are no obvious developmental functions requiring its activity prior to stimulation of S1pr2 in endoderm.

Proper Activity of Histone H3 Lysine 4 (H3K4) Methyltransferase Is Required for Morphogenesis during Zebrafish Cardiogenesis.

While increasing evidence indicates the important function of histone methylation during development, how this process influences cardiac development in vertebrates has not been explored. Here, we elucidate the functions of two histone H3 lysine 4 (H3K4) methylation enzymes, SMYD3 and SETD7, during zebrafish heart morphogenesis using gene expression profiling by whole mount in situ hybridization and antisense morpholino oligonucleotide (MO)-based gene knockdown. We find both smyd3 and setd7 are highly expressed within developing zebrafish heart and knock-down of these genes led to severe defects in cardiac morphogenesis without altering the expressions pattern of heart markers, including cmlc2, vmhc, and amhc. Furthermore, double knock-down by coinjection of smyd3 and setd7 MOs caused the synergistic defects in heart development. As similar to knock-down effect, overexpression of these genes also caused the heart morphogenesis defect in zebrafish. These results indicate that histone modifying enzymes, SMYD3 and SETD7, appear to function synergistically during heart development and their proper functioning is essential for normal heart morphogenesis during development.

Aryl organophosphate flame retardants induced cardiotoxicity during zebrafish embryogenesis: By disturbing expression of the transcriptional regulators

As a result of the ban on some brominated flame retardants (BFRs), the use of organophosphate flame retardants (OPFRs) increases, and they are detected in multi-environment media at higher frequency and concentrations. However, the toxicity data of OPFRs, especially those on developmental toxicology are quite limited, which prevents an accurate evaluation of their environmental and health risk. Because a previous study reported that two aryl-OPFRs induced cardiotoxicity during zebrafish embryogenesis, we designed experiments to compare the heart developmental toxicity of a series of aryl-OPFRs with alkyl-OPFRs and explored possible internal mechanism. First, acute toxicity of 9 frequently used OPFRs were studied with zebrafish embryos (2–96hpf). By comparing the LC50 and EC50 (pericardium edema) data, two aryl-OPFRs, triphenyl phosphate (TPhP) and cresyl diphenyl phosphate (CDP) showed greater heart developmental toxicity than the others. It was also found that the acute toxicity of OPFRs varied mainly depending on their hydrophobicity. Further study on the cardiotoxicty of TPhP and CDP showed that the cardiac looping progress can be impeded by 0.10mg/L TPhP or CDP exposure. Bradycardia and reduction of myocardium were also observed in 0.50 and 1.0mg/L TPhP groups and 0.10, 0.50, and 1.0mg/L CDP groups. 0–48hpf is the vulnerable window of zebrafish cardiogenesis that can be easily affected by TPhP and CDP. RT-qPCR measurement on the expressions of key transcriptional regulators in cardiogenesis showed that BMP4, NKX2–5, and TBX5 were significantly inhibited at the exposure points of 12hpf and 24hpf which may be the internal factors related to the heart developmental toxicity. As zebrafish is a good model organism for human health study, the present results call for a greater attention to the health risk of fetus in pregnant women exposed to such OPFRs.

Tissue factor pathway inhibitor-2 is critical in zebrafish cardiogenesis

Human tissue factor pathway inhibitor-2 (Tfpi-2) is an extracellular matrix-associated Kunitz-type serine proteinase inhibitor and plays an important role in various cellular processes. We have previously shown that zebrafish Tfpi-2 (zTfpi-2) mainly expressed in the brain and heart of zebrafish, and it is involved in the development of central nervous system. Here, we identified zTfpi-2 as an evolutionarily conserved protein essential for zebrafish heart development, as embryos depleted of zTfpi-2 failed to undergo cardiogenesis. Changes of cardiogenic markers, vmhc, amhc and bmp4, confirmed zTfpi-2 knockdown caused cardiac defects, including retrenched ventricle, enlarged atrium and malformation of atrioventricular boundary. The sarcomeric organization was also disrupted by embryonic depletion of zTfpi-2, thus establishing the functional role of zTfpi-2 in cardiac contractility. In addition, hematopoietic defects were detected in the zTfpi-2-deficiency embryos. Importantly, injection of ztfpi-2 mRNA attenuated those cardiac and hematopoietic defects. Taken together, this study demonstrated a critical role of zTfpi-2 during embryonic cardiac development, as well as an important regulator of hematopoiesis.

Cessation of contraction induces cardiomyocyte remodeling during zebrafish cardiogenesis

Contraction regulates heart development via a complex mechanotransduction process controlled by various mechanical forces. Here, we exploit zebrafish embryos as an in vivo animal model to discern the contribution from different mechanical forces and identify the underlying mechanotransductive signaling pathways of cardiogenesis. We treated 2 days postfertilization zebrafish embryos with Blebbistatin, a myosin II inhibitor, to stop cardiac contraction, which induces a response termed cessation of contraction-induced cardiomyocyte (CM) enlargement (CCE). Accompanying the CCE, lateral fusion of myofibrils was attenuated within CMs. The CCE can be blunted by loss of blood in tail-docked zebrafish but not in cloche mutant fish, suggesting that transmural pressure rather than shear stress is accountable for the chamber enlargement. By screening a panel of small molecule inhibitors, our data suggested essential functions of phosphoinositide 3-kinase signaling and protein synthesis in CCE, which are independent of the sarcomere integrity. In summary, we defined a unique CCE response in genetically tractable zebrafish embryos. A panel of assays was established to verify the contribution from extrinsic forces and interrogate underlying signaling pathways.

α‐Actinin2 is required for the lateral alignment ofZdiscs and ventricular chamber enlargement during zebrafish cardiogenesis

α-Actinin2 (Actn2) is a predominant protein in the sarcomere Z disc whose mutation can lead to cardiomyopathy. However, the function of Actn2 in Z-disc assembly and cardiomyopathy in vertebrates remains elusive. We leveraged genetic tools in zebrafish embryos to elucidate the function of Actn2. We identified a single Actn2 homologue expressed in the zebrafish heart and conducted loss-of-function studies by antisense morpholino technology. Although zebrafish Actn2 assembles early into the Z disc, depletion of actn2 did not affect the early steps of sarcomere assembly. Instead, Actn2 is required for Z bodies to register laterally, forming well-aligned Z discs. Presumably as a consequence to this structural defect in the sarcomere, the depletion of Actn2 resulted in reduced cardiac function, primarily characterized as a reduced end-diastolic diameter. The depletion of actn2 also significantly reduced the ventricle chamber size, due to both reduced cardiomyocyte (CM) size and CM number. Interestingly, reduced CM size can be rescued by the cessation of heart contractions. The genetic studies of zebrafish uncovered a function for actn2 in lateral registration of Z body. In actn2 morphant fish, the Z-disc defect sequentially affects cardiac function, which leads to morphological changes in the ventricle through a mechanical force-dependent mechanism.

Abstract P200: Transcriptional Regulation of tcap Conveys an Inhibitory Mechanotransductive Response to Modulate Cardiomyocyte Size

To sense and to respond to mechanical stimuli is a fundamental process of cardiomyocytes during both heart development and diseases. However, how mechanical stimuli control cardiomyocyte size remains elusive. Here, we report a novel inhibitory mechanotransductive response suggested by loss-of-functions studies of α-Actinin2 (Actn2), a predominant sarcomeric Z-disc protein. During zebrafish cardiogenesis, depletion of Actn2 results in a defective Z-disc, while other sarcomeric substructures remain normal. As a consequence, ventricle chamber size is severely reduced. Interestingly, this chamber size reduction can be rescued by the cessation of heart contraction, suggesting the involvement of an inhibitory mechanotransductive reponse. At the molecular level, both tcapa and tcapb, two zebrafish tcap homologues that are transcriptionally responsive to stretch, are transcriptionally activated in actn2 knockdown embryos while activation of these gene results in smaller cardiomyocytes. Depletion of either gene rescues the reduced cardiomyocyte size in actn2 knockdown embryos. Consistent to embryonic studies, tcaps are transcriptionally activated during pathogenesis of anemia-induced cardiomyopathy, while over expression of tcapa via transgenics exerts a cardioprotective effect. Together, we conclude that transcriptional regulation of tcaps conveys a novel inhibitory mechanotransductive response that modulates cardiomyocyte size during both zebrafish cardiogenesis and adult cardiac remodeling.

Regulation of nkx2.5 and mef2C by Hif-1alpha and Hdac9 during zebrafish cardiogenesis

Regulation of nkx2.5 and mef2C by Hif-1alpha and Hdac9 during zebrafish cardiogenesis

Chromatin remodelling complex dosage modulates transcription factor function in heart development

Dominant mutations in cardiac transcription factor genes cause human inherited congenital heart defects (CHDs); however, their molecular basis is not understood. Interactions between transcription factors and the Brg1/Brm-associated factor (BAF) chromatin remodelling complex suggest potential mechanisms; however, the role of BAF complexes in cardiogenesis is not known. In this study, we show that dosage of Brg1 is critical for mouse and zebrafish cardiogenesis. Disrupting the balance between Brg1 and disease-causing cardiac transcription factors, including Tbx5, Tbx20 and Nkx2–5, causes severe cardiac anomalies, revealing an essential allelic balance between Brg1 and these cardiac transcription factor genes. This suggests that the relative levels of transcription factors and BAF complexes are important for heart development, which is supported by reduced occupancy of Brg1 at cardiac gene promoters in Tbx5 haploinsufficient hearts. Our results reveal complex dosage-sensitive interdependence between transcription factors and BAF complexes, providing a potential mechanism underlying transcription factor haploinsufficiency, with implications for multigenic inheritance of CHDs.

Identification and developmental expression of Dec2 in zebrafish

The involvement of Dec2, a member of the basic helix-loop-helix (bHLH) family, in cellular differentiation, hypoxia response, and circadian regulation has been investigated. Here we report the previously unknown spatiotemporal expression of Dec2 in zebrafish embryogenesis. Dec2 is dynamically expressed in zebrafish pineal gland, tract of the postoptic commissure, brain, notochord, heart, common cardinal vein (CCV), axial vein, pronephric duct, swim bladder, and early somites during embryogenesis, which implies that Dec2 is involved in zebrafish central nervous system development, cardiogenesis, and internal organs and somites formation. The embryonic expression patterns of zebrafish Dec2 and its homolog Dec1 partially overlap, but are distinct from each other. The Dec2 expression level was lower than that of Dec1 during zebrafish embryogenesis. Although Dec1 also contributed to zebrafish somites formation, cardiogenesis, and internal organs and central nervous system development, the two Dec genes were not likely to be simply redundant during zebrafish embryogenesis. Our results imply that Dec2, like its homolog Dec1, is involved in zebrafish cardiogenesis, central nervous system development, and internal organs and somites formation with distinct developmental roles.

Depletion of zebrafish essential and regulatory myosin light chains reduces cardiac function through distinct mechanisms

Mutations in the essential myosin light chain (ELC) and regulatory myosin light chain (RLC) genes have been linked to sarcomeric hypertrophic cardiomyopathies in humans; however, the specific functions of the different myosin light chains during cardiogenesis in a vertebrate animal are not well understood. Using zebrafish (Danio rerio) as a model organism, we have identified cmlc1 and cmlc2 as the main ELC and RLC orthologues, respectively, and have furthermore characterized their functions during cardiogenesis by morpholino technology. Depletion of either cmlc1 or cmlc2 using morpholino-modified antisense oligonucleotides leads to a disruption in sarcomere structure and compromises cardiac function as well, although through seemingly distinct mechanisms. While myosin still assembles into a novel rod-like structure in both morphants, the sarcomere length is longer in cmlc1 morphants than that in wild-type embryos, whereas it is shorter in cmlc2 morphants. In addition, cardiomyocyte size and number are increased upon depletion of cmlc1, resulting in a larger ventricular chamber volume; in contrast, depletion of cmlc2 leads to a reduction in cardiomyocyte size and number. Our data have elucidated distinct roles for cmlc1 and cmlc2 during zebrafish cardiogenesis, suggesting that cardiomyopathies resulting from human mutations in ELCs vs. RLCs may have distinct pathological characteristics during disease progression.

Glycogen synthase kinase 3α and 3β have distinct functions during cardiogenesis of zebrafish embryo

BackgroundGlycogen synthase kinase 3 (GSK3) encodes a serine/threonine protein kinase, is known to play roles in many biological processes. Two closely related GSK3 isoforms encoded by distinct genes: GSK3α (51 kDa) and GSK3β (47 kDa). In previously studies, most GSK3 inhibitors are not only inhibiting GSK3, but are also affecting many other kinases. In addition, because of highly similarity in amino acid sequence between GSK3α and GSK3β, making it difficult to identify an inhibitor that can be selective against GSK3α or GSK3β. Thus, it is relatively difficult to address the functions of GSK3 isoforms during embryogenesis. At this study, we attempt to specifically inhibit either GSK3α or GSK3β and uncover the isoform-specific roles that GSK3 plays during cardiogenesis.ResultsWe blocked gsk3α and gsk3β translations by injection of morpholino antisense oligonucleotides (MO). Both gsk3α- and gsk3β-MO-injected embryos displayed similar morphological defects, with a thin, string-like shaped heart and pericardial edema at 72 hours post-fertilization. However, when detailed analysis of the gsk3α- and gsk3β-MO-induced heart defects, we found that the reduced number of cardiomyocytes in gsk3α morphants during the heart-ring stage was due to apoptosis. On the contrary, gsk3β morphants did not exhibit significant apoptosis in the cardiomyocytes, and the heart developed normally during the heart-ring stage. Later, however, the heart positioning was severely disrupted in gsk3β morphants. bmp4 expression in gsk3β morphants was up-regulated and disrupted the asymmetry pattern in the heart. The cardiac valve defects in gsk3β morphants were similar to those observed in axin1 and apcmcr mutants, suggesting that GSK3β might play a role in cardiac valve development through the Wnt/β-catenin pathway. Finally, the phenotypes of gsk3α mutant embryos cannot be rescued by gsk3β mRNA, and vice versa, demonstrating that GSK3α and GSK3β are not functionally redundant.ConclusionWe conclude that (1) GSK3α, but not GSK3β, is necessary in cardiomyocyte survival; (2) the GSK3β plays important roles in modulating the left-right asymmetry and affecting heart positioning; and (3) GSK3α and GSK3β play distinct roles during zebrafish cardiogenesis.

Co-operative and synergistic actions of Tbx5 and MRTF-B in zebrafish cardiogenesis

Co-operative and synergistic actions of Tbx5 and MRTF-B in zebrafish cardiogenesis