Atf7ip and Setdb1 interaction orchestrates the hematopoietic stem and progenitor cell state with diverse lineage differentiation.

Hematopoietic stem and progenitor cells (HSPCs) are a heterogeneous group of cells with expansion, differentiation, and repopulation capacities. How HSPCs orchestrate the stemness state with diverse lineage differentiation at steady condition or acute stress remains largely unknown. Here, we show that zebrafish mutants that are deficient in an epigenetic regulator Atf7ip or Setdb1 methyltransferase undergo excessive myeloid differentiation with impaired HSPC expansion, manifesting a decline in T cells and erythroid lineage. We find that Atf7ip regulates hematopoiesis through Setdb1-mediated H3K9me3 modification and chromatin remodeling. During hematopoiesis, the interaction of Atf7ip and Setdb1 triggers H3K9me3 depositions in hematopoietic regulatory genes including cebpß and cdkn1a, preventing HSPCs from loss of expansion and premature differentiation into myeloid lineage. Concomitantly, loss of Atf7ip or Setdb1 derepresses retrotransposons that instigate the viral sensor Mda5/Rig-I like receptor (RLR) signaling, leading to stress-driven myelopoiesis and inflammation. We find that ATF7IP or SETDB1 depletion represses human leukemic cell growth and induces myeloid differentiation with retrotransposon-triggered inflammation. These findings establish that Atf7ip/Setdb1-mediated H3K9me3 deposition constitutes a genome-wide checkpoint that impedes the myeloid potential and maintains HSPC stemness for diverse blood cell production, providing unique insights into potential intervention in hematological malignancy.

The Gridlock transcriptional repressor impedes vertebrate heart regeneration by restricting expression of lysine methyltransferase.

Teleost zebrafish and neonatal mammalian hearts exhibit the remarkable capacity to regenerate through dedifferentiation and proliferation of pre-existing cardiomyocytes (CMs). Although many mitogenic signals that stimulate zebrafish heart regeneration have been identified, transcriptional programs that restrain injury-induced CM renewal are incompletely understood. Here, we report that mutations in gridlock (grl; also known as hey2), encoding a Hairy-related basic helix-loop-helix transcriptional repressor, enhance CM proliferation and reduce fibrosis following damage. In contrast, myocardial grl induction blunts CM dedifferentiation and regenerative responses to heart injury. RNA sequencing analyses uncover Smyd2 lysine methyltransferase (KMT) as a key transcriptional target repressed by Grl. Reduction in Grl protein levels triggered by injury induces smyd2 expression at the wound myocardium, enhancing CM proliferation. We show that Smyd2 functions as a methyltransferase and modulates the Stat3 methylation and phosphorylation activity. Inhibition of the KMT activity of Smyd2 reduces phosphorylated Stat3 at cardiac wounds, suppressing the elevated CM proliferation in injured grl mutant hearts. Our findings establish an injury-specific transcriptional repression program in governing CM renewal during heart regeneration, providing a potential strategy whereby silencing Grl repression at local regions might empower regeneration capacity to the injured mammalian heart.

Prostaglandin signaling in ciliogenesis and development.

Prostaglandin (PG) signaling regulates a wide variety of physiological and pathological processes, including body temperature, cardiovascular homeostasis, reproduction, and inflammation. Recent studies have revealed that PGs play pivotal roles in embryo development, ciliogenesis, and organ formation. Prostaglandin E2 (PGE2) and its receptor EP4 modulate ciliogenesis by increasing the anterograde intraflagellar transport. Many G-protein-coupled receptors (GPCRs) including EP4 are localized in cilia for modulating cAMP signaling under various conditions. During development, PGE2 signaling regulates embryogenesis, hepatocyte differentiation, hematopoiesis, and kidney formation. Prostaglandins are also essential for skeletal muscle repair. This review outlines recent advances in understanding the functions and mechanisms of prostaglandin signaling in ciliogenesis, embryo development, and organ formation.

Systematic genome editing of the genes on zebrafish Chromosome 1 by CRISPR/Cas9.

Genome editing by the well-established CRISPR/Cas9 technology has greatly facilitated our understanding of many biological processes. However, a complete whole-genome knockout for any species or model organism has rarely been achieved. Here, we performed a systematic knockout of all the genes (1333) on Chromosome 1 in zebrafish, successfully mutated 1029 genes, and generated 1039 germline-transmissible alleles corresponding to 636 genes. Meanwhile, by high-throughput bioinformatics analysis, we found that sequence features play pivotal roles in effective gRNA targeting at specific genes of interest, while the success rate of gene targeting positively correlates with GC content of the target sites. Moreover, we found that nearly one-fourth of all mutants are related to human diseases, and several representative CRISPR/Cas9-generated mutants are described here. Furthermore, we tried to identify the underlying mechanisms leading to distinct phenotypes between genetic mutants and antisense morpholino-mediated knockdown embryos. Altogether, this work has generated the first chromosome-wide collection of zebrafish genetic mutants by the CRISPR/Cas9 technology, which will serve as a valuable resource for the community, and our bioinformatics analysis also provides some useful guidance to design gene-specific gRNAs for successful gene editing.

Wnt Signaling in Heart Development and Regeneration.

PURPOSE OF REVIEW: Cardiovascular diseases are the leading cause of death worldwide, largely due to the limited regenerative capacity of the adult human heart. In contrast, teleost zebrafish hearts possess natural regeneration capacity by proliferation of pre-existing cardiomyocytes after injury. Hearts of mice can regenerate if injured in a few days after birth, which coincides with the transient capacity for cardiomyocyte proliferation. This review tends to elaborate the roles and mechanisms of Wnt/ß-catenin signaling in heart development and regeneration in mammals and non-mammalian vertebrates. RECENT FINDINGS: Studies in zebrafish, mice, and human embryonic stem cells demonstrate the binary effect for Wnt/ß-catenin signaling during heart development. Both Wnts and Wnt antagonists are induced in multiple cell types during cardiac development and injury repair. In this review, we summarize composites of the Wnt signaling pathway and their different action routes, followed by the discussion of their involvements in cardiac specification, proliferation, and patterning. We provide overviews about canonical and non-canonical Wnt activity during heart homeostasis, remodeling, and regeneration. Wnt/ß-catenin signaling exhibits biphasic and antagonistic effects on cardiac specification and differentiation depending on the stage of embryogenesis. Inhibition of Wnt signaling is beneficial for cardiac wound healing and functional recovery after injury. Understanding of the roles and mechanisms of Wnt signaling pathway in injured animal hearts will contribute to the development of potential therapeutics for human diseased hearts.

Requirement of Zebrafish Adcy3a and Adcy5 in Melanosome Dispersion and Melanocyte Stripe Formation.

cAMP-PKA signaling plays a pivotal role in melanin synthesis and melanosome transport by responding to the binding of the α-melanocyte-stimulating hormone (α-MSH) to melanocortin-1 receptor (MC1R). Adenylate cyclases (ADCYs) are the enzymes responsible for the synthesis of cAMP from ATP, which comprises nine transmembrane isoforms (ADCYs 1-9) and one soluble adenylate cyclase (ADCY 10) in mammals. However, little is known about which and how ADCY isoforms regulate melanocyte generation, melanin biosynthesis, and melanosome transport in vivo. In this study, we have generated a series of single and double mutants of Adcy isoforms in zebrafish. Among them, adcy3a-/- and adcy5-/- double mutants cause defects in melanosome dispersion but do not impair melanoblast differentiation and melanocyte regeneration during the embryonic or larval stages. Activation of PKA, the main effector of cAMP signaling, significantly ameliorates the defects in melanosome dispersion in adcy3a-/- and adcy5-/- double mutants. Mechanistically, Adcy3a and Adcy5 regulate melanosome dispersion by activating kinesin-1 while inhibiting cytoplasmic dynein-1. In adult zebrafish, Adcy3a and Adcy5 participate in the regulation of the expression of microphthalmia transcription factor (Mitfa) and melanin synthesis enzymes Tyr, Dct, and Trp1b. The deletion of Adcy3a and Adcy5 inhibits melanin production and reduces pigmented melanocyte numbers, causing a defect in establishing adult melanocyte stripes. Hence, our studies demonstrate that Adcy3a and Adcy5 play essential but redundant functions in mediating α-MSH-MC1R/cAMP-PKA signaling for regulating melanin synthesis and melanosome dispersion.

Disruption of Abcc6 Transporter in Zebrafish Causes Ocular Calcification and Cardiac Fibrosis.

Pseudoxanthoma elasticum (PXE), caused by ABCC6/MRP6 mutation, is a heritable multisystem disorder in humans. The progressive clinical manifestations of PXE are accompanied by ectopic mineralization in various connective tissues. However, the pathomechanisms underlying the PXE multisystem disorder remains obscure, and effective treatment is currently available. In this study, we generated zebrafish abcc6a mutants using the transcription activator-like effector nuclease (TALEN) technique. In young adult zebrafish, abcc6a is expressed in the eyes, heart, intestine, and other tissues. abcc6a mutants exhibit extensive calcification in the ocular sclera and Bruch's membrane, recapitulating part of the PXE manifestations. Mutations in abcc6a upregulate extracellular matrix (ECM) genes, leading to fibrotic heart with reduced cardiomyocyte number. We found that abcc6a mutation reduced levels of both vitamin K and pyrophosphate (PPi) in the serum and diverse tissues. Vitamin K administration increased the gamma-glutamyl carboxylated form of matrix gla protein (cMGP), alleviating ectopic calcification and fibrosis in vertebrae, eyes, and hearts. Our findings contribute to a comprehensive understanding of PXE pathophysiology from zebrafish models.

Photoreceptor cell development requires prostaglandin signaling in the zebrafish retina.

Photoreceptor cells are highly specialized sensory neurons capable of visual phototransduction. The connecting cilia in the retinal photoreceptors link the inner segment to the outer segment and mediate the transport of opsins in vision. Although our previous study demonstrates that Prostaglandin E2 (PGE2) signaling is required for ciliogenesis in zebrafish, its roles in retinal ciliogenesis and photoreceptor cell development remain unknown. Here, we investigated the function of prostaglandin signaling in retina and photoreceptor cell development. We generated zebrafish mutations in prostaglandin endoperoxide synthase 1 (PTGS1) and prostaglandin endoperoxide synthase 2 (PTGS2), two rate-limiting enzymes responsible for prostaglandin production. We found that ptgs2b knockdown in ptgs1-/-ptgs2a-/- double mutants significantly reduced the length of connecting cilia and resulted in severe defects in photoreceptor cell differentiation. Furthermore, mutation in PGE2 transporter Leakytail (Lkt)/ATP-binding cassette transporter 4 (ABCC4) exhibited loss of connecting cilia and outer segment in photoreceptor cells, leading to mislocalization of opsins in the cell bodies of photoreceptors. Together, our findings suggest that PGE2 production and transport are crucial for connecting cilia formation and photoreceptor cell development.

Opposing Roles of Wnt Inhibitors IGFBP-4 and Dkk1 in Cardiac Ischemia by Differential Targeting of LRP5/6 and ß-catenin.

BACKGROUND: Myocardial infarction is one of the leading causes of morbidity and mortality worldwide, triggering irreversible myocardial cell damage and heart failure. The role of low-density lipoprotein receptor-related proteins 5 and 6 (LRP5/6) as coreceptors of the Wnt/ß-catenin pathway in the adult heart remain unknown. Insulin-like growth factor binding protein 4 and dickkopf-related protein 1 (Dkk1) are 2 secreted LRP5/6 binding proteins that play a crucial role in heart development through preventing Wnt/ß-catenin pathway activation. However, their roles in the adult heart remain unexplored. METHODS: To understand the role of LRP5/6 and ß-catenin in the adult heart, we constructed conditional cardiomyocyte-specific LRP5/6 and ß-catenin knockout mice and induced surgical myocardial infarction. We also directly injected recombinant proteins of insulin-like growth factor binding protein 4 and Dkk1 into the heart immediately following myocardial infarction to further examine the mechanisms through which these proteins regulate LRP5/6 and ß-catenin. RESULTS: Deletion of LRP5/6 promoted cardiac ischemic insults. Conversely, deficiency of ß-catenin, a downstream target of LRP5/6, was beneficial in ischemic injury. It is interesting to note that although both insulin-like growth factor binding protein 4 and Dkk1 are secreted Wnt/ß-catenin pathway inhibitors, insulin-like growth factor binding protein 4 protected the ischemic heart by inhibiting ß-catenin, whereas Dkk1 enhanced the injury response mainly through inducing LRP5/6 endocytosis and degradation. CONCLUSIONS: Our findings reveal previously unidentified dual roles of LRP5/6 involved in the cardiomyocyte response to ischemic injury. These findings suggest new therapeutic strategies in ischemic heart disease by fine-tuning LRP5/6 and ß-catenin signaling within the Wnt/ß-catenin pathway.

MAPK/ERK signalling is required for zebrafish cardiac regeneration.

OBJECTIVES: To better understand the molecular mechanisms of regeneration and explore the potential signalling pathways as therapeutic targets for heart attacks. RESULTS: After treatment with the MEK inhibitor AZD6244 upon cardiac injury, the core members in MAPK/ERK signalling-mek and erk-demonstrate elevated expression, and these proteins are deposited at the injury site in zebrafish. pERK is also induced in non-cardiomyocytes near the injury site. Furthermore, the induced expression of a dominant-negative form of MEK1 inhibits zebrafish cardiac regeneration, characterized by increased cardiac fibrosis (a hallmark of regenerative failure), reduced or delayed production of regenerative myocardium, and migration of FLI1+ endothelial cells, without direct inhibition of cardiomyocyte proliferation. CONCLUSION: Appropriate activation of MAPK/ERK signalling is essential for zebrafish cardiac regeneration.

Vegfa Impacts Early Myocardium Development in Zebrafish.

Vascular endothelial growth factor A (Vegfa) signaling regulates cardiovascular development. However, the cellular mechanisms of Vegfa signaling in early cardiogenesis remain poorly understood. The present study aimed to understand the differential functions and mechanisms of Vegfa signaling in cardiac development. A loss-of-function approach was utilized to study the effect of Vegfa signaling in cardiogenesis. Both morphants and mutants for vegfaa display defects in cardiac looping and chamber formation, especially the ventricle. Vegfa regulates the heart morphogenesis in a dose-dependent manner. Furthermore, the initial fusion of the bilateral myocardium population is delayed rather than endocardium. The results demonstrate that Vegfa signaling plays a direct impact on myocardium fusion, indicating that it is the initial cause of the heart defects. The heart morphogenesis is regulated by Vegfa in a dose-dependent manner, and later endocardium defects may be secondary to impaired myocardium-endocardium crosstalk.

Rac1-PAK2 pathway is essential for zebrafish heart regeneration.

P-21 activated kinases, or PAKs, are serine-threonine kinases that play important roles in diverse heart functions include heart development, cardiovascular development and function in a range of models; however, the mechanisms by which PAKs mediate heart regeneration are unknown. Here, we demonstrate that PAK2 and PAK4 expression is induced in cardiomyocytes and vessels, respectively, following zebrafish heart injury. Inhibition of PAK2 and PAK4 using a specific small molecule inhibitor impedes cardiomyocyte proliferation/dedifferentiation and cardiovascular regeneration, respectively. Cdc42 is specifically expressed in the ventricle and may function upstream of PAK2 but not PAK4 under normal conditions and that cardiomyocyte proliferentation during heart regeneration relies on Rac1-mediated activation of Pak2. Our results indicate that PAKs play a key role in heart regeneration.

Trapping cardiac recessive mutants via expression-based insertional mutagenesis screening.

RATIONALE: Mutagenesis screening is a powerful genetic tool for probing biological mechanisms underlying vertebrate development and human diseases. However, the increased colony management efforts in vertebrates impose a significant challenge for identifying genes affecting a particular organ, such as the heart, especially those exhibiting adult phenotypes on depletion. OBJECTIVE: We aim to develop a facile approach that streamlines colony management efforts via enriching cardiac mutants, which enables us to screen for adult phenotypes. METHODS AND RESULTS: The transparency of the zebrafish embryos enabled us to score 67 stable transgenic lines generated from an insertional mutagenesis screen using a transposon-based protein trapping vector. Fifteen lines with cardiac monomeric red fluorescent protein reporter expression were identified. We defined the molecular nature for 10 lines and bred them to homozygosity, which led to the identification of 1 embryonic lethal, 1 larval lethal, and 1 adult recessive mutant exhibiting cardiac hypertrophy at 1 year of age. Further characterization of these mutants uncovered an essential function of methionine adenosyltransferase II, α a (mat2aa) in cardiogenesis, an essential function of mitochondrial ribosomal protein S18B (mrps18b) in cardiac mitochondrial homeostasis, as well as a function of DnaJ (Hsp40) homolog, subfamily B, member 6b (dnajb6b) in adult cardiac hypertrophy. CONCLUSIONS: We demonstrate that transposon-based gene trapping is an efficient approach for identifying both embryonic and adult recessive mutants with cardiac expression. The generation of a zebrafish insertional cardiac mutant collection shall facilitate the annotation of a vertebrate cardiac genome, as well as enable heart-based adult screens.

Conditional control of gene function by an invertible gene trap in zebrafish.

Conditional mutations are essential for determining the stage- and tissue-specific functions of genes. Here we achieve conditional mutagenesis in zebrafish using FT1, a gene-trap cassette that can be stably inverted by both Cre and Flp recombinases. We demonstrate that intronic insertions in the gene-trapping orientation severely disrupt the expression of the host gene, whereas intronic insertions in the neutral orientation do not significantly affect host gene expression. Cre- and Flp-mediated recombination switches the orientation of the gene-trap cassette, permitting conditional rescue in one orientation and conditional knockout in the other. To illustrate the utility of this system we analyzed the functional consequence of intronic FT1 insertion in supv3l1, a gene encoding a mitochondrial RNA helicase. Global supv311 mutants have impaired mitochondrial function, embryonic lethality, and agenesis of the liver. Conditional rescue of supv311 expression in hepatocytes specifically corrected the liver defects. To test whether the liver function of supv311 is required for viability we used Flp-mediated recombination in the germline to generate a neutral allele at the locus. Subsequently, tissue-specific expression of Cre conditionally inactivated the targeted locus. Hepatocyte-specific inactivation of supv311 caused liver degeneration, growth retardation, and juvenile lethality, a phenotype that was less severe than the global disruption of supv311. Thus, supv311 is required in multiple tissues for organismal viability. Our mutagenesis approach is very efficient and could be used to generate conditional alleles throughout the zebrafish genome. Furthermore, because FT1 is based on the promiscuous Tol2 transposon, it should be applicable to many organisms.

Crucial role for phylogenetically conserved cytoplasmic loop 3 in ABCC4 protein expression.

The ABC transporter ABCC4 is recognized as an ATP-dependent exporter of endogenous substances as well as an increasing variety of anionic chemotherapeutics. A loss-of-function variant of zebrafish Abcc4 was identified with a single amino acid substitution in the cytoplasmic loop T804M. Because this substituted amino acid is highly conserved among ABCC4 orthologs and is located in cytoplasmic loop 3 (CL3), we investigated the impact of this mutation on human and zebrafish Abcc4 expression. We demonstrate that zebrafish Abcc4 T804M or human ABCC4 T796M exhibit substantially reduced expression, coupled with impaired plasma membrane localization. To understand the molecular basis for the localization defect, we developed a homology model of zebrafish Abcc4. The homology model suggested that the bulky methionine substitution disrupted side-chain contacts. Molecular dynamic simulations of a fragment of human or zebrafish CL3 containing a methionine substitution indicated altered helicity coupled with reduced thermal stability. Trifluoroethanol challenge coupled with circular dichroism revealed that the methionine substitution disrupted the ability of this fragment of CL3 to readily form an α-helix. Furthermore, expression and plasma membrane localization of these mutant ABCC4/Abcc4 proteins are mostly rescued by growing cells at subphysiological temperatures. Because the cystic fibrosis transmembrane conductance regulator (ABCC7) is closely related to ABCC4, we extended this by engineering certain pathogenic CFTR-CL3 mutations, and we showed they destabilized human and zebrafish ABCC4. Altogether, our studies provide the first evidence for a conserved domain in CL3 of ABCC4 that is crucial in ensuring its proper plasma membrane localization.

Phosphorylation of angiomotin by Lats1/2 kinases inhibits F-actin binding, cell migration, and angiogenesis.

The Hippo tumor suppressor pathway plays important roles in organ size control through Lats1/2 mediated phosphorylation of the YAP/TAZ transcription co-activators. However, YAP/TAZ independent functions of the Hippo pathway are largely unknown. Here we report a novel role of the Hippo pathway in angiogenesis. Angiomotin p130 isoform (AMOTp130) is phosphorylated on a conserved HXRXXS motif by Lats1/2 downstream of GPCR signaling. Phosphorylation disrupts AMOT interaction with F-actin and correlates with reduced F-actin stress fibers and focal adhesions. Furthermore, phosphorylation of AMOT by Lats1/2 inhibits endothelial cell migration in vitro and angiogenesis in zebrafish embryos in vivo. Thus AMOT is a direct substrate of Lats1/2 mediating functions of the Hippo pathway in endothelial cell migration and angiogenesis.

Cardiomyopathy in zebrafish due to mutation in an alternatively spliced exon of titin.

The zebrafish embryo is transparent and can tolerate absence of blood flow because its oxygen is delivered by diffusion rather than by the cardiovascular system. It is therefore possible to attribute cardiac failure directly to particular genes by ruling out the possibility that it is due to a secondary effect of hypoxia. We focus here on pickwickm171 (pikm171), a recessive lethal mutation discovered in a large-scale genetic screen. There are three other alleles in the pik complementation group with this phenotype (pikm242, pikm740, pikm186; ref. 3) and one allele (pikmVO62H) with additional skeletal paralysis. The pik heart develops normally but is poorly contractile from the first beat. Aside from the edema that inevitably accompanies cardiac dysfunction, development is normal during the first three days. We show by positional cloning that the 'causative' mutation is in an alternatively-spliced exon of the gene (ttn) encoding Titin. Titin is the biggest known protein and spans the half-sarcomere from Z-disc to M-line in heart and skeletal muscle. It has been proposed to provide a scaffold for the assembly of thick and thin filaments and to provide elastic recoil engendered by stretch during diastole. We found that nascent myofibrils form in pik mutants, but normal sarcomeres are absent. Mutant cells transplanted to wildtype hearts remain thin and bulge outwards as individual cell aneurysms without affecting nearby wildtype cardiomyocytes, indicating that the contractile deficiency is cell-autonomous. Absence of Titin function thus results in blockage of sarcomere assembly and causes a functional disorder resembling human dilated cardiomyopathies, one form of which is described in another paper in this issue.

Renal interstitial cells promote nephron regeneration by secreting prostaglandin E2.

In organ regeneration, progenitor and stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation, and differentiation. However, the types of cells that form the native microenvironment for renal progenitor cells (RPCs) have not been clarified. Here, single-cell sequencing of zebrafish kidney reveals fabp10a as a principal marker of renal interstitial cells (RICs), which can be specifically labeled by GFP under the control of fabp10a promoter in the fabp10a:GFP transgenic zebrafish. During nephron regeneration, the formation of nephrons is supported by RICs that form a network to wrap the RPC aggregates. RICs that are in close contact with RPC aggregates express cyclooxygenase 2 (Cox2) and secrete prostaglandin E2 (PGE2). Inhibiting PGE2 production prevents nephrogenesis by reducing the proliferation of RPCs. PGE2 cooperates with Wnt4a to promote nephron maturation by regulating ß-catenin stability of RPC aggregates. Overall, these findings indicate that RICs provide a necessary microenvironment for rapid nephrogenesis during nephron regeneration.

Improving adenine and dual base editors through introduction of TadA-8e and Rad51DBD.

Base editors, including dual base editors, are innovative techniques for efficient base conversions in genomic DNA. However, the low efficiency of A-to-G base conversion at positions proximal to the protospacer adjacent motif (PAM) and the A/C simultaneous conversion of the dual base editor hinder their broad applications. In this study, through fusion of ABE8e with Rad51 DNA-binding domain, we generate a hyperactive ABE (hyABE) which offers improved A-to-G editing efficiency at the region (A10-A15) proximal to the PAM, with 1.2- to 7-fold improvement compared to ABE8e. Similarly, we develop optimized dual base editors (eA&C-BEmax and hyA&C-BEmax) with markedly improved simultaneous A/C conversion efficiency (1.2-fold and 1.5-fold improvement, respectively) compared to A&C-BEmax in human cells. Moreover, these optimized base editors catalyze efficiently nucleotide conversions in zebrafish embryos to mirror human syndrome or in human cells to potentially treat genetic diseases, indicating their great potential in broad applications for disease modeling and gene therapy.

Voltage-gated sodium channels are required for heart development in zebrafish.

RATIONALE: Voltage-gated sodium channels initiate action potentials in excitable tissues. Mice in which Scn5A (the predominant sodium channel gene in heart) has been knocked out die early in development with cardiac malformations by mechanisms which have yet to be determined. OBJECTIVE: Here we addressed this question by investigating the role of cardiac sodium channels in zebrafish heart development. METHODS AND RESULTS: Transcripts of the functionally-conserved Scn5a homologs scn5Laa and scn5Lab were detected in the gastrulating zebrafish embryo and subsequently in the embryonic myocardium. Antisense knockdown of either channel resulted in marked cardiac chamber dysmorphogenesis and perturbed looping. These abnormalities were associated with decreased expression of the myocardial precursor genes nkx2.5, gata4, and hand2 in anterior lateral mesoderm and significant deficits in the production of cardiomyocyte progenitors. These early defects did not appear to result from altered membrane electrophysiology, as prolonged pharmacological blockade of sodium current failed to phenocopy channel knockdown. Moreover, embryos grown in calcium channel blocker-containing medium had hearts that did not beat but developed normally. CONCLUSIONS: These findings identify a novel and possibly nonelectrogenic role for cardiac sodium channels in heart development.