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Hox genes orchestrate the segmental specification of the muscular circulatory system in invertebrates but it has not proven straightforward to decipher segmental parallels in the vertebrate heart. Recently, patients with HOXB gene cluster deletion were found to exhibit abnormalities including atrioventricular canal defects. Using CRISPR, we established a mutant with the orthologous hoxbb cluster deletion in zebrafish. The mutant exhibited heart failure and atrioventricular regurgitation at 5 days. Analyzing the four genes in the hoxbb cluster, isolated deletion of hoxb1b -/- recapitulated the cardiac abnormalities, supporting hoxb1b as the causal gene. Both in situ and in vitro data indicated that hoxb1b regulates gata5 to inhibit hand2 expression and ultimately is required to pattern the vertebrate atrioventricular boundary. Together, these data reveal a role for segmental specification in vertebrate cardiac development and highlight the utility of CRISPR techniques for efficiently exploring the function of large structural genomic lesions., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Maintenance of cardiomyocyte identity is vital for normal heart development and function. However, our understanding of cardiomyocyte plasticity remains incomplete. Here, we show that sustained expression of the zebrafish transcription factor Nr2f1a prevents the progressive acquisition of ventricular cardiomyocyte (VC) and pacemaker cardiomyocyte (PC) identities within distinct regions of the atrium. Transcriptomic analysis of flow-sorted atrial cardiomyocytes (ACs) from nr2f1a mutant zebrafish embryos showed increased VC marker gene expression and altered expression of core PC regulatory genes, including decreased expression of nkx2.5 , a critical repressor of PC differentiation. At the arterial (outflow) pole of the atrium in nr2f1a mutants, cardiomyocytes resolve to VC identity within the expanded atrioventricular canal. However, at the venous (inflow) pole of the atrium, there is a progressive wave of AC transdifferentiation into PCs across the atrium toward the arterial pole. Restoring Nkx2.5 is sufficient to repress PC marker identity in nr2f1a mutant atria and analysis of chromatin accessibility identified an Nr2f1a-dependent nkx2.5 enhancer expressed in the atrial myocardium directly adjacent to PCs. CRISPR/Cas9-mediated deletion of the putative nkx2.5 enhancer leads to a loss of Nkx2.5-expressing ACs and expansion of a PC reporter, supporting that Nr2f1a limits PC differentiation within venous ACs via maintaining nkx2.5 expression. The Nr2f-dependent maintenance of AC identity within discrete atrial compartments may provide insights into the molecular etiology of concurrent structural congenital heart defects and associated arrhythmias., Competing Interests: KM, PR, MB, CM, JW No competing interests declared, (© 2023, Martin et al.)

Pharmacology and toxicology are part of a much broader effort to understand the relationship between chemistry and biology. While biomedicine has necessarily focused on specific cases, typically of direct human relevance, there are real advantages in pursuing more systematic approaches to characterizing how health and disease are influenced by small molecules and other interventions. In this context, the zebrafish is now established as the representative screenable vertebrate and, through ongoing advances in the available scale of genome editing and automated phenotyping, is beginning to address systems-level solutions to some biomedical problems. The addition of broader efforts to integrate information content across preclinical model organisms and the incorporation of rigorous analytics, including closed-loop deep learning, will facilitate efforts to create systems pharmacology and toxicology with the ability to continuously optimize chemical biological interactions around societal needs. In this review, we outline progress toward this goal.

Although cyanide's biological effects are pleiotropic, its most obvious effects are as a metabolic poison. Cyanide potently inhibits cytochrome c oxidase and potentially other metabolic enzymes, thereby unleashing a cascade of metabolic perturbations that are believed to cause lethality. From systematic screens of human metabolites using a zebrafish model of cyanide toxicity, we have identified the TCA-derived small molecule glyoxylate as a potential cyanide countermeasure. Following cyanide exposure, treatment with glyoxylate in both mammalian and non-mammalian animal models confers resistance to cyanide toxicity with greater efficacy and faster kinetics than known cyanide scavengers. Glyoxylate-mediated cyanide resistance is accompanied by rapid pyruvate consumption without an accompanying increase in lactate concentration. Lactate dehydrogenase is required for this effect which distinguishes the mechanism of glyoxylate rescue as distinct from countermeasures based solely on chemical cyanide scavenging. Our metabolic data together support the hypothesis that glyoxylate confers survival at least in part by reversing the cyanide-induced redox imbalances in the cytosol and mitochondria. The data presented herein represent the identification of a potential cyanide countermeasure operating through a novel mechanism of metabolic modulation., (© 2022. The Author(s).)

CRISPR-Cas9 can be scaled up for large-scale screens in cultured cells, but CRISPR screens in animals have been challenging because generating, validating, and keeping track of large numbers of mutant animals is prohibitive. Here, we introduce Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform combining droplet microfluidics, single-needle en masse CRISPR ribonucleoprotein injections, and DNA barcoding to enable large-scale functional genetic screens in zebrafish. The platform can efficiently identify genes responsible for morphological or behavioral phenotypes. In one application, we showed that MIC-Drop could identify small-molecule targets. Furthermore, in a MIC-Drop screen of 188 poorly characterized genes, we discovered several genes important for cardiac development and function. With the potential to scale to thousands of genes, MIC-Drop enables genome-scale reverse genetic screens in model organisms.

Background: The turnover of cardiac ion channels underlying action potential duration is regulated by ubiquitination. Genome-wide association studies of QT interval identified several single-nucleotide polymorphisms located in or near genes involved in protein ubiquitination. A genetic variant upstream of LITAF (lipopolysaccharide-induced tumor necrosis factor) gene prompted us to determine its role in modulating cardiac excitation., Methods: Optical mapping was performed in zebrafish hearts to determine Ca 2+ transients. Live-cell confocal calcium imaging was performed on adult rabbit cardiomyocytes to determine intracellular Ca 2+ handling. L-type calcium channel (LTCC) current ( I Ca,L ) was measured using whole-cell recording. To study the effect of LITAF on Cav1.2 (L-type voltage-gated calcium channel 1.2) channel expression, surface biotinylation, and Westerns were performed. LITAF interactions were studied using coimmunoprecipitation and in situ proximity ligation assay., Results: LITAF knockdown in zebrafish resulted in a robust increase in calcium transients. Overexpressed LITAF in 3-week-old rabbit cardiomyocytes resulted in a decrease in I Ca,L and Cavα1c abundance, whereas LITAF knockdown increased I Ca,L and Cavα1c protein. LITAF-overexpressing decreases calcium transients in adult rabbit cardiomyocytes, which was associated with lower Cavα1c levels. In tsA201 cells, overexpressed LITAF downregulated total and surface pools of Cavα1c via increased Cavα1c ubiquitination and its subsequent lysosomal degradation. We observed colocalization between LITAF and LTCC in tsA201 and cardiomyocytes. In tsA201, NEDD ( neural precursor cell expressed developmentally downregulated protein ) 4-1, but not its catalytically inactive form NEDD4-1-C867A, increased Cavα1c ubiquitination. Cavα1c ubiquitination was further increased by coexpressed LITAF and NEDD4-1 but not NEDD4-1-C867A. NEDD4-1 knockdown abolished the negative effect of LITAF on I Ca,L and Cavα1c levels in 3-week-old rabbit cardiomyocytes. Computer simulations demonstrated that a decrease of I Ca,L current associated with LITAF overexpression simultaneously shortened action potential duration and decreased calcium transients in rabbit cardiomyocytes., Conclusions: LITAF acts as an adaptor protein promoting NEDD4-1-mediated ubiquitination and subsequent degradation of LTCC, thereby controlling LTCC membrane levels and function and thus cardiac excitation.

Cyanide is a potent toxic agent, and the few available antidotes are not amenable to rapid deployment in mass exposures. As a result, there are ongoing efforts to exploit different animal models to identify novel countermeasures. We have created a pipeline that combines high-throughput screening in zebrafish with subsequent validation in two mammalian small animal models as well as a porcine large animal model. We found that zebrafish embryos in the first 3 days post fertilization (dpf) are highly resistant to cyanide, becoming progressively more sensitive thereafter. Unbiased analysis of gene expression in response to several hours of ultimately lethal doses of cyanide in both 1 and 7 dpf zebrafish revealed modest changes in iron-related proteins associated with the age-dependent cyanide resistance. Metabolomics measurements demonstrated significant age-dependent differences in energy metabolism during cyanide exposure which prompted us to test modulators of the tricarboxylic acid cycle and related metabolic processes as potential antidotes. In cyanide-sensitive 7 dpf larvae, we identified several such compounds that offer significant protection against cyanide toxicity. Modulators of the pyruvate dehydrogenase complex, as well as the small molecule sodium glyoxylate, consistently protected against cyanide toxicity in 7 dpf zebrafish larvae. Together, our results indicate that the resistance of zebrafish embryos to cyanide toxicity during early development is related to an altered regulation of cellular metabolism, which we propose may be exploited as a potential target for the development of novel antidotes against cyanide poisoning., Competing Interests: I have read the journal's policy and the authors of this manuscript have the following competing interests: a patent application has been submitted for the use of glyoxylate as a cyanide antidote. However, this does not alter the authors' adherence to PLOS ONE policies on sharing data and materials.

Zebrafish provide an excellent model in which to assess the role of the renin-angiotensin system in renal development, injury, and repair. In contrast to mammals, zebrafish kidney organogenesis terminates with the mesonephros. Despite this, the basic functional structure of the nephron is conserved across vertebrates. The relevance of teleosts for studies relating to the regulation of the renin-angiotensin system was established by assessing the phenotype and functional regulation of renin-expressing cells in zebrafish. Transgenic fluorescent reporters for renin ( ren ), smooth muscle actin ( acta2 ), and platelet-derived growth factor receptor-beta ( pdgfrb ) were studied to determine the phenotype and secretory ultrastructure of perivascular renin-expressing cells. Whole kidney ren transcription responded to altered salinity, pharmacological renin-angiotensin system inhibition, and renal injury. Mesonephric ren -expressing cells occupied niches at the preglomerular arteries and afferent arterioles, forming intermittent epithelioid-like multicellular clusters exhibiting a granular secretory ultrastructure. In contrast, renin cells of the efferent arterioles were thin bodied and lacked secretory granules. Renin cells expressed the perivascular cell markers acta2 and pdgfrb Transcriptional responses of ren to physiological challenge support the presence of a functional renin-angiotensin system and are consistent with the production of active renin. The reparative capability of the zebrafish kidney was harnessed to demonstrate that ren transcription is a marker for renal injury and repair. Our studies demonstrate substantive conservation of renin regulation across vertebrates, and ultrastructural studies of renin cells reveal at least two distinct morphologies of mesonephric perivascular ren -expressing cells., (Copyright © 2017 the American Physiological Society.)

Three genes, CCM1, CCM2, and CCM3, interact genetically and biochemically and are mutated in cerebral cavernous malformations (CCM). A recently described member of this CCM family of proteins, CCM2-like (CCM2L), has high homology to CCM2. Here we show that its relative expression in different tissues differs from that of CCM2 and, unlike CCM2, the expression of CCM2L in endothelial cells is regulated by density, flow, and statins. In vitro, both CCM2L and CCM2 bind MEKK3 in a complex with CCM1. Both CCM2L and CCM2 interfere with MEKK3 activation and its ability to phosphorylate MEK5, a downstream target. The in vivo relevance of this regulation was investigated in zebrafish. A knockdown of ccm2l and ccm2 in zebrafish leads to a more severe "big heart" and circulation defects compared with loss of function of ccm2 alone, and also leads to substantial body axis abnormalities. Silencing of mekk3 rescues the big heart and body axis phenotype, suggesting cross-talk between the CCM proteins and MEKK3 in vivo. In endothelial cells, CCM2 deletion leads to activation of ERK5 and a transcriptional program that are downstream of MEKK3. These findings suggest that CCM2L and CCM2 cooperate to regulate the activity of MEKK3.

The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

Although renin is a critical regulatory enzyme of the cardiovascular system, its roles in organogenesis and the establishment of cardiovascular homeostasis remain unclear. Mammalian renin-expressing cells are widespread in embryonic kidneys but are highly restricted, specialized endocrine cells in adults. With a functional pronephros, embryonic zebrafish are ideal for delineating the developmental functions of renin-expressing cells and the mechanisms governing renin transcription. Larval zebrafish renin expression originates in the mural cells of the juxtaglomerular anterior mesenteric artery and subsequently at extrarenal sites. The role of renin was determined by assessing responses to renin-angiotensin system blockade, salinity variation, and renal perfusion ablation. Renin expression did not respond to renal flow ablation but was modulated by inhibition of angiotensin-converting enzyme and altered salinity. Our data in larval fish are consistent with conservation of renin's physiological functions. Using transgenic renin reporter fish, with mindbomb and cloche mutants, we show that Notch signaling and the endothelium are essential for developmental renin expression. After inhibition of angiogenesis, renin-expressing cells precede angiogenic sprouts. Arising from separate lineages, but relying on mutual interplay with endothelial cells, renin-expressing cells are among the earliest mural cells observed in larval fish, performing both endocrine and paracrine functions., (Copyright © 2015 the American Physiological Society.)

Hematopoietic stem cells (HSCs) require multiple molecular inputs for proper specification, including activity of the Notch signaling pathway. A requirement for the Notch1 and dispensability of the Notch2 receptor has been demonstrated in mice, but the role of the remaining Notch receptors has not been investigated. Here, we demonstrate that three of the four Notch receptors are independently required for the specification of HSCs in the zebrafish. The orthologues of the murine Notch1 receptor, Notch1a and Notch1b, are each required intrinsically to fate HSCs, just prior to their emergence from aortic hemogenic endothelium. By contrast, the Notch3 receptor is required earlier within the developing somite to regulate HSC emergence in a non-cell-autonomous manner. Epistatic analyses demonstrate that Notch3 function lies downstream of Wnt16, which is required for HSC specification through its regulation of two Notch ligands, dlc and dld. Collectively, these findings demonstrate for the first time that multiple Notch signaling inputs are required to specify HSCs and that Notch3 performs a novel role within the somite to regulate the neighboring precursors of hemogenic endothelium., (© 2014 The Authors.)

The zebrafish is increasingly used for cardiovascular genetic and functional studies. We present a novel protocol to maintain and monitor whole isolated beating adult zebrafish hearts in culture for long-term experiments. Excised whole adult zebrafish hearts were transferred directly into culture dishes containing optimized L-15 Leibovitz growth medium and maintained for 5 days. Hearts were assessed daily using video-edge analysis of ventricle function using low power microscopy images. High-throughput histology techniques were used to assess changes in myocardial architecture and cell viability. Mean spontaneous Heart rate (HR, min(-1)) declined significantly between day 0 and day 1 in culture (96.7 ± 19.5 to 45.2 ± 8.2 min-1, mean ± SD, p = 0.001), and thereafter declined more slowly to 27.6 ± 7.2 min(-1) on day 5. Ventricle wall motion amplitude (WMA) did not change until day 4 in culture (day 0, 46.7 ± 13.0 µm vs day 4, 16.9 ± 1.9 µm, p = 0.08). Contraction velocity (CV) declined between day 0 and day 3 (35.6 ± 14.8 vs 15.2 ± 5.3 µms(-1), respectively, p = 0.012) while relaxation velocity (RV) declined quite rapidly (day 0, 72.5 ± 11.9 vs day 1, 29.5 ± 5.8 µms(-1), p = 0.03). HR and WMA responded consistently to isoproterenol from day 0 to day 5 in culture while CV and RV showed less consistent responses to beta-agonist. Cellular architecture and cross-striation pattern of cardiomyocytes remained unchanged up to day 3 in culture and thereafter showed significant deterioration with loss of striation pattern, pyknotic nuclei and cell swelling. Apoptotic markers within the myocardium became increasingly frequent by day 3 in culture. Whole adult zebrafish hearts can be maintained in culture-medium for up to 3 days. However, after day-3 there is significant deterioration in ventricle function and heart rate accompanied by significant histological changes consistent with cell death and loss of cardiomyocyte cell integrity. Further studies are needed to assess whether this preparation can be optimised for longer term survival.

The human heart's failure to replace ischemia-damaged myocardium with regenerated muscle contributes significantly to the worldwide morbidity and mortality associated with coronary artery disease. Remarkably, certain vertebrate species, including the zebrafish, achieve complete regeneration of amputated or injured myocardium through the proliferation of spared cardiomyocytes. Nonetheless, the genetic and cellular determinants of natural cardiac regeneration remain incompletely characterized. Here, we report that cardiac regeneration in zebrafish relies on Notch signaling. Following amputation of the zebrafish ventricular apex, Notch receptor expression becomes activated specifically in the endocardium and epicardium, but not the myocardium. Using a dominant negative approach, we discovered that suppression of Notch signaling profoundly impairs cardiac regeneration and induces scar formation at the amputation site. We ruled out defects in endocardial activation, epicardial activation, and dedifferentiation of compact myocardial cells as causative for the regenerative failure. Furthermore, coronary endothelial tubes, which we lineage traced from preexisting endothelium in wild-type hearts, formed in the wound despite the myocardial regenerative failure. Quantification of myocardial proliferation in Notch-suppressed hearts revealed a significant decrease in cycling cardiomyocytes, an observation consistent with a noncell autonomous requirement for Notch signaling in cardiomyocyte proliferation. Unexpectedly, hyperactivation of Notch signaling also suppressed cardiomyocyte proliferation and heart regeneration. Taken together, our data uncover the exquisite sensitivity of regenerative cardiomyocyte proliferation to perturbations in Notch signaling., Competing Interests: The authors declare no conflict of interest.

Comprehensive functional annotation of vertebrate genomes is fundamental to biological discovery. Reverse genetic screening has been highly useful for determination of gene function, but is untenable as a systematic approach in vertebrate model organisms given the number of surveyable genes and observable phenotypes. Unbiased prediction of gene-phenotype relationships offers a strategy to direct finite experimental resources towards likely phenotypes, thus maximizing de novo discovery of gene functions. Here we prioritized genes for phenotypic assay in zebrafish through machine learning, predicting the effect of loss of function of each of 15,106 zebrafish genes on 338 distinct embryonic anatomical processes. Focusing on cardiovascular phenotypes, the learning procedure predicted known knockdown and mutant phenotypes with high precision. In proof-of-concept studies we validated 16 high-confidence cardiac predictions using targeted morpholino knockdown and initial blinded phenotyping in embryonic zebrafish, confirming a significant enrichment for cardiac phenotypes as compared with morpholino controls. Subsequent detailed analyses of cardiac function confirmed these results, identifying novel physiological defects for 11 tested genes. Among these we identified tmem88a, a recently described attenuator of Wnt signaling, as a discrete regulator of the patterning of intercellular coupling in the zebrafish cardiac epithelium. Thus, we show that systematic prioritization in zebrafish can accelerate the pace of developmental gene function discovery.

Organ development is a highly regulated process involving the coordinated proliferation and differentiation of diverse cellular populations. The pathways regulating cell proliferation and their effects on organ growth are complex and for many organs incompletely understood. In all vertebrate species, the cardiac natriuretic peptides (ANP and BNP) are produced by cardiomyocytes in the developing heart. However, their role during cardiogenesis is not defined. Using the embryonic zebrafish and neonatal mammalian cardiomyocytes we explored the natriuretic peptide signaling network during myocardial development. We observed that the cardiac natriuretic peptides ANP and BNP and the guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2 are functionally redundant during early cardiovascular development. In addition, we demonstrate that low levels of the natriuretic peptides preferentially activate Npr3, a receptor with Gi activator sequences, and increase cardiomyocyte proliferation through inhibition of adenylate cyclase. Conversely, high concentrations of natriuretic peptides reduce cardiomyocyte proliferation through activation of the particulate guanylate cyclase-linked natriuretic peptide receptors Npr1 and Npr2, and activation of protein kinase G. These data link the cardiac natriuretic peptides in a complex hierarchy modulating cardiomyocyte numbers during development through opposing effects on cardiomyocyte proliferation mediated through distinct cyclic nucleotide signaling pathways.

Midline convergence of organ primordia is an important mechanism that shapes the vertebrate body plan. Here, we focus on the morphogenetic movements of pronephric glomerular primordia (PGP) occurring during zebrafish embryonic kidney development. To characterize the process of PGP midline convergence, we used Wilms' tumour 1a (wt1a) as a marker to label kidney primordia, and performed quantitative analyses of the migration of the bilateral PGP. The PGP initially are approximately 350 μm apart in a wild type embryo at 10h post fertilization (hpf). The inter-PGP distance decreases exponentially between 10 and 48 hpf, while the anterior-posterior (A-P) dimension of each PGP increases linearly between 10 and 12 hpf, then decreases substantially between 12 and 24 hpf. Using mutants in the Nodal receptor cofactor one-eyed pinhead (oep) and the T-box transcription factors spadetail (spt) and no tail (ntl), we were able to define distinctive regulation underlying these sequential phases of PGP midline migration. Zygotic oep mutants (Zoep(-/-)) exhibited defects in midline convergence after 16 hpf. Spt is necessary for PGP convergence from 10 hpf, whereas ntl's effect on convergence does not begin until 24 hpf. Notably, we observed normal cardiac convergence in spt(-/-) and ntl(-/-) embryos implying that these novel roles of spt and ntl in PGP migration cannot be explained simply by generalised effects on midline convergence. These findings demonstrate that quantitative approaches to developmental migration allow the parsing of early patterning events, and in this instance suggest that the zebrafish may offer insights into midline urogenital migration anomalies in humans., (© 2013 Published by Elsevier Inc.)

Introduction: Our understanding of the complexity of arrhythmogenesis has grown progressively over the last two decades. The range of established arrhythmia mechanisms now includes numerous pathways including ion channels, membrane adaptor proteins, transcription factors and cytoplasmic signaling cascades., Areas Covered: This review outlines the emerging biology underlying potentially lethal rhythm disturbances, and highlights the problems that these novel mechanisms present for rational target identification in the traditional framework of drug discovery. The article describes the fundamental rationale for in vivo screening and highlights the utility of the zebrafish for this approach. The article also outlines initial efforts exploiting in vivo antiarrhythmic discovery and the potential for this approach to be disruptive even in the setting of disease mechanisms that operate across multiple timescales in multiple tissues., Expert Opinion: In vivo screening in genetically faithful model organisms offers access to existing and unimagined arrhythmia pathways. Rate limiting steps are the rigor of the modeling of specific arrhythmias and the downstream identification of the molecular targets once specific disease suppressors have been identified.

Altered mechanical loading of the heart leads to hypertrophy, decompensated heart failure and fatal arrhythmias. However, the molecular mechanisms that link mechanical and electrical dysfunction remain poorly understood. Growing evidence suggest that ventricular electrical remodeling (VER) is a process that can be induced by altered mechanical stress, creating persistent electrophysiological changes that predispose the heart to life-threatening arrhythmias. While VER is clearly a physiological property of the human heart, as evidenced by "T wave memory", it is also thought to occur in a variety of pathological states associated with altered ventricular activation such as bundle branch block, myocardial infarction, and cardiac pacing. Animal models that are currently being used for investigating stretch-induced VER have significant limitations. The zebrafish has recently emerged as an attractive animal model for studying cardiovascular disease and could overcome some of these limitations. Owing to its extensively sequenced genome, high conservation of gene function, and the comprehensive genetic resources that are available in this model, the zebrafish may provide new insights into the molecular mechanisms that drive detrimental electrical remodeling in response to stretch. Here, we have established a zebrafish model to study mechano-electrical feedback in the heart, which combines efficient genetic manipulation with high-precision stretch and high-resolution electrophysiology. In this model, only 90 min of ventricular stretch caused VER and recapitulated key features of VER found previously in the mammalian heart. Our data suggest that the zebrafish model is a powerful platform for investigating the molecular mechanisms underlying mechano-electrical feedback and VER in the heart., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Understanding the developmental basis of cardiac electrical activity has proven technically challenging, largely as a result of the inaccessible nature of the heart during cardiogenesis in many organisms. The emergence of the zebrafish as a model organism has availed the very earliest stages of heart formation to experimental exploration. The zebrafish also offers a robust platform for genetic and chemical screening. These tools have been exploited in screens for modifiers of cardiac electrophysiologic phenotypes and in screens for novel drugs.

Zebrafish, a well-established vertebrate model, offer unique advantages for assessing renal function and physiology. Assays determining renal glomerular function based on cardiovascular erythrocyte flow and reduction of injected FITC-inulin were developed, each validated using the nephrotoxin gentamicin. Bland–Atlman analysis showed a strong association between measurements of the rate of inulin excretion and that of fluorescent reduction from the arterial vasculature. Reduced renal clearance of inulin, resulting from gentamicin or NaCl loading, was concurrent with reduced erythrocyte velocity, and yolk sac and pericardium oedema. These techniques, assessing pronephric function, highlight the potential for in vivo physiological study in this genetically tractable model.

Aims: Despite increased understanding of the fundamental biology regulating cardiomyocyte hypertrophy and heart failure, it has been challenging to find novel chemical or genetic modifiers of these pathways. Traditional cell-based methods do not model the complexity of an intact cardiovascular system and mammalian models are not readily adaptable to chemical or genetic screens. Our objective was to create an in vivo model suitable for chemical and genetic screens for hypertrophy and heart failure modifiers., Methods and Results: Using the developing zebrafish, we established that the cardiac natriuretic peptide genes (nppa and nppb), known markers of cardiomyocyte hypertrophy and heart failure, were induced in the embryonic heart by pathological cardiac stimuli. This pathological induction was distinct from the developmental regulation of these genes. We created a luciferase-based transgenic reporter line that accurately modelled the pathological induction patterns of the zebrafish nppb gene. Utilizing this reporter line, we were able to show remarkable conservation of pharmacological responses between the larval zebrafish heart and adult mammalian models., Conclusion: By performing a focused screen of chemical agents, we were able to show a distinct response of a genetic model of hypertrophic cardiomyopathy to the histone deacetylase inhibitor, Trichostatin A, and the mitogen-activated protein kinase kinase 1/2 inhibitor, U0126. We believe this in vivo reporter line will offer a unique approach to the identification of novel chemical or genetic regulators of myocardial hypertrophy and heart failure.

The vertebrate heart is one of the first organs to form, and its early function and morphogenesis are crucial for continued embryonic development. Here we analyze the effects of loss of Heart adaptor protein 1 (Hadp1), which we show is required for normal function and morphogenesis of the embryonic zebrafish heart. Hadp1 is a pleckstrin homology (PH)-domain-containing protein whose expression is enriched in embryonic cardiomyocytes. Knockdown of hadp1 in zebrafish embryos reduced cardiac contractility and altered late myocyte differentiation. By using optical mapping and submaximal levels of hadp1 knockdown, we observed profound effects on Ca(2+) handling and on action potential duration in the absence of morphological defects, suggesting that Hadp1 plays a major role in the regulation of intracellular Ca(2+) handling in the heart. Hadp1 interacts with phosphatidylinositol 4-phosphate [PI4P; also known as PtdIns(4)P] derivatives via its PH domain, and its subcellular localization is dependent upon this motif. Pharmacological blockade of the synthesis of PI4P derivatives in vivo phenocopied the loss of hadp1 in zebrafish. Collectively, these results demonstrate that hadp1 is required for normal cardiac function and morphogenesis during embryogenesis, and suggest that hadp1 modulates Ca(2+) handling in the heart through its interaction with phosphatidylinositols.

Natural models of heart regeneration in lower vertebrates such as zebrafish are based on invasive surgeries causing mechanical injuries that are limited in size. Here, we created a genetic cell ablation model in zebrafish that facilitates inducible destruction of a high percentage of cardiomyocytes. Cell-specific depletion of over 60% of the ventricular myocardium triggered signs of cardiac failure that were not observed after partial ventricular resection, including reduced animal exercise tolerance and sudden death in the setting of stressors. Massive myocardial loss activated robust cellular and molecular responses by endocardial, immune, epicardial and vascular cells. Destroyed cardiomyocytes fully regenerated within several days, restoring cardiac anatomy, physiology and performance. Regenerated muscle originated from spared cardiomyocytes that acquired ultrastructural and electrophysiological characteristics of de-differentiation and underwent vigorous proliferation. Our study indicates that genetic depletion of cardiomyocytes, even at levels so extreme as to elicit signs of cardiac failure, can be reversed by natural regenerative capacity in lower vertebrates such as zebrafish.

To assess the effects during cardiac development of mutations that cause human cardiomyopathy, we modeled a sarcomeric gene mutation in the embryonic zebrafish. We designed morpholino antisense oligonucleotides targeting the exon 13 splice donor site in the zebrafish cardiac troponin T (tnnt2) gene, in order to precisely recapitulate a human TNNT2 mutation that causes hypertrophic cardiomyopathy (HCM). HCM is a disease characterized by myocardial hypertrophy, myocyte and myofibrillar disarray, as well as an increased risk of sudden death. Similar to humans with HCM, the morphant zebrafish embryos displayed sarcomere disarray and there was a robust induction of myocardial hypertrophic pathways. Microarray analysis uncovered a number of shared transcriptional responses between this zebrafish model and a well-characterized mouse model of HCM. However, in contrast to adult hearts, these embryonic hearts developed cardiomyocyte hyperplasia in response to this genetic perturbation. The re-creation of a human disease-causing TNNT2 splice variant demonstrates that sarcomeric mutations can alter cardiomyocyte biology at the earliest stages of heart development with distinct effects from those observed in adult hearts despite shared transcriptional responses.

The zebrafish offers a scalable vertebrate model for many areas of biologic investigation. There is substantial conservation of genetic and genomic features and, at a higher order, conservation of intermolecular networks, as well as physiologic systems and phenotypes. We highlight recent work demonstrating the extent of this homology, and efforts to develop high-throughput phenotyping strategies suited to genetic or chemical screening on a scale compatible with in vivo validation for systems biology. We discuss the implications of these approaches for functional annotation of the genome, elucidation of multicellular processes in vivo, and mechanistic exploration of hypotheses generated by a broad range of 'unbiased' 'omic technologies such as expression profiling and genome-wide association. Finally, we outline potential strategies for the application of the zebrafish to the systematic study of phenotypic architecture, disease heterogeneity and drug responses., (Copyright © 2010 John Wiley & Sons, Inc.)

Electrical gradients are critical for many biological processes, including the normal function of excitable tissues, left-right patterning, organogenesis and wound healing. The fundamental mechanisms that regulate the establishment and maintenance of such electrical polarities are poorly understood. Here we identify a gradient of electrical coupling across the developing ventricular myocardium using high-speed optical mapping of transmembrane potentials and calcium concentrations in the zebrafish heart. We excluded a role for differences in cellular excitability, connexin localization, tissue geometry and mechanical inputs, but in contrast we were able to demonstrate that non-canonical Wnt11 signals are required for the genesis of this myocardial electrical gradient. Although the traditional planar cell polarity pathway is not involved, we obtained evidence that Wnt11 acts to set up this gradient of electrical coupling through effects on transmembrane Ca(2+) conductance mediated by the L-type calcium channel. These data reveal a previously unrecognized role for Wnt/Ca(2+) signalling in establishing an electrical gradient in the plane of the developing cardiac epithelium through modulation of ion-channel function. The regulation of cellular coupling through such mechanisms may be a general property of non-canonical Wnt signals.

Phenotype-driven screens in larval zebrafish have transformed our understanding of the molecular basis of cardiovascular development. Screens to define the genetic determinants of physiological phenotypes have been slow to materialize as a result of the limited number of validated in vivo assays with relevant dynamic range. To enable rigorous assessment of cardiovascular physiology in living zebrafish embryos, we developed a suite of software tools for the analysis of high-speed video microscopic images and validated these, using established cardiomyopathy models in zebrafish as well as modulation of the nitric oxide (NO) pathway. Quantitative analysis in wild-type fish exposed to NO or in a zebrafish model of dilated cardiomyopathy demonstrated that these tools detect significant differences in ventricular chamber size, ventricular performance, and aortic flow velocity in zebrafish embryos across a large dynamic range. These methods also were able to establish the effects of the classic pharmacological agents isoproterenol, ouabain, and verapamil on cardiovascular physiology in zebrafish embryos. Sequence conservation between zebrafish and mammals of key amino acids in the pharmacological targets of these agents correlated with the functional orthology of the physiological response. These data provide evidence that the quantitative evaluation of subtle physiological differences in zebrafish can be accomplished at a resolution and with a dynamic range comparable to those achieved in mammals and provides a mechanism for genetic and small-molecule dissection of functional pathways in this model organism.

The molecular pathways by which newly formed, immature endothelial cell tubes remodel to form a mature network of vessels supported by perivascular mural cells are not well understood. The zebrafish iguana (igu) genetic mutant has a mutation in the daz-interacting protein 1 (dzip1), a member of the hedgehog signaling pathway. Loss of dzip1 results in decreased size of the cranial dorsal aortae, ultrastructural defects in perivascular mural cell recruitment and subsequent hemorrhage. Although hedgehog signaling is disrupted in igu mutants, we find no defects in vessel patterning or artery-vein specification. Rather, we show that the loss of angiopoietin1 (angpt1) expression in ventral perivascular mesenchyme is responsible for vascular instability in igu mutants. Over-expression of angpt1 or partial down-regulation of the endogenous Angpt1 antagonist angpt2 rescues hemorrhage. This is the first direct in vivo link between hedgehog signaling and the induction of vascular stability by recruitment of perivascular mural cells through angiopoietin signaling., (Copyright 2010 Elsevier Ireland Ltd. All rights reserved.)

Recent studies indicate that mammals, including humans, maintain some capacity to renew cardiomyocytes throughout postnatal life. Yet, there is little or no significant cardiac muscle regeneration after an injury such as acute myocardial infarction. By contrast, zebrafish efficiently regenerate lost cardiac muscle, providing a model for understanding how natural heart regeneration may be blocked or enhanced. In the absence of lineage-tracing technology applicable to adult zebrafish, the cellular origins of newly regenerated cardiac muscle have remained unclear. Using new genetic fate-mapping approaches, here we identify a population of cardiomyocytes that become activated after resection of the ventricular apex and contribute prominently to cardiac muscle regeneration. Through the use of a transgenic reporter strain, we found that cardiomyocytes throughout the subepicardial ventricular layer trigger expression of the embryonic cardiogenesis gene gata4 within a week of trauma, before expression localizes to proliferating cardiomyocytes surrounding and within the injury site. Cre-recombinase-based lineage-tracing of cells expressing gata4 before evident regeneration, or of cells expressing the contractile gene cmlc2 before injury, each labelled most cardiac muscle in the ensuing regenerate. By optical voltage mapping of surface myocardium in whole ventricles, we found that electrical conduction is re-established between existing and regenerated cardiomyocytes between 2 and 4 weeks post-injury. After injury and prolonged fibroblast growth factor receptor inhibition to arrest cardiac regeneration and enable scar formation, experimental release of the signalling block led to gata4 expression and morphological improvement of the injured ventricular wall without loss of scar tissue. Our results indicate that electrically coupled cardiac muscle regenerates after resection injury, primarily through activation and expansion of cardiomyocyte populations. These findings have implications for promoting regeneration of the injured human heart.

Over the last decade the zebrafish has emerged as a major genetic model organism. While stimulated originally by the utility of its transparent embryos for the study of vertebrate organogenesis, the success of the zebrafish was consolidated through multiple genetic screens, sequencing of the fish genome by the Sanger Center, and the advent of extensive genomic resources. In the last few years the potential of the zebrafish for in vivo cell biology, physiology, disease modeling and drug discovery has begun to be realized. This review will highlight work on cardiac electrophysiology, emphasizing the arenas in which the zebrafish complements other in vivo and in vitro models; developmental physiology, large-scale screens, high-throughput disease modeling and drug discovery. Much of this work is at an early stage, and so the focus will be on the general principles, the specific advantages of the zebrafish and on future potential.

To investigate the mechanisms by which mutations in the human transcriptional co-activator EYA4 gene cause sensorineural hearing loss that can occur in association with dilated cardiomyopathy, we studied eya4 expression during zebrafish development and characterized eya4 deficiency. eya4 morphant fish embryos had reduced numbers of hair cells in the otic vesicle and lateral line neuromasts with impaired sensory responses. Analyses of candidate genes that are known to be expressed in a temporal and spatial pattern comparable to eya4 focused our analyses on atp1b2b, which encodes the beta2b subunit of the zebrafish Na+/K+-ATPase. We demonstrate atp1b2b levels are reduced in eya4 morphant fish and that morpholino oligonucleotides targeting the atp1b2b gene recapitulated the eya4 deficiency phenotypes, including heart failure, decreased sensory hair cell numbers in the otic vesicle and neuromasts, and abnormal sensory responses. Furthermore, atp1b2b overexpression rescued these phenotypes in eya4 morphant fish. We conclude that eya4 regulation of Na+/K+-ATPase is crucial for the development of mechanosensory cells and the maintenance of cardiac function in zebrafish.

AML1-ETO is one of the most common chromosomal translocation products associated with acute myelogenous leukemia (AML). Patients carrying the AML1-ETO fusion gene exhibit an accumulation of granulocyte precursors in the bone marrow and the blood. Here, we describe a transgenic zebrafish line that enables inducible expression of the human AML1-ETO oncogene. Induced AML1-ETO expression in embryonic zebrafish causes a phenotype that recapitulates some aspects of human AML. Using this highly tractable model, we show that AML1-ETO redirects myeloerythroid progenitor cells that are developmentally programmed to adopt the erythroid cell fate into the granulocytic cell fate. This fate change is characterized by a loss of gata1 expression and an increase in pu.1 expression in myeloerythroid progenitor cells. Moreover, we identify scl as an early and essential mediator of the effect of AML1-ETO on hematopoietic cell fate. AML1-ETO quickly shuts off scl expression, and restoration of scl expression rescues the effects of AML1-ETO on myeloerythroid progenitor cell fate. These results demonstrate that scl is an important mediator of the ability of AML1-ETO to reprogram hematopoietic cell fate decisions, suggesting that scl may be an important contributor to AML1-ETO-associated leukemia. In addition, treatment of AML1-ETO transgenic zebrafish embryos with a histone deacetylase inhibitor, Trichostatin A, restores scl and gata1 expression, and ameliorates the accumulation of granulocytic cells caused by AML1-ETO. Thus, this zebrafish model facilitates in vivo dissection of AML1-ETO-mediated signaling, and will enable large-scale chemical screens to identify suppressors of the in vivo effects of AML1-ETO.

In the last decade the zebrafish has become a major model organism for the study of development and organogenesis. To maximize the experimental utility of this organism, it will be important to establish methods for adult phenotyping. We previously proposed that the embryonic zebrafish may be useful in high-throughput screening for drug-induced cardiotoxicity. We now describe a method for the reproducible recording of the adult zebrafish ECG and illustrate its application in the investigation of QT-prolonging drugs. Zebrafish ECGs were obtained by inserting two needle electrodes through the ventral epidermis. Fish were perfused orally, and motion artifacts were eliminated with a paralytic dose of mu-conotoxin GIIIB. Test compounds were delivered via the perfusion system. Without a means of hydration and oxygenation, the fish succumb rapidly. The use of a perfusion system allowed stable recording for > 6 h. Baseline conduction intervals were as follows: PR, 66 ms (SD 14); QRS, 34 ms (SD 11); QT, 242 ms (SD 54); and R-R, 398 ms (SD 77). The known QT-prolonging agents astemizole, haloperidol, pimozide, and terfenadine caused corrected QT increases of 18% (SD 9), 16% (SD 11), 17% (SD 9), and 11% (SD 6), respectively. The control drugs clonidine, penicillin and propranolol did not prolong the corrected QT interval. In conclusion, perfusion and muscular paralysis allows stable, low-noise recording of zebrafish ECGs. Agents known to cause QT prolongation in humans caused QT prolongation in fish in each case. The development of rigorous tools for the phenotyping of adult zebrafish will complement the high-throughput assays currently under development for embryonic and larval fish.

Whole genome comparisons of distantly related species effectively predict biologically important sequences--core genes and cis-acting regulatory elements (REs)--but require experimentation to verify biological activity. To examine the efficacy of comparative genomics in identification of active REs from anonymous, non-coding (NC) sequences, we generated a novel alignment of the human and draft zebrafish genomes, and contrasted this set to existing human and fugu datasets. We tested the transcriptional regulatory potential of candidate sequences using two in vivo assays. Strict selection of non-genic elements which are deeply conserved in vertebrate evolution identifies 1744 core vertebrate REs in human and two fish genomes. We tested 16 elements in vivo for cis-acting gene regulatory properties using zebrafish transient transgenesis and found that 10 (63%) strongly modulate tissue-specific expression of a green fluorescent protein reporter vector. We also report a novel quantitative enhancer assay with potential for increased throughput based on normalized luciferase activity in vivo. This complementary system identified 11 (69%; including 9 of 10 GFP-confirmed elements) with cis-acting function. Together, these data support the utility of comparative genomics of distantly related vertebrate species to identify REs and provide a scaleable, in vivo quantitative assay to define functional activity of candidate REs.

We identified a human mutation that causes dilated cardiomyopathy and heart failure preceded by sensorineural hearing loss (SNHL). Unlike previously described mutations causing dilated cardiomyopathy that affect structural proteins, this mutation deletes 4,846 bp of the human transcriptional coactivator gene EYA4. To elucidate the roles of eya4 in heart function, we studied zebrafish embryos injected with antisense morpholino oligonucleotides. Attenuated eya4 transcript levels produced morphologic and hemodynamic features of heart failure. To determine why previously described mutated EYA4 alleles cause SNHL without heart disease, we examined biochemical interactions of mutant Eya4 peptides. Eya4 peptides associated with SNHL, but not the shortened 193-amino acid peptide associated with dilated cardiomyopathy and SNHL, bound wild-type Eya4 and associated with Six proteins. These data define unrecognized and crucial roles for Eya4-Six-mediated transcriptional regulation in normal heart function.

The development of rapidly acting cyanide countermeasures using intramuscular injection (IM) represents an unmet medical need to mitigate toxicant exposures in mass casualty settings. Previous work established that cisplatin and other platinum(II) or platinum(IV)-based agents effectively mitigate cyanide toxicity in zebrafish. Cyanide's in vivo reaction with platinum-containing materials was proposed to reduce the risk of acute toxicities. However, cyanide antidote activity depended on a formulation of platinum-chloride salts with dimethyl sulfoxide (DMSO) followed by dilution in phosphate-buffered saline (PBS). A working hypothesis to explain the DMSO requirement is that the formation of platinum-sulfoxide complexes activates the cyanide scavenging properties of platinum. Preparations of isolated NaPtCl5-DMSO and Na (NH3)2PtCl-DMSO complexes in the absence of excess DMSO provided agents with enhanced reactivity toward cyanide in vitro and fully recapitulated in vivo cyanide rescue in zebrafish and mouse models. The enhancement of the cyanide scavenging effects of the DMSO ligand could be attributed to the activation of platinum(IV) and (II) with a sulfur ligand. Unfortunately, the efficacy of DMSO complexes was not robust when administered IM. Alternative Pt(II) materials containing sulfide and amine ligands in bidentate complexes show enhanced reactivity toward cyanide addition. The cyanide addition products yielded tetracyanoplatinate(II), translating to a stoichiometry of 1:4 Pt to each cyanide scavenger. These new agents demonstrate a robust and enhanced potency over the DMSO-containing complexes using IM administration in mouse and rabbit models of cyanide toxicity. Using the zebrafish model with these Pt(II) complexes, no acute cardiotoxicity was detected, and dose levels required to reach lethality exceeded 100 times the effective dose. Data are presented to support a general chemical design approach that can expand a new lead candidate series for developing next-generation cyanide countermeasures.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

Cisplatin holds an illustrious position in the history of chemistry most notably for its role in the virtual cure of testicular cancer. Here we describe a role for this small molecule in cyanide detoxification in vivo. Cyanide kills organisms as diverse as insects, fish, and humans within seconds to hours. Current antidotes exhibit limited efficacy and are not amenable to mass distribution requiring the development of new classes of antidotes. The binding affinity of the cyanide anion for the positively charged metal platinum is known to create an extremely stable complex in vitro. We therefore screened a panel of diverse cisplatin analogs and identified compounds that conferred protection from cyanide poisoning in zebrafish, mice, and rabbits. Cumulatively, this discovery pipeline begins to establish the characteristics of platinum ligands that influence their solubility, toxicity, and efficacy, and provides proof of concept that platinum-based complexes are effective antidotes for cyanide poisoning.

RATIONALE: The rapid induction and orchestration of new blood vessels are critical for tissue repair in response to injury, such as myocardial infarction, and for physiological angiogenic responses, such as embryonic development and exercise. OBJECTIVE: We aimed to identify and characterize microRNAs (miR) that regulate pathological and physiological angiogenesis. METHODS AND RESULTS: We show that miR-26a regulates pathological and physiological angiogenesis by targeting endothelial cell (EC) bone morphogenic protein/SMAD1 signaling in vitro and in vivo. MiR-26a expression is increased in a model of acute myocardial infarction in mice and in human subjects with acute coronary syndromes. Ectopic expression of miR-26a markedly induced EC cycle arrest and inhibited EC migration, sprouting angiogenesis, and network tube formation in matrigel, whereas blockade of miR-26a had the opposite effects. Mechanistic studies demonstrate that miR-26a inhibits the bone morphogenic protein/SMAD1 signaling pathway in ECs by binding to the SMAD1 3-untranslated region, an effect that decreased expression of Id1 and increased p21(WAF/CIP) and p27. In zebrafish, miR-26a overexpression inhibited formation of the caudal vein plexus, a bone morphogenic protein-responsive process, an effect rescued by ectopic SMAD1 expression. In mice, miR-26a overexpression inhibited EC SMAD1 expression and exercise-induced angiogenesis. Furthermore, systemic intravenous administration of an miR-26a inhibitor, locked nucleic acid-anti-miR-26a, increased SMAD1 expression and rapidly induced robust angiogenesis within 2 days, an effect associated with reduced myocardial infarct size and improved heart function. CONCLUSIONS: These findings establish miR-26a as a regulator of bone morphogenic protein/SMAD1-mediated EC angiogenic responses, and that manipulating miR-26a expression could provide a new target for rapid angiogenic therapy in ischemic disease states.

Exposure to cyanide causes a spectrum of cardiac, neurological, and metabolic dysfunctions that can be fatal. Improved cyanide antidotes are needed, but the ideal biological pathways to target are not known. To understand better the metabolic effects of cyanide and to discover novel cyanide antidotes, we developed a zebrafish model of cyanide exposure and scaled it for high-throughput chemical screening. In a screen of 3120 small molecules, we discovered 4 novel antidotes that block cyanide toxicity. The most potent antidote was riboflavin. Metabolomic profiling of cyanide-treated zebrafish revealed changes in bile acid and purine metabolism, most notably by an increase in inosine levels. Riboflavin normalizes many of the cyanide-induced neurological and metabolic perturbations in zebrafish. The metabolic effects of cyanide observed in zebrafish were conserved in a rabbit model of cyanide toxicity. Further, humans treated with nitroprusside, a drug that releases nitric oxide and cyanide ions, display increased circulating bile acids and inosine. In summary, riboflavin may be a novel treatment for cyanide toxicity and prophylactic measure during nitroprusside treatment, inosine may serve as a biomarker of cyanide exposure, and metabolites in the bile acid and purine metabolism pathways may shed light on the pathways critical to reversing cyanide toxicity. [ABSTRACT FROM AUTHOR]

Organophosphates are a class of highly toxic chemicals that includes many pesticides and chemical weapons. Exposure to organophosphates, either through accidents or acts of terrorism, poses a significant risk to human health and safety. Existing antidotes, in use for over 50 years, have modest efficacy and undesirable toxicities. Therefore, discovering new organophosphate antidotes is a high priority. Early life stage zebrafish exposed to organophosphates exhibit several phenotypes that parallel the human response to organophosphates, including behavioral deficits, paralysis, and eventual death. Here, we have developed a high-throughput zebrafish screen in a 96-well plate format to find new antidotes that counteract organophosphate-induced lethality. In a pilot screen of 1200 known drugs, we identified 16 compounds that suppress organophosphate toxicity in zebrafish. Several in vitro assays coupled with liquid chromatography/tandem mass spectrometry–based metabolite profiling enabled determination of mechanisms of action for several of the antidotes, including reversible acetylcholinesterase inhibition, cholinergic receptor antagonism, and inhibition of bioactivation. Therefore, the in vivo screen is capable of discovering organophosphate antidotes that intervene in distinct pathways. These findings suggest that zebrafish screens might be a broadly applicable approach for discovering compounds that counteract the toxic effects of accidental or malicious poisonous exposures. [ABSTRACT FROM PUBLISHER]

Normal heart function is critically dependent on the timing and coordination provided by a complex network of specialized cells: the cardiac conduction system. We have employed functional assays in zebrafish to explore early steps in the patterning of the conduction system that previously have been inaccessible. We demonstrate that a ring of atrioventricular conduction tissue develops at 40 hours post-fertilization in the zebrafish heart. Analysis of the mutant cloche reveals a requirement for endocardial signals in the formation of this tissue. The differentiation of these specialized cells, unlike that of adjacent endocardial cushions and valves, is not dependent on blood flow or cardiac contraction. Finally, both neuregulin and notch1b are necessary for the development of atrioventricular conduction tissue. These results are the first demonstration of the endocardial signals required for patterning central `slow' conduction tissue, and they reveal the operation of distinct local endocardial-myocardial interactions within the developing heart tube. [ABSTRACT FROM AUTHOR]

Background: Defining conserved molecular pathways in animal models of successful cardiac regeneration could yield insight into why adult mammals have inadequate cardiac regeneration after injury. Insight into the transcriptomic landscape of early cardiac regeneration from model organisms will shed light on evolutionarily conserved pathways in successful cardiac regeneration.Methods: Here we describe a cross-species transcriptomic screen in 3 model organisms for cardiac regeneration: axolotl, neonatal mice, and zebrafish. Apical resection to remove ≈10% to 20% of ventricular mass was carried out in these model organisms. RNA-sequencing analysis was performed on the hearts harvested at 3 time points: 12, 24, and 48 hours after resection. Sham surgery was used as internal control.Results: Genes associated with inflammatory processes were found to be upregulated in a conserved manner. Complement receptors (activated by complement components, part of the innate immune system) were found to be highly upregulated in all 3 species. This approach revealed induction of gene expression for complement 5a receptor 1 in the regenerating hearts of zebrafish, axolotls, and mice. Inhibition of complement 5a receptor 1 significantly attenuated the cardiomyocyte proliferative response to heart injury in all 3 species. Furthermore, after left ventricular apical resection, the cardiomyocyte proliferative response was diminished in mice with genetic deletion of complement 5a receptor 1.Conclusions: These data reveal that activation of complement 5a receptor 1 mediates an evolutionarily conserved response that promotes cardiomyocyte proliferation after cardiac injury and identify complement pathway activation as a common pathway of successful heart regeneration. [ABSTRACT FROM AUTHOR]