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The innate immune response is triggered rapidly after injury and its spatiotemporal dynamics are critical for regeneration; however, many questions remain about its exact role. Here we show that MyD88, a key component of the innate immune response, controls not only the inflammatory but also the fibrotic response during zebrafish cardiac regeneration. We find in cryoinjured myd88 -/- ventricles a significant reduction in neutrophil and macrophage numbers and the expansion of a collagen-rich endocardial population. Further analyses reveal compromised PI3K/AKT pathway activation in the myd88 -/- endocardium and increased myofibroblasts and scarring. Notably, endothelial-specific overexpression of myd88 reverses these neutrophil, fibrotic and scarring phenotypes. Mechanistically, we identify the endocardial-derived chemokine gene cxcl18b as a target of the MyD88 signaling pathway, and using loss-of-function and gain-of-function tools, we show that it controls neutrophil recruitment. Altogether, these findings shed light on the pivotal role of MyD88 in modulating inflammation and fibrosis during tissue regeneration., (© 2024. The Author(s).)

Background: Ischemic heart disease following the obstruction of coronary vessels leads to the death of cardiac tissue and the formation of a fibrotic scar. In contrast to adult mammals, zebrafish can regenerate their heart after injury, enabling the study of the underlying mechanisms. One of the earliest responses following cardiac injury in adult zebrafish is coronary revascularization. Defects in this process lead to impaired cardiomyocyte repopulation and scarring. Hence, identifying and investigating factors that promote coronary revascularization holds great therapeutic potential., Methods: We used wholemount imaging, immunohistochemistry and histology to assess various aspects of zebrafish cardiac regeneration. Deep transcriptomic analysis allowed us to identify targets and potential effectors of Vegfc (vascular endothelial growth factor C) signaling. We used newly generated loss- and gain-of-function genetic tools to investigate the role of Emilin2a (elastin microfibril interfacer 2a) and Cxcl8a (chemokine (C-X-C) motif ligand 8a)-Cxcr1 (chemokine (C-X-C) motif receptor 1) signaling in cardiac regeneration., Results: We first show that regenerating coronary endothelial cells upregulate vegfc upon cardiac injury in adult zebrafish and that Vegfc signaling is required for their proliferation during regeneration. Notably, blocking Vegfc signaling also significantly reduces cardiomyocyte dedifferentiation and proliferation. Using transcriptomic analyses, we identified emilin2a as a target of Vegfc signaling and found that manipulation of emilin2a expression can modulate coronary revascularization as well as cardiomyocyte proliferation. Mechanistically, Emilin2a induces the expression of the chemokine gene cxcl8a in epicardium-derived cells, while cxcr1 , the Cxcl8a receptor gene, is expressed in coronary endothelial cells. We further show that Cxcl8a-Cxcr1 signaling is also required for coronary endothelial cell proliferation during cardiac regeneration., Conclusions: These data show that after cardiac injury, coronary endothelial cells upregulate vegfc to promote coronary network reestablishment and cardiac regeneration. Mechanistically, Vegfc signaling upregulates epicardial emilin2a and cxcl8a expression to promote cardiac regeneration. These studies aid in understanding the mechanisms underlying coronary revascularization in zebrafish, with potential therapeutic implications to enhance revascularization and regeneration in injured human hearts.

Over the last decades, myocardial infarction and heart failure have accounted every year for millions of deaths worldwide. After a coronary occlusion, the lack of blood supply to downstream muscle leads to cell death and scarring. To date, several pro-angiogenic factors have been tested to stimulate reperfusion of the affected myocardium, VEGFA being one of the most extensively studied. Given the unsuccessful outcomes of clinical trials, understanding how cardiac revascularization takes place in models with endogenous regenerative capacity holds the key to devising more efficient therapies. Here, we summarize the main findings on VEGFA's role during cardiac repair and regeneration, with a particular focus on zebrafish as a regenerative model. Moreover, we provide a comprehensive overview of available tools to modulate Vegfa expression and action in zebrafish regeneration studies. Understanding the role of Vegfa during zebrafish heart regeneration may help devise efficient therapies and circumvent current limitations in using VEGFA for therapeutic angiogenesis approaches., (© 2022. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Ankyrin repeat domain 1 (ANKRD1) is a functionally pleiotropic protein found in the nuclei and sarcomeres of cardiac and skeletal muscles, with a proposed role in linking myofibrilar stress and transcriptional regulation. Rapid upregulation of its expression in response to both physiological and pathological stress supports the involvement of ANKRD1 in muscle tissue adaptation and remodeling. However, the exact role of ANKRD1 remains poorly understood. To begin to investigate its function at higher resolution, we have generated and characterized a TgBAC(ankrd1a:EGFP) zebrafish line. This reporter line displays transgene expression in slow skeletal muscle fibers during development and exercise responsiveness in adult cardiac muscle. To better understand the role of Ankrd1a in pathological conditions in adult zebrafish, we assessed ankrd1a expression after cardiac ventricle cryoinjury and observed localized upregulation in cardiomyocytes in the border zone. We show that this expression in injured hearts is recapitulated by the TgBAC(ankrd1a:EGFP) reporter. Our results identify novel expression domains of ankrd1a and suggest an important role for Ankrd1a in the early stress response and regeneration of cardiac tissue. This new reporter line will help decipher the role of Ankrd1a in striated muscle stress response, including after cardiac injury., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish., Competing Interests: Declaration of competing interest The authors declare no competing or financial interests., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Cardiac metabolism plays a crucial role in producing sufficient energy to sustain cardiac function. However, the role of metabolism in different aspects of cardiomyocyte regeneration remains unclear. Working with the adult zebrafish heart regeneration model, we first find an increase in the levels of mRNAs encoding enzymes regulating glucose and pyruvate metabolism, including pyruvate kinase M1/2 (Pkm) and pyruvate dehydrogenase kinases (Pdks), especially in tissues bordering the damaged area. We further find that impaired glycolysis decreases the number of proliferating cardiomyocytes following injury. These observations are supported by analyses using loss-of-function models for the metabolic regulators Pkma2 and peroxisome proliferator-activated receptor gamma coactivator 1 alpha. Cardiomyocyte-specific loss- and gain-of-function manipulations of pyruvate metabolism using Pdk3 as well as a catalytic subunit of the pyruvate dehydrogenase complex (PDC) reveal its importance in cardiomyocyte dedifferentiation and proliferation after injury. Furthermore, we find that PDK activity can modulate cell cycle progression and protrusive activity in mammalian cardiomyocytes in culture. Our findings reveal new roles for cardiac metabolism and the PDK-PDC axis in cardiomyocyte behavior following cardiac injury., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

Tuberculosis is a highly infectious and potentially fatal disease accompanied by wasting symptoms, which cause severe metabolic changes in infected people. In this study we have compared the effect of mycobacteria infection on the level of metabolites in blood of humans and mice and whole zebrafish larvae using one highly standardized mass spectrometry pipeline, ensuring technical comparability of the results. Quantification of a range of circulating small amines showed that the levels of the majority of these compounds were significantly decreased in all three groups of infected organisms. Ten of these metabolites were common between the three different organisms comprising: methionine, asparagine, cysteine, threonine, serine, tryptophan, leucine, citrulline, ethanolamine and phenylalanine. The metabolomic changes of zebrafish larvae after infection were confirmed by nuclear magnetic resonance spectroscopy. Our study identified common biomarkers for tuberculosis disease in humans, mice and zebrafish, showing across species conservation of metabolic reprogramming processes as a result of disease. Apparently, the mechanisms underlying these processes are independent of environmental, developmental and vertebrate evolutionary factors. The zebrafish larval model is highly suited to further investigate the mechanism of metabolic reprogramming and the connection with wasting syndrome due to infection by mycobacteria.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia. The major AF susceptibility locus 4q25 establishes long-range interactions with the promoter of PITX2 , a transcription factor gene with critical functions during cardiac development. While many AF-linked loci have been identified in genome-wide association studies, mechanistic understanding into how genetic variants, including those at the 4q25 locus, increase vulnerability to AF is mostly lacking. Here, we show that loss of pitx2c in zebrafish leads to adult cardiac phenotypes with substantial similarities to pathologies observed in AF patients, including arrhythmia, atrial conduction defects, sarcomere disassembly, and altered cardiac metabolism. These phenotypes are also observed in a subset of pitx2c +/- fish, mimicking the situation in humans. Most notably, the onset of these phenotypes occurs at an early developmental stage. Detailed analyses of pitx2c loss- and gain-of-function embryonic hearts reveal changes in sarcomeric and metabolic gene expression and function that precede the onset of cardiac arrhythmia first observed at larval stages. We further find that antioxidant treatment of pitx2c -/- larvae significantly reduces the incidence and severity of cardiac arrhythmia, suggesting that metabolic dysfunction is an important driver of conduction defects. We propose that these early sarcomere and metabolic defects alter cardiac function and contribute to the electrical instability and structural remodeling observed in adult fish. Overall, these data provide insight into the mechanisms underlying the development and pathophysiology of some cardiac arrhythmias and importantly, increase our understanding of how developmental perturbations can predispose to functional defects in the adult heart., Competing Interests: The authors declare no competing interest.

Background: The function of Toll-like receptor 2 (TLR2) in host defense against pathogens, especially Mycobacterium tuberculosis (Mtb) is poorly understood. To investigate the role of TLR2 during mycobacterial infection, we analyzed the response of tlr2 zebrafish mutant larvae to infection with Mycobacterium marinum (Mm), a close relative to Mtb, as a model for tuberculosis. We measured infection phenotypes and transcriptome responses using RNA deep sequencing in mutant and control larvae., Results: tlr2 mutant embryos at 2 dpf do not show differences in numbers of macrophages and neutrophils compared to control embryos. However, we found substantial changes in gene expression in these mutants, particularly in metabolic pathways, when compared with the heterozygote tlr2 +/- control. At 4 days after Mm infection, the total bacterial burden and the presence of extracellular bacteria were higher in tlr2 -/- larvae than in tlr2 +/- , or tlr2 +/+ larvae, whereas granuloma numbers were reduced, showing a function of Tlr2 in zebrafish host defense. RNAseq analysis of infected tlr2 -/- versus tlr2 +/- shows that the number of up-regulated and down-regulated genes in response to infection was greatly diminished in tlr2 mutants by at least 2 fold and 10 fold, respectively. Analysis of the transcriptome data and qPCR validation shows that Mm infection of tlr2 mutants leads to decreased mRNA levels of genes involved in inflammation and immune responses, including il1b, tnfb, cxcl11aa/ac, fosl1a, and cebpb. Furthermore, RNAseq analyses revealed that the expression of genes for Maf family transcription factors, vitamin D receptors, and Dicps proteins is altered in tlr2 mutants with or without infection. In addition, the data indicate a function of Tlr2 in the control of induction of cytokines and chemokines, such as the CXCR3-CXCL11 signaling axis., Conclusion: The transcriptome and infection burden analyses show a function of Tlr2 as a protective factor against mycobacteria. Transcriptome analysis revealed tlr2-specific pathways involved in Mm infection, which are related to responses to Mtb infection in human macrophages. Considering its dominant function in control of transcriptional processes that govern defense responses and metabolism, the TLR2 protein can be expected to be also of importance for other infectious diseases and interactions with the microbiome.

Defective coronary network function and insufficient blood supply are both cause and consequence of myocardial infarction. Efficient revascularization after infarction is essential to support tissue repair and function. Zebrafish hearts exhibit a remarkable ability to regenerate, and coronary revascularization initiates within hours of injury, but how this process is regulated remains unknown. Here, we show that revascularization requires a coordinated multi-tissue response culminating with the formation of a complex vascular network available as a scaffold for cardiomyocyte repopulation. During a process we term "coronary-endocardial anchoring," new coronaries respond by sprouting (1) superficially within the regenerating epicardium and (2) intra-ventricularly toward the activated endocardium. Mechanistically, superficial revascularization is guided by epicardial Cxcl12-Cxcr4 signaling and intra-ventricular sprouting by endocardial Vegfa signaling. Our findings indicate that the injury-activated epicardium and endocardium support cardiomyocyte replenishment initially through the guidance of coronary sprouting. Simulating this process in the injured mammalian heart should help its healing., (Copyright © 2019 Elsevier Inc. All rights reserved.)

In recent years, there has been increasing interest in the role of lymphatics in organ repair and regeneration, due to their importance in immune surveillance and fluid homeostasis. Experimental approaches aimed at boosting lymphangiogenesis following myocardial infarction in mice, were shown to promote healing of the heart. Yet, the mechanisms governing cardiac lymphatic growth remain unclear. Here, we identify two distinct lymphatic populations in the hearts of zebrafish and mouse, one that forms through sprouting lymphangiogenesis, and the other by coalescence of isolated lymphatic cells. By tracing the development of each subset, we reveal diverse cellular origins and differential response to signaling cues. Finally, we show that lymphatic vessels are required for cardiac regeneration in zebrafish as mutants lacking lymphatics display severely impaired regeneration capabilities. Overall, our results provide novel insight into the mechanisms underlying lymphatic formation during development and regeneration, opening new avenues for interventions targeting specific lymphatic populations., Competing Interests: DG, BR, GP, RM, JS, RM, RK, HR, NM, YA, OG, KP, KR, KY No competing interests declared, DS Senior editor, eLife, (© 2019, Gancz et al.)

Immediately after cardiac injury, the immune system plays major roles in repair and regeneration as it becomes involved in a number of processes including damage-associated signaling, inflammation, revascularization, cardiomyocyte dedifferentiation and replenishment, and fibrotic scar formation/resolution. Recent studies have revealed that different immune responses occur in the various experimental models capable or incapable of cardiac regeneration, and that harnessing these immune responses might improve cardiac repair. In light of this concept, this review analyzes current knowledge about the immune responses to cardiac injury from a comparative perspective. Insights gained from such comparative analyses may provide ways to modulate the immune response as a potential therapeutic strategy for cardiac disease.

Many eukaryotic genes play essential roles in multiple biological processes in several different tissues. Conditional mutants are needed to analyze genes with such pleiotropic functions. In vertebrates, conditional gene inactivation has only been feasible in the mouse, leaving other model systems to rely on surrogate experimental approaches such as overexpression of dominant negative proteins and antisense-based tools. Here, we have developed a simple and straightforward method to integrate loxP sequences at specific sites in the zebrafish genome using the CRISPR/Cas9 technology and oligonucleotide templates for homology directed repair. We engineered conditional (floxed) mutants of tbx20 and fleer, and demonstrate excision of exons flanked by loxP sites using tamoxifen-inducible CreERT2 recombinase. To demonstrate broad applicability of our method, we also integrated loxP sites into two additional genes, aldh1a2 and tcf21. The ease of this approach will further expand the use of zebrafish to study various aspects of vertebrate biology, especially post-embryonic processes such as regeneration., Competing Interests: The authors have declared that no competing interests exist.

Muscle proteins with ankyrin repeats (MARPs) ANKRD1 and ANKRD2 are titin-associated proteins with a putative role as transcriptional co-regulators in striated muscle, involved in the cellular response to mechanical, oxidative and metabolic stress. Since many aspects of the biology of MARPs, particularly exact mechanisms of their action, in striated muscle are still elusive, research in this field will benefit from novel animal model system. Here we investigated the MARPs found in zebrafish for protein structure, evolutionary conservation, spatiotemporal expression profiles and response to increased muscle activity. Ankrd1 and Ankrd2 show overall moderate conservation at the protein level, more pronounced in the region of ankyrin repeats, motifs indispensable for their function. The two zebrafish genes, ankrd1a and ankrd1b, counterparts of mammalian ANKRD1/Ankrd1, have different expression profiles during first seven days of development. Mild increase of ankrd1a transcript levels was detected at 72 hpf (1.74±0.24 fold increase relative to 24 hpf time point), while ankrd1b expression was markedly upregulated from 24 hpf onward and peaked at 72 hpf (92.18±36.95 fold increase relative to 24 hpf time point). Spatially, they exhibited non-overlapping expression patterns during skeletal muscle development in trunk (ankrd1a) and tail (ankrd1b) somites. Expression of ankrd2 was barely detectable. Zebrafish MARPs, expressed at a relatively low level in adult striated muscle, were found to be responsive to endurance exercise training consisting of two bouts of 3 hours of forced swimming daily, for five consecutive days. Three hours after the last exercise bout, ankrd1a expression increased in cardiac muscle (6.19±5.05 fold change), while ankrd1b and ankrd2 were upregulated in skeletal muscle (1.97±1.05 and 1.84±0.58 fold change, respectively). This study provides the foundation to establish zebrafish as a novel in vivo model for further investigation of MARPs function in striated muscle., Competing Interests: The authors have declared that no competing interests exist.

Formation of the lymphatic system requires the coordinated expression of several key regulators: vascular endothelial growth factor C (VEGFC), its receptor FLT4, and a key transcriptional effector, PROX1. Yet, how expression of these signaling components is regulated remains poorly understood. Here, using a combination of genetic and molecular approaches, we identify the transcription factor hematopoietically expressed homeobox (HHEX) as an upstream regulator of VEGFC, FLT4, and PROX1 during angiogenic sprouting and lymphatic formation in vertebrates. By analyzing zebrafish mutants, we found that hhex is necessary for sprouting angiogenesis from the posterior cardinal vein, a process required for lymphangiogenesis. Furthermore, studies of mammalian HHEX using tissue-specific genetic deletions in mouse and knockdowns in cultured human endothelial cells reveal its highly conserved function during vascular and lymphatic development. Our findings that HHEX is essential for the regulation of the VEGFC/FLT4/PROX1 axis provide insights into the molecular regulation of lymphangiogenesis.

Zebrafish display a distinct ability to regenerate their heart following injury. However, this ability is not shared by another teleost, the medaka. In order to identify cellular and molecular bases for this difference, we performed comparative transcriptomic analyses following cardiac cryoinjury. This comparison points to major differences in immune cell dynamics between these models. Upon closer examination, we observed delayed and reduced macrophage recruitment in medaka, along with delayed neutrophil clearance. To investigate the role of immune responses in cardiac regeneration, we delayed macrophage recruitment in zebrafish and observed compromised neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. In contrast, stimulating Toll-like receptor signaling in medaka enhanced immune cell dynamics and promoted neovascularization, neutrophil clearance, cardiomyocyte proliferation and scar resolution. Altogether, these data provide further insight into the complex role of the immune response during regeneration, and serve as a platform to identify and test additional regulators of cardiac repair.

Macrophages are known to interact with endothelial cells during developmental and pathological angiogenesis but the molecular mechanisms modulating these interactions remain unclear. Here, we show a role for the Hif-1α transcription factor in this cellular communication. We generated hif-1aa;hif-1ab double mutants in zebrafish, hereafter referred to as hif-1α mutants, and find that they exhibit impaired macrophage mobilization from the aorta-gonad-mesonephros (AGM) region as well as angiogenic defects and defective vascular repair. Importantly, macrophage ablation is sufficient to recapitulate the vascular phenotypes observed in hif-1α mutants, revealing for the first time a macrophage-dependent angiogenic process during development. Further substantiating our observations of vascular repair, we find that most macrophages closely associated with ruptured blood vessels are Tnfα-positive, a key feature of classically activated macrophages. Altogether, our data provide genetic evidence that Hif-1α regulates interactions between macrophages and endothelial cells starting with the mobilization of macrophages from the AGM.

Zebrafish have a remarkable capacity to regenerate their heart. Efficient replenishment of lost tissues requires the activation of different cell types including the epicardium and endocardium. A complex set of processes is subsequently needed to support cardiomyocyte repopulation. Previous studies have identified important determinants of heart regeneration; however, to date, how revascularization of the damaged area happens remains unknown. Here, we show that angiogenic sprouting into the injured area starts as early as 15 h after injury. To analyze the role of vegfaa in heart regeneration, we used vegfaa mutants rescued to adulthood by vegfaa mRNA injections at the one-cell stage. Surprisingly, vegfaa mutants develop coronaries and revascularize after injury. As a possible explanation for these observations, we find that vegfaa mutant hearts up-regulate the expression of potentially compensating genes. Therefore, to overcome the lack of a revascularization phenotype in vegfaa mutants, we generated fish expressing inducible dominant negative Vegfaa. These fish displayed minimal revascularization of the damaged area. In the absence of fast angiogenic revascularization, cardiomyocyte proliferation did not occur, and the heart failed to regenerate, retaining a fibrotic scar. Hence, our data show that a fast endothelial invasion allows efficient revascularization of the injured area, which is necessary to support replenishment of new tissue and achieve efficient heart regeneration. These findings revisit the model where neovascularization is considered to happen concomitant with the formation of new muscle. Our work also paves the way for future studies designed to understand the molecular mechanisms that regulate fast revascularization., Competing Interests: The authors declare no conflict of interest.

Background: Although the responses to many pathogen associated molecular patterns (PAMPs) in cell cultures and extracted organs are well characterized, there is little known of transcriptome responses to PAMPs in whole organisms. To characterize this in detail, we have performed RNAseq analysis of responses of zebrafish embryos to injection of PAMPs in the caudal vein at one hour after exposure. We have compared two ligands that in mammals have been shown to specifically activate the TLR2 and TLR5 receptors: Pam3CSK4 and flagellin, respectively., Results: We identified a group of 80 common genes that respond with high stringency selection to stimulations with both PAMPs, which included several well-known immune marker genes such as il1b and tnfa. Surprisingly, we also identified sets of 48 and 42 genes that specifically respond to either Pam3CSK4 or flagellin, respectively, after a comparative filtering approach. Remarkably, in the Pam3CSK4 specific set, there was a set of transcription factors with more than 2 fold-change, as confirmed by qPCR analyses, including cebpb, fosb, nr4a1 and egr3. We also showed that the regulation of the Pam3CSK4 and flagellin specifically responding sets is inhibited by knockdown of tlr2 or tlr5, respectively., Conclusions: Our studies show that Pam3CSK4 and flagellin can stimulate the Tlr2 and Tlr5 signaling pathways leading to common and specific responses in the zebrafish embryo system.

Glucose transporter 2 (GLUT2; gene name SLC2A2) has a key role in the regulation of glucose dynamics in organs central to metabolism. Although GLUT2 has been studied in the context of its participation in peripheral and central glucose sensing, its role in the brain is not well understood. To decipher the role of GLUT2 in brain development, we knocked down slc2a2 (glut2), the functional ortholog of human GLUT2, in zebrafish. Abrogation of glut2 led to defective brain organogenesis, reduced glucose uptake and increased programmed cell death in the brain. Coinciding with the observed localization of glut2 expression in the zebrafish hindbrain, glut2 deficiency affected the development of neural progenitor cells expressing the proneural genes atoh1b and ptf1a but not those expressing neurod. Specificity of the morphant phenotype was demonstrated by the restoration of brain organogenesis, whole-embryo glucose uptake, brain apoptosis, and expression of proneural markers in rescue experiments. These results indicate that glut2 has an essential role during brain development by facilitating the uptake and availability of glucose and support the involvement of glut2 in brain glucose sensing.

Cardiomyopathies-associated metabolic pathologies (e.g., type 2 diabetes and insulin resistance) are a leading cause of mortality. It is known that the association between these pathologies works in both directions, for which heart failure can lead to metabolic derangements such as insulin resistance. This intricate crosstalk exemplifies the importance of a fine coordination between one of the most energy-demanding organs and an equilibrated carbohydrate metabolism. In this light, to assist in the understanding of the role of insulin-regulated glucose transporters (GLUTs) and the development of cardiomyopathies, we have developed a model for glut12 deficiency in zebrafish. GLUT12 is a novel insulin-regulated GLUT expressed in the main insulin-sensitive tissues, such as cardiac muscle, skeletal muscle, and adipose tissue. In this study, we show that glut12 knockdown impacts the development of the embryonic heart resulting in abnormal valve formation. Moreover, glut12-deficient embryos also exhibited poor glycemic control. Glucose measurements showed that these larvae were hyperglycemic and resistant to insulin administration. Transcriptome analysis demonstrated that a number of genes known to be important in cardiac development and function as well as metabolic mediators were dysregulated in these larvae. These results indicate that glut12 is an essential GLUT in the heart where the reduction in glucose uptake due to glut12 deficiency leads to heart failure presumably due to the lack of glucose as energy substrate. In addition, the diabetic phenotype displayed by these larvae after glut12 abrogation highlights the importance of this GLUT during early developmental stages., (© 2015 Society for Endocrinology.)

Type 2 diabetes, obesity, and metabolic syndrome are pathologies where insulin resistance plays a central role, and that affect a large population worldwide. These pathologies are usually associated with a dysregulation of insulin secretion leading to a chronic exposure of the tissues to high insulin levels (i.e. hyperinsulinemia), which diminishes the concentration of key downstream elements, causing insulin resistance. The complexity of the study of insulin resistance arises from the heterogeneity of the metabolic states where it is observed. To contribute to the understanding of the mechanisms triggering insulin resistance, we have developed a zebrafish model to study insulin metabolism and its associated disorders. Zebrafish larvae appeared to be sensitive to human recombinant insulin, becoming insulin-resistant when exposed to a high dose of the hormone. Moreover RNA-seq-based transcriptomic profiling of these larvae revealed a strong downregulation of a number of immune-relevant genes as a consequence of the exposure to hyperinsulinemia. Interestingly, as an exception, the negative immune modulator protein tyrosine phosphatase nonreceptor type 6 (ptpn6) appeared to be upregulated in insulin-resistant larvae. Knockdown of ptpn6 was found to counteract the observed downregulation of the immune system and insulin signaling pathway caused by hyperinsulinemia. These results indicate that ptpn6 is a mediator of the metabolic switch between insulin-sensitive and insulin-resistant states. Our zebrafish model for hyperinsulinemia has therefore demonstrated its suitability for discovery of novel regulators of insulin resistance. In addition, our data will be very useful in further studies of the function of immunological determinants in a non-obese model system., (© 2014 Society for Endocrinology.)

Zebrafish are becoming a valuable tool in the preclinical phase of drug discovery screenings as a whole animal model with high throughput screening possibilities. They can be used to bridge the gap between cell based assays at earlier stages and in vivo validation in mammalian models, reducing, in this way, the number of compounds passing through to testing on the much more expensive rodent models. In this light, in the present manuscript is described a new high throughput pipeline using zebrafish as in vivo model system for the study of Staphylococcus epidermidis and Mycobacterium marinum infection. This setup allows the generation and analysis of large number of synchronous embryos homogenously infected. Moreover the flexibility of the pipeline allows the user to easily implement other platforms to improve the resolution of the analysis when needed. The combination of the zebrafish together with innovative high throughput technologies opens the field of drug testing and discovery to new possibilities not only because of the strength of using a whole animal model but also because of the large number of transgenic lines available that can be used to decipher the mode of action of new compounds.

[This corrects the article DOI: 10.1371/journal.pone.0080628.].

The glucose transporter 4 (GLUT4) plays a key role in glucose uptake in insulin target tissues. This transporter has been extensively studied in many species in terms of its function, expression and cellular traffic and complex mechanisms are involved in its regulation at many different levels. However, studies investigating the transcription of the GLUT4 gene and its regulation are scarce. In this study, we have identified the GLUT4 gene in a teleost fish, the Fugu (Takifugu rubripes), and have cloned and characterized a functional promoter of this gene for the first time in a non-mammalian vertebrate. In silico analysis of the Fugu GLUT4 promoter identified potential binding sites for transcription factors such as SP1, C/EBP, MEF2, KLF, SREBP-1c and GC-boxes, as well as a CpG island, but failed to identify a TATA box. In vitro analysis revealed three transcription start sites, with the main residing 307 bp upstream of the ATG codon. Deletion analysis determined that the core promoter was located between nucleotides -132/+94. By transfecting a variety of 5´deletion constructs into L6 muscle cells we have determined that Fugu GLUT4 promoter transcription is regulated by insulin, PG-J2, a PPARγ agonist, and electrical pulse stimulation. Furthermore, our results suggest the implication of motifs such as PPARγ/RXR and HIF-1α in the regulation of Fugu GLUT4 promoter activity by PPARγ and contractile activity, respectively. These data suggest that the characteristics and regulation of the GLUT4 promoter have been remarkably conserved during the evolution from fish to mammals, further evidencing the important role of GLUT4 in metabolic regulation in vertebrates.

The increasing use of zebrafish larvae for biomedical research applications is resulting in versatile models for a variety of human diseases. These models exploit the optical transparency of zebrafish larvae and the availability of a large genetic tool box. Here we present detailed protocols for the robotic injection of zebrafish embryos at very high accuracy with a speed of up to 2000 embryos per hour. These protocols are benchmarked for several applications: (1) the injection of DNA for obtaining transgenic animals, (2) the injection of antisense morpholinos that can be used for gene knock-down, (3) the injection of microbes for studying infectious disease, and (4) the injection of human cancer cells as a model for tumor progression. We show examples of how the injected embryos can be screened at high-throughput level using fluorescence analysis. Our methods open up new avenues for the use of zebrafish larvae for large compound screens in the search for new medicines., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Interleukin-6 (IL-6) has been identified and characterized from several fish species and its mRNA expression is induced by pathogen-associated molecular patterns (PAMPs) and cytokines in immune cells and tissues. However, the transcriptional regulation of the IL-6 gene in fish is not well understood. In the present study, we have cloned and sequenced a 1028 bp 5'-flanking DNA region from the IL-6 gene in seabream (Sparus aurata). Sequence analysis of the seabream IL-6 promoter (sbIL-6P) evidenced the presence of a conserved TATA motif and conserved response elements for NF-κB, C/EBPβ (NF-IL6), AP-1 and GRE, similar to other vertebrate IL-6 promoters. Functional characterization of sbIL-6P was performed by cloning sbIL-6P into a luciferase expression vector and by transfecting it into L6 muscle cells, a mammalian cell line shown previously to express IL-6 in response to pro-inflammatory stimuli. We show here that the activity of sbIL-6P was significantly induced by pro-inflammatory cytokines such as tumor necrosis factor alpha (TNFα), IL-6 and IL-2, as well as by lipopolysaccharide (LPS), but significantly repressed by dexamethasone. In addition, the stimulatory effects of TNFα on sbIL-6P activity appeared to be mediated by the NF-κB, p38 MAPK and JNK signaling pathways. Deletion analyses of sbIL-6P suggested that activation of sbIL-6P by TNFα and IL-6 required the presence of binding motifs present in the proximal promoter (-171 to -84) whereas activation by IL-2 required binding motifs present in the distal promoter (-1024 to -864). The results from this study indicate, for the first time in fish, that pro-inflammatory cytokines, LPS and glucocorticoids can regulate the activity of the IL-6 gene at a transcriptional level and identify important regions in its response to cytokines., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

Spermatogenesis is a complex process where hormonal signals regulate the interaction of different cell types in a tight spatial and temporal fashion. The Senegalese sole (Solea senegalensis) is a marine flatfish that, in contrast to many fish, exhibits a semi-cystic, asynchronous pattern of spermatogenesis progression. This pattern is characterized by the release of spermatids into the tubule lumen, where they transform into spermatozoa. In this study, we used laser capture microdissection (LCM) to isolate cells from cysts containing spermatogonia, spermatocytes, spermatids or spermatozoa in order to investigate developmental patterns of gene expression. Furthermore, we also analyzed the stage-specific expression of the same set of genes throughout spermatogenesis (early-mid, late and maturing spermatogenic stages) in tissue fragments of the Senegalese sole testis. Genes analyzed by absolute qPCR in cysts isolated by LCM and stage-specific testis samples included genes involved in steroid synthesis and action (3β-hsd, 17β-hsd, 20β-hsd, star, star-like, progesterone receptor), gonadotropin action (fshr, lhr), the kisspeptin system (kiss2, kiss2r) and other genes important for the production of mature gametes (zona pellucida 2.2, claudin and clusterin). Our results show that, in general, steroidogenesis-related genes tended to increase with spermatogenesis progression and that 3β-hsd and 20β-hsd were expressed in germ cells but 17β-hsd was not. Our results also show that fshr is expressed in most testicular cell types, including germ cells. In contrast, lhr is expressed only in late spermatogenesis and is not expressed in any of the germ cell types examined, indicating that, in contrast to fshr, lhr may be primarily expressed in non-germinal cells (e.g. Leydig cells). Furthermore, kisspeptin and its receptor were expressed in all germ cell types examined and, as expected, gamete maturation-related genes were more expressed in mature stages. These results illustrate that key factors that participate in the hormonal regulation of spermatogenesis in the Senegalese sole testis show complex cell type- and stage-specific patterns of gene expression., (Copyright © 2013 Elsevier Inc. All rights reserved.)

The Senegalese sole (Solea senegalensis) is a marine flatfish of high economic value and a target species for aquaculture. The efforts to reproduce this species in captivity have been hampered by the fact that farmed males (F1) often show lower sperm production and fertilization capacity than wild-type males (F0). Our knowledge on spermatogenesis is however limited to a few studies. In a previous work, we identified by 2-D DIGE several potential protein markers in testis for the poor reproductive performance of F1 males. Therefore, the objectives of the present study were, first, to investigate changes in genes and proteins expressed in the testis throughout spermatogenesis in F0 males by using a combination of transcriptomic and proteomic approaches and, second, to further compare the testis proteome between late spermatogenic stages of F0 and F1 fish to identify potential indicators of hampered reproductive performance in F1 fish. We identified approximately 400 genes and 49 proteins that are differentially expressed during the progression of spermatogenesis and that participate in processes such as transcriptional activation, the ubiquitin-proteasome system, sperm maturation and motility or cytoskeletal remodeling. Interestingly, a number of these proteins differed in abundance between F0 and F1 fish, pointing toward alterations in cytoskeleton, sperm motility, the ubiquitin-proteasome system and the redox state during spermiogenesis as possible causes for the decreased fertility of F1 fish., (Copyright © 2011 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In male teleosts, testicular steroids are essential hormones for the regulation of spermatogenesis and their production is regulated by pituitary gonadotropins. In the Senegalese sole (Solea senegalensis), an economically important flatfish with semi-cystic and asynchronous spermatogenesis, the gonadotropic regulation of spermatogenesis, particularly regarding the production and regulation of testicular steroids, are not well understood. For this reason, we first cloned and characterized the response of several key genes for the production and action of testicular steroids to the in vivo administration of human chorionic gonadotropin (hCG) and, second, we investigated the transcriptomic effects of hCG in the Senegalese sole testis. We succeeded in cloning the full-length cDNAs for Steroidogenic Acute Regulatory protein (StAR), 3β-hydroxysteroid dehydrogenase (3β-HSD), 17β-HSD and 20β-HSD and a partial cDNA for the nuclear progesterone receptor. In this study we also identified a transcript encoding a protein with homology to StAR, which we named StAR-like, that could represent a new member of the StAR-related lipid transfer (START) family. All the cloned genes were expressed in the testis and their expression levels were significantly increased by the in vivo administration of hCG. The plasma levels of testosterone and 11-ketotestosterone also increased in response to hCG administration, likely as a result of the induction of the expression of steroidogenic enzymes by hCG. Furthermore, gene expression analysis by microarray identified 90 differentially expressed genes in the testis in response to hCG administration, including genes potentially involved in steroidogenesis, progression of spermatogenesis and germ cell maturation and cytoskeletal organization. Our results have identified for the first time a number of key genes involved in the regulation of steroid production and spermatogenesis in the Senegalese sole testis that are under gonadotropic control., (Copyright © 2011 Elsevier Inc. All rights reserved.)