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Clustered regularly interspaced palindromic repeats (CRISPR)/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio) to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible., Summary: A new CRISPR/Cas9-based method of fluorescent reporter technology in zebrafish to confirm loss of gene function in this model.

The exocrine pancreas displays a significant capacity for regeneration and renewal. In humans and mammalian model systems, the partial loss of exocrine tissue, such as after acute pancreatitis or partial pancreatectomy induces rapid recovery via expansion of surviving acinar cells. In mouse it was further found that an almost complete removal of acinar cells initiates regeneration from a currently not well-defined progenitor pool. Here, we used the zebrafish as an alternative model to study cellular mechanisms of exocrine regeneration following an almost complete removal of acinar cells. We introduced and validated two novel transgenic approaches for genetically encoded conditional cell ablation in the zebrafish, either by caspase-8-induced apoptosis or by rendering cells sensitive to diphtheria toxin. By using the ela3l promoter for exocrine-specific expression, we show that both approaches allowed cell-type-specific removal of >95% of acinar tissue in larval and adult zebrafish without causing any signs of unspecific side effects. We find that zebrafish larvae are able to recover from a virtually complete acinar tissue ablation within 2 weeks. Using short-term lineage-tracing experiments and EdU incorporation assays, we exclude duct-associated Notch-responsive cells as the source of regeneration. Rather, a rare population of slowly dividing ela3l-negative cells expressing ptf1a and CPA was identified as the origin of the newly forming exocrine cells. Cells are actively maintained, as revealed by a constant number of these cells at different larval stages and after repeated cell ablation. These cells establish ela3l expression about 4-6 days after ablation without signs of increased proliferation in between. With onset of ela3l expression, cells initiate rapid proliferation, leading to fast expansion of the ela3l-positive population. Finally, we show that this proliferation is blocked by overexpression of the Wnt-signaling antagonist dkk1b. In conclusion, we show a conserved requirement for Wnt signaling in exocrine tissue expansion and reveal a potential novel progenitor or stem cell population as a source for exocrine neogenesis after complete loss of acinar cells., Summary: New transgenic models for exocrine pancreas regeneration in zebrafish reveal a novel potential exocrine progenitor as the source for a two-phase regeneration process after complete acinar cell loss.

The specific roles of the two major innate immune cell types – neutrophils and macrophages – in response to infection and sterile inflammation are areas of great interest. The larval zebrafish model of innate immunity, and the imaging capabilities it provides, is a source of new research and discoveries in this field. Multiple methods have been developed in larval zebrafish to specifically deplete functional macrophages or neutrophils. Each of these has pros and cons, as well as caveats, that often make it difficult to directly compare results from different studies. The purpose of this Review is to (1) explore the pros, cons and caveats of each of these immune cell-depleted models; (2) highlight and place into a broader context recent key findings on the specific functions of innate immune cells using these models; and (3) explore future directions in which immune cell depletion methods are being expanded., Summary: Macrophages and neutrophils are distinct innate immune cells with diverse roles in diverse inflammatory contexts. Recent research in larval zebrafish using cell-specific depletion methods has revealed new insights into these cells' functions.

Induction of endoplasmic reticulum (ER) stress is associated with diverse developmental and degenerative diseases. Modified ER homeostasis causes activation of conserved stress pathways at the ER called the unfolded protein response (UPR). ATF6 is a transcription factor activated during ER stress as part of a coordinated UPR. ATF6 resides at the ER and, upon activation, is transported to the Golgi apparatus, where it is cleaved by proteases to create an amino-terminal cytoplasmic fragment (ATF6f). ATF6f translocates to the nucleus to activate transcriptional targets. Here, we describe the establishment and validation of zebrafish reporter lines for ATF6 activity. These transgenic lines are based on a defined and multimerized ATF6 consensus site, which drives either eGFP or destabilized eGFP, enabling dynamic study of ATF6 activity during development and disease. The results show that the reporter is specific for the ATF6 pathway, active during development and induced in disease models known to engage UPR. Specifically, during development, ATF6 activity is highest in the lens, skeletal muscle, fins and gills. The reporter is also activated by common chemical inducers of ER stress, including tunicamycin, thapsigargin and brefeldin A, as well as by heat shock. In models for amyotrophic lateral sclerosis and cone dystrophy, ATF6 reporter expression is induced in spinal cord interneurons or photoreceptors, respectively, suggesting a role for ATF6 response in multiple neurodegenerative diseases. Collectively our results show that these ATF6 reporters can be used to monitor ATF6 activity changes throughout development and in zebrafish models of disease. This article has an associated First Person interview with the first author of the paper., Summary: In this study, we validate transgenic zebrafish generated to specifically report the activity of ATF6, representing a major branch of the endoplasmic reticulum stress pathway with functions in development and disease.

Maladaptive signaling by pro-inflammatory cytokines (PICs), such as TNFα, IL1β and IFNɣ, can activate downstream signaling cascades that are implicated in the development and progression of multiple inflammatory diseases. Despite playing critical roles in pathogenesis, the availability of in vivo models in which to model tissue-specific induction of PICs is limited. To bridge this gap, we have developed a novel multi-gene expression system dubbed Cre-enabled and tetracycline-inducible transgenic system for conditional, tissue-specific expression of pro-inflammatory cytokines (CETI-PIC3). This binary transgenic system permits the stoichiometric co-expression of proteins Tumor necrosis factor a (Tnfa), Interleukin-1 beta (Il1b) and Interferon gamma (Ifng1), and H2B-GFP fluorescent reporter in a dose-dependent manner. Furthermore, cytokine misexpression is enabled only in tissue domains that can be defined by Cre recombinase expression. We have validated this system in zebrafish using an insulin:cre line. In doubly transgenic fish, quantitative real-time polymerase chain reaction demonstrated increased expression levels of tnfa, il1b and ifng1 mRNA. Moreover, specific expression in pancreatic β cells was demonstrated by both Tnfa immunofluorescence and GFP fluorescence. Cytokine-overexpressing islets elicited specific responses: β cells exhibited increased expression of genes associated with reactive oxidative species-mediated stress and endoplasmic reticulum stress, surveilling and infiltrating macrophages were increased, and β cell death was promoted. This powerful and versatile model system can be used for modeling, analysis and therapy development of diseases with an underlying inflammatory etiology. This article has an associated First Person interview with the first author of the paper., Summary: Here, we report a novel transgenic zebrafish system that permits tissue-specific, inducible and tunable expression of pro-inflammatory cytokines for modeling inflammatory diseases.

Mutations in the gene AK2 are responsible for reticular dysgenesis (RD), a rare and severe form of primary immunodeficiency in children. RD patients have a severely shortened life expectancy and without treatment die, generally from sepsis soon after birth. The only available therapeutic option for RD is hematopoietic stem cell transplantation (HSCT). To gain insight into the pathophysiology of RD, we previously created zebrafish models for Ak2 deficiencies. One of the clinical features of RD is hearing loss, but its pathophysiology and causes have not been determined. In adult mammals, sensory hair cells of the inner ear do not regenerate; however, their regeneration has been observed in several non-mammalian vertebrates, including zebrafish. Therefore, we used our RD zebrafish models to determine whether Ak2 deficiency affects sensory organ development and/or hair cell regeneration. Our studies indicated that Ak2 is required for the correct development, survival and regeneration of sensory hair cells. Interestingly, Ak2 deficiency induces the expression of several oxidative stress markers and it triggers an increased level of cell death in the hair cells. Finally, we show that glutathione treatment can partially rescue hair cell development in the sensory organs in our RD models, pointing to the potential use of antioxidants as a therapeutic treatment supplementing HSCT to prevent or ameliorate sensorineural hearing deficits in RD patients., Summary: A zebrafish model of reticular dysgenesis reveals hair cell developmental deficits that can be partially rescued by antioxidants, pointing to their potential use as a therapeutic treatment for reticular dysgenesis patients.

The mariner (myo7aa−/−) mutant is a zebrafish model for Usher syndrome type 1 (USH1). To further characterize hair cell synaptic elements in myo7aa−/− mutants, we focused on the ribbon synapse and evaluated ultrastructure, number and distribution of immunolabeled ribbons, and postsynaptic densities. By transmission electron microscopy, we determined that myo7aa−/− zebrafish have fewer glutamatergic vesicles tethered to ribbon synapses, yet maintain a comparable ribbon area. In myo7aa−/− hair cells, immunolocalization of Ctbp2 showed fewer ribbon-containing cells in total and an altered distribution of Ctbp2 puncta compared to wild-type hair cells. myo7aa−/− mutants have fewer postsynaptic densities – as assessed by MAGUK immunolabeling – compared to wild-type zebrafish. We quantified the circular swimming behavior of myo7aa−/− mutant fish and measured a greater turning angle (absolute smooth orientation). It has previously been shown that L-type voltage-gated calcium channels are necessary for ribbon localization and occurrence of postsynaptic density; thus, we hypothesized and observed that L-type voltage-gated calcium channel agonists change behavioral and synaptic phenotypes in myo7aa−/− mutants in a drug-specific manner. Our results indicate that treatment with L-type voltage-gated calcium channel agonists alter hair cell synaptic elements and improve behavioral phenotypes of myo7aa−/− mutants. Our data support that L-type voltage-gated calcium channel agonists induce morphological changes at the ribbon synapse – in both the number of tethered vesicles and regarding the distribution of Ctbp2 puncta – shift swimming behavior and improve acoustic startle response., Summary: We quantified behavioral and synaptic morphology differences between wild-type zebrafish larvae and the mariner (myo7aa−/−) mutant, finding that these differences can be modified by L-type voltage-gated calcium channel agonists.

Dreissenids are established model species for ecological and ecotoxicological studies, since they are sessile and filter feeder organisms and reflect in situ freshwater quality. Despite this strong interest for hydrosystem biomonitoring, omics data are still scarce. In the present study, we achieved full de novo assembly transcriptomes of digestive glands to gain insight into Dreissena polymorpha and D. rostriformis bugensis molecular knowledge. Transcriptomes were obtained by Illumina RNA sequencing of seventy-nine organisms issued from fifteen populations inhabiting sites that exhibits multiple freshwater contamination levels and different hydrosystem topographies (open or closed systems). Based on a recent de novo assembly algorithm, we carried out a complete, quality-checked and annotated transcriptomes. The power of the present study lies in the completeness of transcriptomes gathering multipopulational organisms sequencing and its full availability through an open access interface that gives a friendly and ready-to-use access to data. The use of such data for proteogenomic and targeted biological pathway investigations purpose is promising as they are first full transcriptomes for this two Dreissena species., Measurement(s)transcriptomeTechnology Type(s)RNA sequencingFactor Type(s)sampling locationSample Characteristic - OrganismDreissena polymorpha • Dreissena rostriformis bugensisSample Characteristic - Environmentfreshwater biomeSample Characteristic - LocationMetropolitan France Machine-accessible metadata file describing the reported data: 10.6084/m9.figshare.9934226

Toxoplasma gondii is an obligate intracellular parasite capable of invading any nucleated cell. Three main clonal lineages (type I, II, III) exist and murine models have driven the understanding of general and strain-specific immune mechanisms underlying Toxoplasma infection. However, murine models are limited for studying parasite-leukocyte interactions in vivo, and discrepancies exist between cellular immune responses observed in mouse versus human cells. Here, we developed a zebrafish infection model to study the innate immune response to Toxoplasma in vivo. By infecting the zebrafish hindbrain ventricle, and using high-resolution microscopy techniques coupled with computer vision-driven automated image analysis, we reveal that Toxoplasma invades brain cells and replicates inside a parasitophorous vacuole to which type I and III parasites recruit host cell mitochondria. We also show that type II and III strains maintain a higher infectious burden than type I strains. To understand how parasites are cleared in vivo, we further analyzed Toxoplasma-macrophage interactions using time-lapse microscopy and three-dimensional correlative light and electron microscopy (3D CLEM). Time-lapse microscopy revealed that macrophages are recruited to the infection site and play a key role in Toxoplasma control. High-resolution 3D CLEM revealed parasitophorous vacuole breakage in brain cells and macrophages in vivo, suggesting that cell-intrinsic mechanisms may be used to destroy the intracellular niche of tachyzoites. Together, our results demonstrate in vivo control of Toxoplasma by macrophages, and highlight the possibility that zebrafish may be further exploited as a novel model system for discoveries within the field of parasite immunity. This article has an associated First Person interview with the first author of the paper., Summary: We have established a novel zebrafish infection model to investigate Toxoplasma replication in vivo, and demonstrate a key role for macrophages in parasite control.

Tumors are frequently dependent on primary oncogenes to maintain their malignant properties (known as ‘oncogene addiction’). We have previously established several inducible hepatocellular carcinoma (HCC) models in zebrafish by transgenic expression of an oncogene. These tumor models are strongly oncogene addicted, as the induced and histologically proven liver tumors regress after suppression of oncogene expression by removal of a chemical inducer. However, the question of whether the liver tumor cells are eliminated or revert to normal cells remains unanswered. In the present study, we generated a novel Cre/loxP transgenic zebrafish line, Tg(fabp10: loxP-EGFP-stop-loxP-DsRed; TRE: CreERT2) (abbreviated to CreER), in order to trace tumor cell lineage during tumor regression after crossing with the xmrk (activated EGFR homolog) oncogene transgenic line, Tg(fabp10: rtTA; TRE: xmrk; krt4: EGFP). We found that, during HCC regression, restored normal liver contained both reverted tumor hepatocytes (RFP+) and newly differentiated hepatocytes (GFP+). RNA sequencing (RNA-seq) analyses of the RFP+ and GFP+ hepatocyte populations after tumor regression confirmed the conversion of tumor cells to normal hepatocytes, as most of the genes and pathways that were deregulated in the tumor stages were found to have normal regulation in the tumor-reverted hepatocytes. Thus, our lineage-tracing studies demonstrated the potential for transformed tumor cells to revert to normal cells after suppression of expression of a primary oncogene. This observation may provide a basis for the development of a therapeutic approach targeting addicted oncogenes or oncogenic pathways., Summary: In an oncogene-addicted liver cancer zebrafish model, transformed cancer hepatocytes could be reverted to normal hepatocytes, as shown by Cre-loxP-mediated cell-lineage tracing.

Although the zebrafish embryo is a powerful animal model of human heart failure, the methods routinely employed to monitor cardiac function produce rough approximations that are susceptible to bias and inaccuracies. We developed and validated a deep learning-based image-analysis platform for automated extraction of volumetric parameters of cardiac function from dynamic light-sheet fluorescence microscopy (LSFM) images of embryonic zebrafish hearts. This platform, the Cardiac Functional Imaging Network (CFIN), automatically delivers rapid and accurate assessments of cardiac performance with greater sensitivity than current approaches. This article has an associated First Person interview with the first author of the paper., Summary: The authors present CFIN, a deep learning-based image-analysis platform to automatically analyze dynamic light-sheet fluorescence microscopy images and determine volumetric indices of cardiac function in embryonic zebrafish.

Fibrolamellar carcinoma (FLC) is a rare liver cancer that affects adolescents and young adults. Genomic analysis of FLC has revealed a 400 kb deletion in chromosome 19 that leads to the chimeric transcript DNAJB1-PRKACA (DnaJ-PKAc), comprised of the first exon of heat shock protein 40 (DNAJB1) and exons 2-10 of the catalytic subunit of protein kinase A (PRKACA). Here, we report a new zebrafish model of FLC induced by ectopic expression of zebrafish Dnaja-Pkaca (zfDnaJa-Pkaca) in hepatocytes that is amenable to live imaging of early innate immune inflammation. Expression of zfDnaJa-Pkaca in hepatocytes induces hepatomegaly and increased hepatocyte size. In addition, FLC larvae exhibit early innate immune inflammation characterized by early infiltration of neutrophils and macrophages into the liver microenvironment. Increased Caspase-a (the zebrafish homolog for human caspase-1) activity was also found in the liver of FLC larvae, and pharmacological inhibition of Tnfα and caspase-a decreased liver size and inflammation. Overall, these findings show that innate immune inflammation is an early feature in a zebrafish model of FLC and that pharmacological inhibition of TNFα or caspase-1 activity might be targets to treat inflammation and progression in FLC patients. This article has an associated First Person interview with the first author of the paper., Summary: Inflammation and Caspase-a activation occur early in a new zebrafish model for fibrolamellar carcinoma (FLC). Pharmacological inhibition of TNFα secretion and caspase-1 might be useful approaches to treat FLC patients.

Reduced bone quality or mineral density predict susceptibility to fracture and also attenuate subsequent repair. Bone regrowth is also compromised by bacterial infection, which exacerbates fracture site inflammation. Because of the cellular complexity of fracture repair, as well as genetic and environmental influences, there is a need for models that permit visualisation of the fracture repair process under clinically relevant conditions. To characterise the process of fracture repair in zebrafish, we employed a crush fracture of fin rays, coupled with histological and transgenic labelling of cellular responses; the results demonstrate a strong similarity to the phased response in humans. We applied our analysis to a zebrafish model of osteogenesis imperfecta (OI), which shows reduced bone quality, spontaneous fractures and propensity for non-unions. We found deficiencies in the formation of a bone callus during fracture repair in our OI model and showed that clinically employed antiresorptive bisphosphonates can reduce spontaneous fractures in OI fish and also measurably reduce fracture callus remodelling in wild-type fish. The csf1ra mutant, which has reduced osteoclast numbers, also showed reduced callus remodelling. Exposure to excessive bisphosphonate, however, disrupted callus repair. Intriguingly, neutrophils initially colonised the fracture site, but were later completely excluded. However, when fractures were infected with Staphylococcus aureus, neutrophils were retained and compromised repair. This work elevates the zebrafish bone fracture model and indicates its utility in assessing conditions of relevance to an orthopaedic setting with medium throughput. This article has an associated First Person interview with the first author of the paper., Summary: The effect of osteogenesis imperfecta, bisphosphonate treatment and bacterial infection on phases of bone fracture repair are determined using a zebrafish fracture model.

Zebrafish offer a powerful vertebrate model for studies of development and disease. The major advantages of this model include the possibilities of conducting reverse and forward genetic screens and of observing cellular processes by in vivo imaging of single cells. Moreover, pathways regulating blood development are highly conserved between zebrafish and mammals, and several discoveries made in fish were later translated to murine and human models. This review and accompanying poster provide an overview of zebrafish hematopoiesis and discuss the existing zebrafish models of blood disorders, such as myeloid and lymphoid malignancies, bone marrow failure syndromes and immunodeficiencies, with a focus on how these models were generated and how they can be applied for translational research., Summary: This At A Glance article and poster summarize the last 20 years of research in zebrafish models for hematopoietic disorders, highlighting how these models were created and are being applied for translational research.

Diabetes is associated with dysfunction of the neurovascular unit, although the mechanisms of this are incompletely understood and currently no treatment exists to prevent these negative effects. We previously found that the nitric oxide (NO) donor sodium nitroprusside (SNP) prevents the detrimental effect of glucose on neurovascular coupling in zebrafish. We therefore sought to establish the wider effects of glucose exposure on both the neurovascular unit and on behaviour in zebrafish, and the ability of SNP to prevent these. We incubated 4-days post-fertilisation (dpf) zebrafish embryos in 20 mM glucose or mannitol for 5 days until 9 dpf, with or without 0.1 mM SNP co-treatment for 24 h (8-9 dpf), and quantified vascular NO reactivity, vascular mural cell number, expression of a klf2a reporter, glial fibrillary acidic protein (GFAP) and transient receptor potential cation channel subfamily V member 4 (TRPV4), as well as spontaneous neuronal activation at 9 dpf, all in the optic tectum. We also assessed the effect on light/dark preference and locomotory characteristics during free-swimming studies. We find that glucose exposure significantly reduced NO reactivity, klf2a reporter expression, vascular mural cell number and TRPV4 expression, while significantly increasing spontaneous neuronal activation and GFAP expression (all in the optic tectum). Furthermore, when we examined larval behaviour, we found that glucose exposure significantly altered light/dark preference and high and low speed locomotion while in light. Co-treatment with SNP reversed all these molecular and behavioural effects of glucose exposure. Our findings comprehensively describe the negative effects of glucose exposure on the vascular anatomy, molecular phenotype and function of the optic tectum, and on whole-organism behaviour. We also show that SNP or other NO donors may represent a therapeutic strategy to ameliorate the complications of diabetes on the neurovascular unit. This article has an associated First Person interview with the first author of the paper., Summary: Diabetes is associated with vascular and neurological impairments. The authors show that an NO donor ameliorates the glucose-exposure-induced dysfunction in the tectal neurovascular unit and whole-organism behaviour.

Hypertrophic cardiomyopathy (HCM) is a hereditary disease characterized by cardiac hypertrophy with diastolic dysfunction. Gene mutations causing HCM have been found in about half of HCM patients, while the genetic etiology and pathogenesis remain unknown for many cases of HCM. To identify novel mechanisms underlying HCM pathogenesis, we generated a cardiovascular-mutant medaka fish, non-spring heart (nsh), which showed diastolic dysfunction and hypertrophic myocardium. The nsh homozygotes had fewer myofibrils, disrupted sarcomeres and expressed pathologically stiffer titin isoforms. In addition, the nsh heterozygotes showed M-line disassembly that is similar to the pathological changes found in HCM. Positional cloning revealed a missense mutation in an immunoglobulin (Ig) domain located in the M-line–A-band transition zone of titin. Screening of mutations in 96 unrelated patients with familial HCM, who had no previously implicated mutations in known sarcomeric gene candidates, identified two mutations in Ig domains close to the M-line region of titin. In vitro studies revealed that the mutations found both in medaka fish and in familial HCM increased binding of titin to muscle-specific ring finger protein 1 (MURF1) and enhanced titin degradation by ubiquitination. These findings implicate an impaired interaction between titin and MURF1 as a novel mechanism underlying the pathogenesis of HCM., Summary: The authors identified and characterized a medaka mutation in titin that leads to a phenotype similar to hypertrophic cardiomyopathy. Similar mutations were also observed in human patients.

Acute kidney injury (AKI) is a serious disorder for which there are limited treatment options. Following injury, native nephrons display limited regenerative capabilities, relying on the dedifferentiation and proliferation of renal tubular epithelial cells (RTECs) that survive the insult. Previously, we identified 4-(phenylthio)butanoic acid (PTBA), a histone deacetylase inhibitor (HDI), as an enhancer of renal recovery, and showed that PTBA treatment increased RTEC proliferation and reduced renal fibrosis. Here, we investigated the regenerative mechanisms of PTBA in zebrafish models of larval renal injury and adult cardiac injury. With respect to renal injury, we showed that delivery of PTBA using an esterified prodrug (UPHD25) increases the reactivation of the renal progenitor gene Pax2a, enhances dedifferentiation of RTECs, reduces Kidney injury molecule-1 (Kim-1) expression, and lowers the number of infiltrating macrophages. Further, we found that the effects of PTBA on RTEC proliferation depend upon retinoic acid signaling and demonstrate that the therapeutic properties of PTBA are not restricted to the kidney but also increase cardiomyocyte proliferation and decrease fibrosis following cardiac injury in adult zebrafish. These studies provide key mechanistic insights into how PTBA enhances tissue repair in models of acute injury and lay the groundwork for translating this novel HDI into the clinic. This article has an associated First Person interview with the joint first authors of the paper., Summary: Mortality associated with AKI is in part due to limited treatments available to ameliorate injury. The authors identify a compound that accelerates AKI recovery and promotes cellular dedifferentiation.

The zebrafish model system offers significant utility for in vivo imaging of adipose tissue (AT) dynamics and for screening to identify chemical and genetic modifiers of adiposity. In particular, AT can be quantified accurately in live zebrafish using fluorescent lipophilic dyes. Although this methodology offers considerable promise, the comprehensive identification and classification of zebrafish ATs has not been performed. Here, we use fluorescent lipophilic dyes and in vivo imaging systematically to identify, classify and quantify the zebrafish AT pool. We identify 34 regionally distinct zebrafish ATs, including five visceral ATs and 22 subcutaneous ATs. For each of these ATs, we describe detailed morphological characteristics to aid their identification in future studies. Furthermore, we quantify the areas for each AT and construct regression models to allow prediction of expected AT size and variation across a range of developmental stages. Finally, we demonstrate the utility of this resource for identifying effects of strain variation and high-fat diet on AT growth. Altogether, this resource provides foundational information on the identity, dynamics and expected quantities of zebrafish ATs for use as a reference for future studies., Summary: A standardized nomenclature and classification system for zebrafish adipose tissues and regression models to predict expected adipose size during the course of zebrafish development.

The zebrafish is a prominent vertebrate model for low-cost in vivo whole organism screening. In our recent screening of the distribution patterns of fluorescent compounds in live zebrafish larvae, fifteen compounds with tissue-specific distributions were identified. Several compounds were observed to accumulate in tissues where they were reported to induce side-effects, and compounds with similar structures tended to be enriched in the same tissues, with minor differences. In particular, we found three novel red fluorescent bone-staining dyes: purpurin, lucidin and 3-hydroxy-morindone; purpurin can effectively label bones in both larval and adult zebrafish, as well as in postnatal mice, without significantly affecting bone mass and density. Moreover, two structurally similar chemotherapeutic compounds, doxorubicin and epirubicin, were observed to have distinct distribution preferences in zebrafish. Epirubicin maintained a relatively higher concentration in the liver, and performed better in inhibiting hepatic hyperplasia caused by the over-expression of krasG12V. In total, our study suggests that the transparent zebrafish larvae serve as valuable tools for identifying tissue-specific distributions of fluorescent compounds., Summary: Fifteen fluorescent compounds have tissue-specific distributions in zebrafish larvae: three anthraquinones were identified as bone dyes in live vertebrates, and two chemotherapeutic drugs were shown to have distinct preferences for the liver.

Patients with von Hippel–Lindau (VHL) disease harbor a germline mutation in the VHL gene leading to the development of several tumor types including clear cell renal cell carcinoma (ccRCC). In addition, the VHL gene is inactivated in over 90% of sporadic ccRCC cases. ‘Clear cell’ tumors contain large, proliferating cells with ‘clear cytoplasm’, and a reduced number of cilia. VHL inactivation leads to the stabilization of hypoxia inducible factors 1a and 2a [HIF1a and HIF2a (HIF2a is also known as EPAS1)] with consequent up-regulation of specific target genes involved in cell proliferation, angiogenesis and erythropoiesis. A zebrafish model with a homozygous inactivation in the VHL gene (vhl−/−) recapitulates several aspects of the human disease, including development of highly vascular lesions in the brain and the retina and erythrocytosis. Here, we characterize for the first time the epithelial abnormalities present in the kidney of the vhl−/− zebrafish larvae as a first step in building a model of ccRCC in zebrafish. Our data show that the vhl−/− zebrafish kidney is characterized by an increased tubule diameter, disorganized cilia, the dramatic formation of cytoplasmic lipid vesicles, glycogen accumulation, aberrant cell proliferation and abnormal apoptosis. This phenotype of the vhl−/− pronephros is reminiscent of clear cell histology, indicating that the vhl−/− mutant zebrafish might serve as a model of early stage RCC. Treatment of vhl−/− zebrafish embryos with a small-molecule HIF2a inhibitor rescued the pronephric abnormalities, underscoring the value of the zebrafish model in drug discovery for treatment of VHL disease and ccRCC., Summary: Zebrafish with an inactivating mutation in the vhl gene can be used as a model of early stage clear cell renal cell carcinoma, with applications for genetic studies and drug screens.

Age is the highest risk factor for some of the most prevalent human diseases, including cancer. Telomere shortening is thought to play a central role in the aging process in humans. The link between telomeres and aging is highlighted by the fact that genetic diseases causing telomerase deficiency are associated with premature aging and increased risk of cancer. For the last two decades, this link has been mostly investigated using mice that have long telomeres. However, zebrafish has recently emerged as a powerful and complementary model system to study telomere biology. Zebrafish possess human-like short telomeres that progressively decline with age, reaching lengths in old age that are observed when telomerase is mutated. The extensive characterization of its well-conserved molecular and cellular physiology makes this vertebrate an excellent model to unravel the underlying relationship between telomere shortening, tissue regeneration, aging and disease. In this Review, we explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease., Summary: In this Review, the authors explore the advantages of using zebrafish in telomere research and discuss the primary discoveries made in this model that have contributed to expanding our knowledge of how telomere attrition contributes to cellular senescence, organ dysfunction and disease.

Zebrafish are a major model for chemical genetics, and most studies use embryos when investigating small molecules that cause interesting phenotypes or that can rescue disease models. Limited studies have dosed adults with small molecules by means of water-borne exposure or injection techniques. Challenges in the form of drug delivery-related trauma and anesthesia-related toxicity have excluded the adult zebrafish from long-term drug efficacy studies. Here, we introduce a novel anesthetic combination of MS-222 and isoflurane to an oral gavage technique for a non-toxic, non-invasive and long-term drug administration platform. As a proof of principle, we established drug efficacy of the FDA-approved BRAF [superscript V600E] inhibitor, Vemurafenib, in adult zebrafish harboring BRAF[superscript V600E] melanoma tumors. In the model, adult casper zebrafish intraperitoneally transplanted with a zebrafish melanoma cell line (ZMEL1) and exposed to daily sub-lethal dosing at 100 mg/kg of Vemurafenib for 2 weeks via oral gavage resulted in an average 65% decrease in tumor burden and a 15% mortality rate. In contrast, Vemurafenib-resistant ZMEL1 cell lines, generated in culture from low-dose drug exposure for 4 months, did not respond to the oral gavage treatment regimen. Similarly, this drug treatment regimen can be applied for treatment of primary melanoma tumors in the zebrafish. Taken together, we developed an effective long-term drug treatment system that will allow the adult zebrafish to be used to identify more effective anti-melanoma combination therapies and opens up possibilities for treating adult models of other diseases., National Institutes of Health (U.S.), National Cancer Institute (U.S.) (grant CA103846-13)

Neutrophils are essential for host defence and are recruited to sites of inflammation in response to tissue injury or infection. For inflammation to resolve, these cells must be cleared efficiently and in a controlled manner, either by apoptosis or reverse migration. If the inflammatory response is not well-regulated, persistent neutrophils can cause damage to host tissues and contribute to the pathogenesis of chronic inflammatory diseases, which respond poorly to current treatments. It is therefore important to develop drug discovery strategies that can identify new therapeutics specifically targeting neutrophils, either by promoting their clearance or by preventing their recruitment. Our recent in vivo chemical genetic screen for accelerators of inflammation resolution identified a subset of compounds sharing a common chemical signature, the bicyclic benzopyrone rings. Here, we further investigate the mechanisms of action of the most active of this chemical series, isopimpinellin, in our zebrafish model of neutrophilic inflammation. We found that this compound targets both the recruitment and resolution phases of the inflammatory response. Neutrophil migration towards a site of injury is reduced by isopimpinellin and this occurs as a result of PI3K inhibition. We also show that isopimpinellin induces neutrophil apoptosis to drive inflammation resolution in vivo using a new zebrafish reporter line detecting in vivo neutrophil caspase-3 activity and allowing quantification of flux through the apoptotic pathway in real time. Finally, our studies reveal that clinically available ‘cromones’ are structurally related to isopimpinellin and have previously undescribed pro-resolution activity in vivo. These findings could have implications for the therapeutic use of benzopyrones in inflammatory disease., Summary: Zebrafish inflammation screen identifies a new series of structurally related compounds with combined anti-inflammatory and pro-resolution activity, and reveals a previously unknown mechanism of action of clinical cromones.

Traditionally, most investigators in the biomedical arena exploit one model system in the course of their careers. Occasionally, an investigator will switch models. The selection of a suitable model system is a crucial step in research design. Factors to consider include the accuracy of the model as a reflection of the human disease under investigation, the numbers of animals needed and ease of husbandry, its physiology and developmental biology, and the ability to apply genetics and harness the model for drug discovery. In my lab, we have primarily used the zebrafish but combined it with other animal models and provided a framework for others to consider the application of developmental biology for therapeutic discovery. Our interdisciplinary approach has led to many insights into human diseases and to the advancement of candidate drugs to clinical trials. Here, I draw on my experiences to highlight the importance of combining multiple models, establishing infrastructure and genetic tools, forming collaborations, and interfacing with the medical community for successful translation of basic findings to the clinic., Summary: Animal models have greatly facilitated the process of drug discovery. Len Zon draws on his own experiences to highlight how a cross-model, interdisciplinary approach can bring basic findings to the clinic.

話し合い、問題解決をすることは中学生である学習者に対して容易なことではない。そこで、「ゼブラ」を教材として、物語の内容や話の構成について話し合い、考えを深め合うブッククラブ・メソッドに基づいた物語文の授業を実践した。学習者はブッククラブ・メソッドにある、シーケンスチャート作成の課題に対し、話し合いの中で積極的に意見を出すことができていた。また意見の交流の中で新たな考えに気付き、話し合うことの価値に気付くことができた。本実践を通して、ブッククラブ・メソッドを用いた物語の授業の提案をしたい。

Mutations in GNB5, encoding the G-protein β5 subunit (Gβ5), have recently been linked to a multisystem disorder that includes severe bradycardia. Here, we investigated the mechanism underlying bradycardia caused by the recessive p.S81L Gβ5 variant. Using CRISPR/Cas9-based targeting, we generated an isogenic series of human induced pluripotent stem cell (hiPSC) lines that were either wild type, heterozygous or homozygous for the GNB5 p.S81L variant. These were differentiated into cardiomyocytes (hiPSC-CMs) that robustly expressed the acetylcholine-activated potassium channel [I(KACh); also known as IK,ACh]. Baseline electrophysiological properties of the lines did not differ. Upon application of carbachol (CCh), homozygous p.S81L hiPSC-CMs displayed an increased acetylcholine-activated potassium current (IK,ACh) density and a more pronounced decrease of spontaneous activity as compared to wild-type and heterozygous p.S81L hiPSC-CMs, explaining the bradycardia in homozygous carriers. Application of the specific I(KACh) blocker XEN-R0703 resulted in near-complete reversal of the phenotype. Our results provide mechanistic insights and proof of principle for potential therapy in patients carrying GNB5 mutations. This article has an associated First Person interview with the first author of the paper., Summary: The authors show hiPSC-cardiomyocyte-based modeling of a mutation in a G-protein β-subunit that affects the electrophysiological response of cardiomyocytes to the cholinergic regulation of the heart rate, uncovering a potential therapy for severe bradycardia in humans carrying these mutations.