Zebrafish System Research Articles

Double-modified, thio and methylene ATP analogue facilitates wound healing in vitro and in vivo

Recent data indicate that extracellular ATP affects wound healing efficacy via P2Y2-dependent signaling pathway. In the current work, we propose double-modified ATP analogue—alpha-thio-beta,gamma-methylene-ATP as a potential therapeutic agent for a skin regeneration. For the better understanding of structure–activity relationship, beside tested ATP analogues, the appropriate single-modified derivatives of target compound, such as alpha-thio-ATP and beta,gamma-methylene-ATP, were also tested in the context of their involvement in the activation of ATP-dependent purinergic signaling pathway via the P2Y2 receptor. The diastereomerically pure alpha-thio-modified-ATP derivatives were obtained using the oxathiaphospholane method as separate SP and RP diastereomers. Both the single- and double- modified ATP analogues were then tested for their impact on the viability and migration of human keratinocytes. The involvement of P2Y2-dependent purinergic signaling was analyzed in silico by molecular docking of the tested compounds to the P2Y2 receptor and experimentally by studying intracellular calcium mobilization in the human keratinocytes HaCaT. The effects obtained for ATP analogues were compared with the results for ATP as a natural P2Y2 agonist. To confirm the contribution of the P2Y2 receptor to the observed effects, the tests were also performed in the presence of the selective P2Y2 antagonist—AR-C118925XX. The ability of the alpha-thio-beta,gamma-methylene-ATP to influence cell migration was analyzed in vitro on the model HaCaT and MDA-MB-231 cells by wound healing assay and transwell migration test as well as in vivo using zebrafish system. The impact on tissue regeneration was estimated based on the regrowth rate of cut zebrafish tails. The in vitro and in vivo studies have shown that the SP-alpha-thio-beta,gamma-methylene-ATP analogue promotes regeneration-related processes, making it a suitable agent for enhance wound healing. Performed studies indicated its impact on the cell migration, induction of epithelial–mesenchymal transition and intracellular calcium mobilization. The enhanced regeneration of cut zebrafish tails confirmed the pro-regenerative activity of this ATP analogue. Based on the performed studies, the SP-alpha-thio-beta,gamma-methylene-ATP is proposed as a potential therapeutic agent for wound healing and skin regeneration treatment.

Abstract 6621: Development of a melanocyte lineage-specific knockout system in zebrafish

Abstract Zebrafish have emerged as an ideal model organism for investigating melanocyte and melanoma biology. While CRISPR-Cas9 technology has significantly advanced our understanding of gene functions in zebrafish, two challenges remain: 1) how do we knock out embryonic lethal genes in adult melanocytes? and 2) how do we knock out tumor promoting genes (rather than tumor suppressors)? To address this, we have developed a new transgenic line in which Cas9 is knocked into the melanocyte-specific endogenous mitfa locus. To test this system in normal melanocytes, Tg(mitfa:Cas9) embryos were injected with a plasmid containing albino sgRNA. Cells with integration of this plasmid exhibited almost complete loss of pigmentation, demonstrating the high efficiency of our approach. This system can now be rapidly used to knock out embryonic essential genes specifically in melanocytes and evaluate the phenotype of adult fish. In melanoma, the commonly available zebrafish lines overexpress the oncogene BRAFV600E in melanocytes with germline knockout of tp53. This is not comparable to the situation in humans, where global p53 loss is rare. By utilizing our mitfa:Cas9 system, we have generated tumors with melanoma-specific loss of tp53 or ptena/b, thereby eliminating non-specific tumors that originate from knockout of tumor suppressors in other cell types. Using this system, we are now testing whether knockout of tumor promoting genes delays melanoma onset and progression, something that has previously been difficult to efficiently achieve in zebrafish. In addition to specificity, an additional advantage of our system is that we can create nearly complete knockout of genes in the melanocytes by the F1, rather than the F2 generation. This saves several months of time and reduces the number of uninformative animals without the relevant genotype. Collectively, our system provides a highly efficient mechanism to study essential genes in melanocytes and melanoma in the zebrafish system. Citation Format: Sarah Perlee, Yilun Ma, Jacob Swanson, Zhitao Ming, Julia Xia, Timothée Lionnet, Maura McGrail, Richard White. Development of a melanocyte lineage-specific knockout system in zebrafish [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 6621.

Functional Genomics of Novel Rhabdomyosarcoma Fusion-Oncogenes Using Zebrafish.

Clinical sequencing efforts continue to identify novel putative oncogenes with limited strategies to perform functional validation in vivo and study their role in tumorigenesis. Here, we present a pipeline for fusion-driven rhabdomyosarcoma (RMS) in vivo modeling using transgenic zebrafish systems. This strategy originates with novel fusion-oncogenes identified from patient samples that require functional validation in vertebrate systems, integrating these genes into the zebrafish genome, and then characterizing that they indeed drive rhabdomyosarcoma tumor formation. In this scenario, the human form of the fusion-oncogene is inserted into the zebrafish genome to understand if it is an oncogene, and if so, the underlying mechanisms of tumorigenesis. This approach has been successful in our models of infantile rhabdomyosarcoma and alveolar rhabdomyosarcoma, both driven by respective fusion-oncogenes, VGLL2-NCOA2 and PAX3-FOXO1. Our described zebrafish platform is a rapid method to understand the impact of fusion-oncogene activity, divergent and shared fusion-oncogenebiology, and whether any analyzed pathways converge for potential clinically actionable targets.

Abstract P2176: Development Of Integrated Transparent Transgenic Zebrafish Modeling System For Experimental Cardio-oncology Therapeutics

The zebrafish model has provided an excellent platform to study the genetic and molecular approach of basic and translational cardiac research. Zebrafish heart cells are similar to human heart cells at the molecular level and determine the function of genes that control cardiac function and dysfunction. In zebrafish, myl7 is myosin light chain-7 gene and identified as a regulatory gene of heart orthologs to human MYL7. In vertebrates, including zebrafish, murine and human systems, the in-vivo spatial resolution is limited due to the normal opacification of skin and subdermal structures. For in-vivo imaging the skin transparency is primary requirement and to maintain the transparency, blocking the pigmentation needs to maintain. Chemical inhibition treatment is temporary and possible till the organism treated with the chemical inhibitor agent. Zebrafish casper mutant maintain transparency throughout the life and serve as ideal combination of sensitivity and resolution for in-vivo stem cell analyses and in-vivo imaging. To maintain the skin transparency, in this study, we have developed transparent transgenic zebrafish model and established of time lapse in-vivo confocal microscopy to study of cellular phenotype/pathologies of the cardiomyocytes to quantify changes in cardiomyocyte morphology and function overtime by comparing control and cardiac injury by generating casper/ myl7:RFP; annexin-5:YFP transgenic zebrafish. In the field of cardio-oncology, we are developing in-vivo transparent transgenic cellular phenotype model of zebrafish to study of the cell death process (Annexin5) and Inflammatory activity (NFkB) in the microglia, and the resident macrophage of the brain and cardiomyocytes) homozygous casper/ myl7:RFP; annexin-5:YFP/NF-kB:GFP/mpeg1:mcherryFP transgenic strain zebrafish. By using this novel model our lab establishing the role of NFkB and microglia function under normal healthy status and in glioblastomas Our strategist approach to yield crucial new insights into in-vivo cardiomyocyte imaging by confocal microscopy to observe and track the cell death pattern and cardio inflammatory pathways in cardiomyocyte and develop novel therapeutic approaches to treat cardio inflammatory pathology and glioblastoma

Fish Hacks: Hybridizing Stand-Alone Zebrafish System Plumbing and Pumps to Extend and Improve Function.

One of the greatest expenses in running a zebrafish laboratory is the aquatic systems used for housing. These critical pieces of equipment are essential and incorporate components undergoing constant activity in pumping water, monitoring, dosing, and filtration. The systems available on the market are robust, yet ongoing activity eventually leads to the need for repair or replacement. Moreover, some systems are no longer commercially available, impairing the ability to service this critical infrastructure. In this study, we demonstrate a do it yourself (DIY) method to re-engineer an aquatic system's pumps and plumbing, which hybridizes a system no longer commercially available with components used by active vendors. This transition from a two external pump Aquatic Habitat/Pentair design to an individual submerged pump Aquaneering-like plan saves funds by expanding infrastructure longevity. Our hybridized configuration has been in uninterrupted use for >3 years, supporting zebrafish health and high fecundity.

RNU12 inhibits gastric cancer progression via sponging miR-575 and targeting BLID

Gastric cancer (GC) is one of the major causes of cancer deaths with 5-year survival ratio of 20%. RNU12 is one of long noncoding RNAs (lncRNAs) regulating the tumor progression. However, how RNU12 affecting GC is not clear. qRT-PCR was utilized for determining the RNU12 expression in cell lines, 113 cases of paired gastric cancer (GC) and their adjacent normal gastric tissues. The biofunction alterations of RNU12 were assessed by its overexpression or knockdown in GC cells. MTT and cloning assay were assayed for the cell proliferation, the flow cytometry for the detection of cell cycle and the wound healing assay (WHA) and transwell invasion assay (TIA) for examining the migration and invasion of cells. The expressions of a set of genes related proliferation and migration were investigated with the Western Blotting (WB). RNA immunoprecipitation (RIP), biotinylated RNA pull-down and dual luciferase reporter tests were used to detect the interactions of RNU12 with miR-575/BLID. The in vivo proliferation and migration ability of RNU12 infected cells were determined in zebrafish system. This study revealed that RNU12 inhibited proliferation, invasion and metastasis by sponging of miR-575 and regulating the downstream BLID and modulated EMT of GC cells. The RNU12/miR-575/BLID axis is likely to be the prognosis biomarkers and drug targets of GC.

High resolution protein in-cell NMR in zebrafish embryos

In-cell NMR spectroscopy has emerged as a powerful tool to evaluate protein conformations and dynamics in the native environment of live cells. Here we extend these studies to a multicellular developing vertebrate. Zebrafish (Danio rerio) embryos are complex organisms with dynamic tissue organization and may be well suited for the high resolution NMR analysis of microinjected, isotopically enriched proteins. We used the intrinsically disordered protein Alpha-synuclein (aSyn) as a test model. aSyn has been thoroughly evaluated inside bacterial and mammalian cells, providing good reference points for NMR comparisons and the critical analysis of the advantages and disadvantages of the zebrafish system. High resolution 2D 1H-15N NMR showed that aSyn in zebrafish embryos had the same conformational and biological features previously observed in mammalian cells, including conserved interactions with cellular biomolecules and the establishment of physiological protein post-translational modifications. A direct comparative analysis of gamma-synuclein (gSyn), a naturally occurring homolog of aSyn, in bacteria, mammalian cells and zebrafish embryos confirmed these observations. Our results showed that high resolution in-cell NMR is attainable in embryonic cells within the native environment of a live animal. This system provides more physiological cellular environments for high resolution, in situ protein biophysical studies.

Transcriptomic and Metabolomic Studies Reveal That Toll-like Receptor 2 Has a Role in Glucose-Related Metabolism in Unchallenged Zebrafish Larvae (Danio rerio).

Toll-like receptors (TLRs) have been implicated in the regulation of various metabolism pathways, in addition to their function in innate immunity. Here, we investigate the metabolic function of TLR2 in a larval zebrafish system. We studied larvae from a tlr2 mutant and the wild type sibling controls in an unchallenged normal developmental condition using transcriptomic and metabolomic analyses methods. RNAseq was used to evaluate transcriptomic differences between the tlr2 mutant and wild-type control zebrafish larvae and found a signature set of 149 genes to be significantly altered in gene expression. The expression level of several genes was confirmed by qPCR analyses. Gene set enrichment analysis (GSEA) revealed differential enrichment of genes between the two genotypes related to valine, leucine, and isoleucine degradation and glycolysis and gluconeogenesis. Using 1H nuclear magnetic resonance (NMR) metabolomics, we found that glucose and various metabolites related with glucose metabolism were present at higher levels in the tlr2 mutant. Furthermore, we confirmed that the glucose level is higher in tlr2 mutants by using a fluorometric assay. Therefore, we have shown that TLR2, in addition to its function in immunity, has a function in controlling metabolism during vertebrate development. The functions are associated with transcriptional regulation of various enzymes involved in glucose metabolism that could explain the different levels of glucose, lactate, succinate, and malate in larvae of a tlr2 mutant.

Neurodevelopmental Toxicity of Emamectin Benzoate to the Early Life Stage of Zebrafish Larvae (Danio rerio).

Emamectin benzoate (EMB) is a widely used pesticide and feed additive in agriculture and aquaculture. It easily enters the aquatic environment through various pathways, thus causing adverse effects on aquatic organisms. However, there are no systematic studies regarding the effects of EMB on the developmental neurotoxicity of aquatic organisms. Therefore, the aim of this study was to evaluate the neurotoxic effects and mechanisms of EMB at different concentrations (0.1, 0.25, 0.5, 1, 2, 4 and 8 μg/mL) using zebrafish as a model. The results showed that EMB significantly inhibited the hatching rate, spontaneous movement, body length, and swim bladder development of zebrafish embryos, as well as significantly increased the malformation rate of zebrafish larvae. In addition, EMB adversely affected the axon length of motor neurons in Tg (hb9: eGFP) zebrafish and central nervous system (CNS) neurons in Tg (HuC: eGFP) zebrafish and significantly inhibited the locomotor behavior of zebrafish larvae. Meanwhile, EMB induced oxidative damage and was accompanied by increasing reactive oxygen species in the brains of zebrafish larvae. In addition, gene expression involvement in oxidative stress-related (cat, sod and Cu/Zn-sod), GABA neural pathway-related (gat1, gabra1, gad1b, abat and glsa), neurodevelopmental-related (syn2a, gfap, elavl3, shha, gap43 and Nrd) and swim bladder development-related (foxa3, pbxla, mnx1, has2 and elovlla) genes was significantly affected by EMB exposure. In conclusion, our study shows that exposure to EMB during the early life stages of zebrafish significantly increases oxidative damage and inhibits early central neuronal development, motor neuron axon growth and swim bladder development, ultimately leading to neurobehavioral changes in juvenile zebrafish.

Lack of Paxillin phosphorylation promotes single-cell migration in vivo.

Focal adhesions are structures that physically link the cell to the extracellular matrix for cell migration. Although cell culture studies have provided a wealth of information regarding focal adhesion biology, it is critical to understand how focal adhesions are dynamically regulated in their native environment. We developed a zebrafish system to visualize focal adhesion structures during single-cell migration in vivo. We find that a key site of phosphoregulation (Y118) on Paxillin exhibits reduced phosphorylation in migrating cells in vivo compared to in vitro. Furthermore, expression of a non-phosphorylatable version of Y118-Paxillin increases focal adhesion disassembly and promotes cell migration in vivo, despite inhibiting cell migration in vitro. Using a mouse model, we further find that the upstream kinase, focal adhesion kinase, is downregulated in cells in vivo, and cells expressing non-phosphorylatable Y118-Paxillin exhibit increased activation of the CRKII-DOCK180/RacGEF pathway. Our findings provide significant new insight into the intrinsic regulation of focal adhesions in cells migrating in their native environment.

A Critical Review of Zebrafish Neurological Disease Models-1. The Premise: Neuroanatomical, Cellular and Genetic Homology and Experimental Tractability.

The last decade has seen a dramatic rise in the number of genes linked to neurological disorders, necessitating new models to explore underlying mechanisms and to test potential therapies. Over a similar period, many laboratories adopted zebrafish as a tractable model for studying brain development, defining neural circuits and performing chemical screens. Here we discuss strengths and limitations of using the zebrafish system to model neurological disorders. The underlying premise for many disease models is the high degree of homology between human and zebrafish genes, coupled with the conserved vertebrate Bauplan and repertoire of neurochemical signaling molecules. Yet, we caution that important evolutionary divergences often limit the extent to which human symptoms can be modeled meaningfully in zebrafish. We outline advances in genetic technologies that allow human mutations to be reproduced faithfully in zebrafish. Together with methods that visualize the development and function of neuronal pathways at the single cell level, there is now an unprecedented opportunity to understand how disease-associated genetic changes disrupt neural circuits, a level of analysis that is ideally suited to uncovering pathogenic changes in human brain disorders.

Modeling the effects of genetic- and diet-induced obesity on melanoma progression in zebrafish.

Obesity is a rising concern and associated with an increase in numerous cancers, often in a sex-specific manner. Preclinical models are needed to deconvolute the intersection between obesity, sex and melanoma. Here, we generated a zebrafish system that can be used as a platform for studying these factors. We studied how germline overexpression of Agrp along with a high-fat diet affects melanomas dependent on BRAFV600E and loss of p53. This revealed an increase in tumor incidence and area in male, but not female, obese fish, consistent with the clinical literature. We then determined whether this was further affected by additional somatic mutations in the clinically relevant genes rb1 or ptena/b. We found that the male obesogenic effect on melanoma was present with tumors generated with BRAF;p53;Rb1 but not BRAF;p53;Pten. These data indicate that both germline (Agrp) and somatic (BRAF, Rb1) mutations contribute to obesity-related effects in melanoma. Given the rapid genetic tools available in the zebrafish, this provides a high-throughput system to dissect the interactions of genetics, diet, sex and host factors in obesity-related cancers.

Strategies for Alleviating Tyrosine Kinase Resistance in Acute Myeloid Leukemia By Modulating p53 Signaling Pathway with Small Molecules

Background:FLT3-ITD can be found in up to 30% of acute myeloid leukemia (AML) patients and confers a poor prognosis. While midostaurin and gilteritinib are FDA-approved for these high-risk patients, they only result in a temporary response without transplantation, suggesting the importance of identifying the acquired resistance. Previously, we demonstrated that cabozantinib, an oral multi-target tyrosine kinase inhibitor, was selectively cytotoxic to AML cells with FLT3-ITD (MV4-11 and Molm13). Subsequently, we established cabozantinib-resistant human leukemic MV4-11 derivative (MV4-11-XR) and Molm13 derivative (Molm13-XR) after gradual escalating concentration of cabozantinib incubation from parental MV4-11 (MV4-11-P) and Molm13 (Molm13-P) cells, respectively. Subsequently, cell cytotoxicity assay showed these two cabozantinib-resistant cells were also resistant to quizartinib, sorafenib and gilteritinib. RNA-seq analysis revealed that MV4-11-XR and Molm13-XR cells shared 87 identical differentially down-regulated genes compared to their respective parental cells. Further Metascape for gene enriched pathway analysis demonstrated the most affected was p53 signaling pathway. Aim: To investigate whether modulation with p53 activator RITA or MDM2 inhibitor RG7388 could alleviate the drug resistance and their underlying molecular mechanism. Materials and Methods: Human leukemia MV4-11 and Molm13 cells were used in this study. The differential expression genes (DEGs) were examined using RNA-seq (Illumina NextSeq-500). Metascape enrichment pathway analyses were preformed to predict the biological functions of DEGs. Cabozantinib, RITA, and RG7388 (Idasanutlin) were used. MTS assay was performed to the effects of various drug on cell growth. Western blotting, flow cytometry and quantitative real-time PCR were applied to elucidate potential molecular mechanism. Fluorescent leukemic cell xenografts in zebrafish and high content imaging system were used for investigating the efficacy of various drugs in vivo. Results: The gene expression profile in MV4-11-P and Molm13-P cells was used as a baseline to establish the up- and down-regulated genes in MV4-11-XR and Molm13-XR cells. The FPKM were estimated with the selection criteria of p value <0.05 and [log2 (fold change)] <1 for significantly differential expression for downregulation. We identified a total of 747 DEGs between the MV4-11-P and -XR cells and 407 DEGs between the Molm13-P and -XR cells. Among them, 87 DEGs were shared by these two cohorts. Metascape enrichment analysis revealed that those down-regulated DEGs were enriched in p53-signaling pathway, in line with decreased expression of P53-associated genes, BBC3, CCNG2, CKDN1A, ZMAT3, confirmed by using q-RT-PCR. IGV (Integrative Genomics Viewer) analysis showed that the variant allele frequency of R248W progressed from 57% in MV4-11-P to 100% in MV4-11-XR cells, a similar phenomenon also appeared in P72R (65% to 98%). Microsatellite analysis confirmed the loss of heterozygosity in the TP53 gene of MV4-11-XR. On the other hand, there were not TP53 variants in both Molm13-P and Molm13-XR cells, but increased level of MDM2 in Molm13-XR cells was noted. Further study revealed that p53 activator RITA with 77.6nM could reduce IC50 of cabozantinib from 1026nM to 310.7nM, coupled with increased gene expression and protein levels of BBC3 (PUMA), CCNG2 (cyclin G2), CDKN1A (p21) and ZMAT3. In addition, RITA had synergistic effect with cabozantinib on cell cycle arrest on G2/M phase, increased apoptosis and decreased colony formation and proliferation. On the other hand, MDM2 inhibitor RG7388 reduced the IC50 of cabozantinib from 517nM to 101nM with only 50.6 nM in Molm13-XR cells. Similarly, RG7388 could increase those P53-associated gene expression and protein levels, and had synergistic effect with cabozantinib on cell cycle arrest on G0/G1 phase, increased apoptosis and decreased colony formation and proliferation. In addition, in vivo zebrafish experiment demonstrated that the combination of RG7388 and cabozantinib had a synergistic and significant inhibitory effect compared with single use. Conclusion: These findings suggested that reactivation of p53 is a potential therapeutic option to alleviate drug resistance in FLT3-mutated AML, provided that AML patients are tested for p53 dysfunction, including TP53 mutational status and downregulation.

Expanding allelic and phenotypic spectrum of ZC4H2-related disorder: A novel hypomorphic variant and high prevalence of tethered cord.

ZC4H2 (MIM# 300897) is a nuclear factor involved in various cellular processes including proliferation and differentiation of neural stem cells, ventral spinal patterning and osteogenic and myogenic processes. Pathogenic variants in ZC4H2 have been associated with Wieacker-Wolff syndrome (MIM# 314580), an X-linked neurodevelopmental disorder characterized by arthrogryposis, development delay, hypotonia, feeding difficulties, poor growth, skeletal abnormalities, and dysmorphic features. Zebrafish zc4h2 null mutants recapitulated the human phenotype, showed complete loss of vsx2 expression in brain, and exhibited abnormal swimming and balance problems. Here we report 7 new patients (four males and three females) with ZC4H2-related disorder from six unrelated families. Four of the 6 ZC4H2 variants are novel: three missense variants, designated as c.142T>A (p.Tyr48Asn), c.558G>A (p.Met186Ile) and c.602C>T (p.Pro201Leu), and a nonsense variant, c.618C>A (p.Cys206*). Two variants were previously reported : a nonsense variant c.199C>T (p.Arg67*) and a splice site variant (c.225+5G>A). Five patients were on the severe spectrum of clinical findings, two of whom had early death. The male patient harboring the p.Met186Ile variant and the female patient that carries the p.Pro201Leu variant have a relatively mild phenotype. Of note, 4/7 patients had a tethered cord that required a surgical repair. We also demonstrate and discuss previously under-recognized phenotypic features including sleep apnea, arrhythmia, hypoglycemia, and unexpected early death. To study the effect of the missense variants, we performed microinjection of human ZC4H2 wild-type or variant mRNAs into zc4h2 null mutant zebrafish embryos. The p.Met186Ile mRNA variant was able to partially rescue vsx2 expression while p.Tyr48Asn and p.Pro201Leu mRNA variants were not. However, swimming and balance problems could not be rescued by any of these variants. These results suggest that the p.Met186Ile is a hypomorphic allele. Our work expands the genotypes and phenotypes associated with ZC4H2-related disorder and demonstrates that the zebrafish system is a reliable method to determine the pathogenicity of ZC4H2 variants.

How to Generate a Vascular-Labelled Transgenic Zebrafish Model to Study Tumor Angiogenesis and Extravasation.

The use of transgenic animals carrying exogenous DNA integrated in their genome is a routine in modern-day laboratories. Nowadays, the zebrafish system represents the most useful tool for transgenesis studies mainly due to easy accessibility and manipulation of the eggs, which are produced in high numbersand overa relatively short generation time. The zebrafish transgenic technology is very straightforward when coupled with angiogenesis studies allowing easy in vivo observation of the vertebrate embryonic vasculature. Here, we describe the most common technique to generate vascular-labelled transgenic zebrafish embryos and their applications to study tumor angiogenesis and visualize tumor extravasation.

A Primer Genetic Toolkit for Exploring Mitochondrial Biology and Disease Using Zebrafish.

Mitochondria are a dynamic eukaryotic innovation that play diverse roles in biology and disease. The mitochondrial genome is remarkably conserved in all vertebrates, encoding the same 37-gene set and overall genomic structure, ranging from 16,596 base pairs (bp) in the teleost zebrafish (Danio rerio) to 16,569 bp in humans. Mitochondrial disorders are amongst the most prevalent inherited diseases, affecting roughly 1 in every 5000 individuals. Currently, few effective treatments exist for those with mitochondrial ailments, representing a major unmet patient need. Mitochondrial dysfunction is also a common component of a wide variety of other human illnesses, ranging from neurodegenerative disorders such as Huntington’s disease and Parkinson’s disease to autoimmune illnesses such as multiple sclerosis and rheumatoid arthritis. The electron transport chain (ETC) component of mitochondria is critical for mitochondrial biology and defects can lead to many mitochondrial disease symptoms. Here, we present a publicly available collection of genetic mutants created in highly conserved, nuclear-encoded mitochondrial genes in Danio rerio. The zebrafish system represents a potentially powerful new opportunity for the study of mitochondrial biology and disease due to the large number of orthologous genes shared with humans and the many advanced features of this model system, from genetics to imaging. This collection includes 15 mutant lines in 13 different genes created through locus-specific gene editing to induce frameshift or splice acceptor mutations, leading to predicted protein truncation during translation. Additionally, included are 11 lines created by the random insertion of the gene-breaking transposon (GBT) protein trap cassette. All these targeted mutant alleles truncate conserved domains of genes critical to the proper function of the ETC or genes that have been implicated in human mitochondrial disease. This collection is designed to accelerate the use of zebrafish to study many different aspects of mitochondrial function to widen our understanding of their role in biology and human disease.

Zebrafish behavior as a gateway to nervous system assembly and plasticity.

Nervous system assembly relies on a diversity of cellular processes ranging from dramatic tissue reorganization to local, subcellular changes all driven by precise molecular programs. Combined, these processes culminate in an animal's ability to plan and execute behaviors. Animal behavior can, therefore, serve as a functional readout of nervous system development. Benefitting from an expansive and growing set of molecular and imaging tools paired with an ever-growing number of assays of diverse behaviors, the zebrafish system has emerged as an outstanding platform at the intersection of nervous system assembly, plasticity and behavior. Here, we summarize recent advancements in the field, including how developing neural circuits are refined to shape complex behaviors and plasticity.

Nitroreductase/Metronidazole-Mediated Ablation and a MATLAB Platform (RpEGEN) for Studying Regeneration of the Zebrafish Retinal Pigment Epithelium.

The retinal pigment epithelium (RPE) resides at the back of the eye and performs functions essential for maintaining the health and integrity of adjacent retinal and vascular tissues. At present, the limited reparative capacity of mammalian RPE, which is restricted to small injuries, has hindered progress to understanding in vivo RPE regenerative processes. Here, a detailed methodology is provided to facilitate the study of in vivo RPE repair utilizing the zebrafish, avertebrate model capable of robust tissue regeneration. This protocol describes a transgenic nitroreductase/metronidazole (NTR/MTZ)-mediated injury paradigm (rpe65a:nfsB-eGFP), which results in ablation of the central two-thirds of the RPE after 24 h treatment with MTZ, with subsequent tissue recovery. Focus is placed on RPE ablations in larval zebrafish and methods for testing the effects of pharmacological compounds on RPE regeneration are also outlined. Generation and validation of RpEGEN, a MATLAB script created to automate quantification of RPE regeneration based on pigmentation, is also discussed. Beyond active RPE repair mechanisms, this protocol can be expanded to studies of RPE degeneration and injury responses as well as the effects of RPE damage on adjacent retinal and vascular tissues, among other cellular and molecular processes. This zebrafish system holds significant promise in identifying genes, networks, and processes that drive RPE regeneration and RPE disease-related mechanisms, with the long-term goal of applying this knowledge to mammalian systems and, ultimately, toward therapeutic development.

Toxic Effects and Mechanisms of Silver and Zinc Oxide Nanoparticles on Zebrafish Embryos in Aquatic Ecosystems.

The global application of engineered nanomaterials and nanoparticles (ENPs) in commercial products, industry, and medical fields has raised some concerns about their safety. These nanoparticles may gain access into rivers and marine environments through industrial or household wastewater discharge and thereby affect the ecosystem. In this study, we investigated the effects of silver nanoparticles (AgNPs) and zinc oxide nanoparticles (ZnONPs) on zebrafish embryos in aquatic environments. We aimed to characterize the AgNP and ZnONP aggregates in natural waters, such as lakes, reservoirs, and rivers, and to determine whether they are toxic to developing zebrafish embryos. Different toxic effects and mechanisms were investigated by measuring the survival rate, hatching rate, body length, reactive oxidative stress (ROS) level, apoptosis, and autophagy. Spiking AgNPs or ZnONPs into natural water samples led to significant acute toxicity to zebrafish embryos, whereas the level of acute toxicity was relatively low when compared to Milli-Q (MQ) water, indicating the interaction and transformation of AgNPs or ZnONPs with complex components in a water environment that led to reduced toxicity. ZnONPs, but not AgNPs, triggered a significant delay of embryo hatching. Zebrafish embryos exposed to filtered natural water spiked with AgNPs or ZnONPs exhibited increased ROS levels, apoptosis, and lysosomal activity, an indicator of autophagy. Since autophagy is considered as an early indicator of ENP interactions with cells and has been recognized as an important mechanism of ENP-induced toxicity, developing a transgenic zebrafish system to detect ENP-induced autophagy may be an ideal strategy for predicting possible ecotoxicity that can be applied in the future for the risk assessment of ENPs.

Regeneration in the phylogenetic empire: an interview with Peter Currie.

Regeneration in the phylogenetic empire: an interview with Peter Currie.