ZenoFishDb v1.1: A Database for Xenotransplantation Studies in Zebrafish.

Rapidly accumulating literature has proven feasibility of the zebrafish xenograft models in cancer research. Nevertheless, online databases for searching the current zebrafish xenograft literature are in great demand. Herein, we have developed a manually curated database, called ZenoFishDb v1.1 (https://konulab.shinyapps.io/zenofishdb), based on R Shiny platform aiming to provide searchable information on ever increasing collection of zebrafish studies for cancer cell line transplantation and patient-derived xenografts (PDXs). ZenoFishDb v1.1 user interface contains four modules: DataTable, Visualization, PDX Details, and PDX Charts. The DataTable and Visualization pages represent xenograft study details, including injected cell lines, PDX injections, molecular modifications of cell lines, zebrafish strains, as well as technical aspects of the xenotransplantation procedures in table, bar, and/or pie chart formats. The PDX Details module provides comprehensive information on the patient details in table format and can be searched and visualized. Overall, ZenoFishDb v1.1 enables researchers to effectively search, list, and visualize different technical and biological attributes of zebrafish xenotransplantation studies particularly focusing on the new trends that make use of reporters, RNA interference, overexpression, or mutant gene constructs of transplanted cancer cells, stem cells, and PDXs, as well as distinguished host modifications.

Protocol for Dissection and Dissociation of Zebrafish Telencephalon for Single-Cell Sequencing.

Single-cell sequencing (sc-Seq) is a powerful tool to investigate the molecular signatures of cell types in a complex mixture of cells. A critical step in sc-Seq is preparing a single-cell suspension with a high number of viable cells. Here, we show how to dissect zebrafish telencephalon and how to dissociate it into a single-cell suspension. This is followed by flow cytometry-based sorting to enrich for neural progenitor stem cells. Our technique typically yields 70,000 live cells from one zebrafish telencephalon. For complete details on the use and execution of this protocol, please refer to Cosacak et al. (2019).

Effects of 17α­ethinylestradiol on caudal fin regeneration in zebrafish larvae.

The ability to restore tissue function and morphology after injury is a key advantage of many fish for a greater chance of survival. The tissue regeneration process is regulated by multiple pathways, and it can therefore be hypothesized that environmental contaminants targeting components of these signaling pathways, may disrupt the fish's capability to repair or regenerate. This could lead to higher mortality and eventually even to a decline in populations. In this study, the effects of 17α­ethinylestradiol (EE2), a synthetic estrogen, were assessed on the regenerative capacity of larval zebrafish. Zebrafish aged 2 hour post fertilization (hpf) were exposed to 1, 10, or 100 ng/L EE2, and the caudal fins were amputated at 72 hpf. It was found that EE2 exposure significantly inhibited fin regeneration and changed locomotor behavior. The transcription levels for most of the genes involved in the signaling networks regulating the fin regeneration, such as axin2, fgfr1, bmp2b and igf2b, were down-regulated in the amputated fish in response to EE2 exposure, which was in contrast to their increased patterns in the vehicle-exposed control fish. Additionally, the mRNA levels of several immune-related genes, such as il-1ß, il-6, il-10 and nf-κb2, were significantly decreased after EE2 exposure, accompanied by a lower density of neutrophils migrated into the wound site. In conclusion, the present study indicated for the first time that estrogenic endocrine disrupting chemicals (EEDCs) could inhibit the regenerative capacity of zebrafish, and this effect was speculated to be mediated through the alteration in regeneration-related signaling pathways and immune competence. This work expands our knowledge of the potential effects of EEDCs on injured aquatic organisms, and highlights the ecotoxicological significance of relationships between regenerative process and endocrine system. This study also implies the potential application of fin regeneration assay for assessing immunotoxicity in ecotoxicological risk assessment.

Differential oxidation of protein-tyrosine phosphatases during zebrafish caudal fin regeneration.

Zebrafish have the capacity to regenerate lost tissues and organs. Amputation of the caudal fin results in a rapid, transient increase in H2O2 levels emanating from the wound margin, which is essential for regeneration, because quenching of reactive oxygen species blocks regeneration. Protein-tyrosine phosphatases (PTPs) have a central role in cell signalling and are susceptible to oxidation, which results in transient inactivation of their catalytic activity. We hypothesized that PTPs may become oxidized in response to amputation of the caudal fin. Using the oxidized PTP-specific (ox-PTP) antibody and liquid chromatography-mass spectrometry, we identified 33 PTPs in adult zebrafish fin clips of the total of 44 PTPs that can theoretically be detected based on sequence conservation. Of these 33 PTPs, 8 were significantly more oxidized 40 min after caudal fin amputation. Surprisingly, Shp2, one of the PTPs that were oxidized in response to caudal fin amputation, was required for caudal fin regeneration. In contrast, Rptpα, which was not oxidized upon amputation, was dispensable for caudal fin regeneration. Our results demonstrate that PTPs are differentially oxidized in response to caudal fin amputation and that there is a differential requirement for PTPs in regeneration.

Behavioral Characteristics of Adult Zebrafish (Danio rerio) after MS222 Anesthesia for Fin Excision.

The health of laboratory animals is an ethical responsibility of researchers and a critical determinant of experimental outcome. Therefore, all husbandry procedures should be evaluated for their effects on mortality, behavior, and physiology to maximize animal welfare and minimize experimental variability. For adult zebrafish, the excision of a small portion of the caudal fin (that is, 'fin clipping') under MS222 anesthesia is a common procedure to obtain tissue for genotyping. The potential effect of this procedure on behavioral and physiologic assays of feeding, anxiety, and stress has not previously been assessed. Here, we evaluated feeding behavior, anxiety-associated behaviors, and physiologic indicators of stress at multiple time points within 24 h after performing a standard fin-clip procedure under MS222 anesthesia. Within 1 h of the procedure, fin-clipped fish showed a mild increase in anxiety and exhibited reduced feeding; however, these effects were short-lived, and the fish exhibited baseline levels of anxiety and feeding by 6 and 24 h after fin clipping. Together with the zebrafish's ability to regenerate fin tissue and the low mortality associated with fin clipping, our data support the continued practice of this technique under MS222 anesthesia as a routine husbandry procedure that is unlikely to alter experimental outcomes related to feeding, anxiety, or stress.

Developing a Novel Embryo-Larval Zebrafish Xenograft Assay to Prioritize Human Glioblastoma Therapeutics.

Glioblastoma is an aggressive brain cancer requiring improved treatments. Existing methods of drug discovery and development require years before new therapeutics become available to patients. Zebrafish xenograft models hold promise for prioritizing drug development. We have developed an embryo-larval zebrafish xenograft assay in which cancer cells are implanted in a brain microenvironment to discover and prioritize compounds that impact glioblastoma proliferation, migration, and invasion. We illustrate the utility of our assay by evaluating the well-studied, phosphatidylinositide 3-kinase inhibitor LY294002 and zinc oxide nanoparticles (ZnO NPs), which demonstrate selective cancer cytotoxicity in cell culture, but the in vivo effectiveness has not been established. Exposures of 3.125-6.25 µM LY294002 significantly decreased proliferation up to 34% with concentration-dependent trends. Exposure to 6.25 µM LY294002 significantly inhibited migration/invasion by ∼27% within the glioblastoma cell mass (0-80 µm) and by ∼32% in the next distance region (81-160 µm). Unexpectedly, ZnO enhanced glioblastoma proliferation by ∼19% and migration/invasion by ∼35% at the periphery of the cell mass (161+ µm); however, dissolution of these NPs make it difficult to discern whether this was a nano or ionic effect. These results demonstrate that we have a short, relevant, and sensitive zebrafish-based assay to aid glioblastoma therapeutic development.

Microbead Implantation in the Zebrafish Embryo.

The zebrafish has emerged as a valuable genetic model system for the study of developmental biology and disease. Zebrafish share a high degree of genomic conservation, as well as similarities in cellular, molecular, and physiological processes, with other vertebrates including humans. During early ontogeny, zebrafish embryos are optically transparent, allowing researchers to visualize the dynamics of organogenesis using a simple stereomicroscope. Microbead implantation is a method that enables tissue manipulation through the alteration of factors in local environments. This allows researchers to assay the effects of any number of signaling molecules of interest, such as secreted peptides, at specific spatial and temporal points within the developing embryo. Here, we detail a protocol for how to manipulate and implant beads during early zebrafish development.

Large-scale zebrafish embryonic heart dissection for transcriptional analysis.

The zebrafish embryonic heart is composed of only a few hundred cells, representing only a small fraction of the entire embryo. Therefore, to prevent the cardiac transcriptome from being masked by the global embryonic transcriptome, it is necessary to collect sufficient numbers of hearts for further analyses. Furthermore, as zebrafish cardiac development proceeds rapidly, heart collection and RNA extraction methods need to be quick in order to ensure homogeneity of the samples. Here, we present a rapid manual dissection protocol for collecting functional/beating hearts from zebrafish embryos. This is an essential prerequisite for subsequent cardiac-specific RNA extraction to determine cardiac-specific gene expression levels by transcriptome analyses, such as quantitative real-time polymerase chain reaction (RT-qPCR). The method is based on differential adhesive properties of the zebrafish embryonic heart compared with other tissues; this allows for the rapid physical separation of cardiac from extracardiac tissue by a combination of fluidic shear force disruption, stepwise filtration and manual collection of transgenic fluorescently labeled hearts.

Recovery of adult zebrafish hearts for high-throughput applications.

Use of the zebrafish model system for studying development, regeneration, and disease is expanding toward use of adult hearts for cell dissociation and purification of RNA, DNA, and proteins. All of these applications demand the rapid recovery of significant numbers of zebrafish hearts to avoid gene regulatory, metabolic, and other changes that begin after death. Adult zebrafish hearts are also required for studying heart structure for a variety of mutants and for studying heart regeneration. However, the traditional zebrafish heart dissection is slow and difficult and requires specialized tools, making large-scale dissection of adult zebrafish hearts tedious. Traditional methods also harbor the risk of damaging the heart during the dissection. Here, we describe a method for dissection of adult zebrafish hearts that is fast, reproducible, and preserves heart architecture. Furthermore, this method does not require specialized tools, is painless for the zebrafish, can be performed on fresh or fixed specimens, and can be performed on zebrafish as young as one month old. The approach described expands the use of adult zebrafish for cardiovascular research.

Spinal cord transection in the larval zebrafish.

Mammals fail in sensory and motor recovery following spinal cord injury due to lack of axonal regrowth below the level of injury as well as an inability to reinitiate spinal neurogenesis. However, some anamniotes including the zebrafish Danio rerio exhibit both sensory and functional recovery even after complete transection of the spinal cord. The adult zebrafish is an established model organism for studying regeneration following spinal cord injury, with sensory and motor recovery by 6 weeks post-injury. To take advantage of in vivo analysis of the regenerative process available in the transparent larval zebrafish as well as genetic tools not accessible in the adult, we use the larval zebrafish to study regeneration after spinal cord transection. Here we demonstrate a method for reproducibly and verifiably transecting the larval spinal cord. After transection, our data shows sensory recovery beginning at 2 days post-injury (dpi), with the C-bend movement detectable by 3 dpi and resumption of free swimming by 5 dpi. Thus we propose the larval zebrafish as a companion tool to the adult zebrafish for the study of recovery after spinal cord injury.

Dissection and lateral mounting of zebrafish embryos: analysis of spinal cord development.

The zebrafish spinal cord is an effective investigative model for nervous system research for several reasons. First, genetic, transgenic and gene knockdown approaches can be utilized to examine the molecular mechanisms underlying nervous system development. Second, large clutches of developmentally synchronized embryos provide large experimental sample sizes. Third, the optical clarity of the zebrafish embryo permits researchers to visualize progenitor, glial, and neuronal populations. Although zebrafish embryos are transparent, specimen thickness can impede effective microscopic visualization. One reason for this is the tandem development of the spinal cord and overlying somite tissue. Another reason is the large yolk ball, which is still present during periods of early neurogenesis. In this article, we demonstrate microdissection and removal of the yolk in fixed embryos, which allows microscopic visualization while preserving surrounding somite tissue. We also demonstrate semipermanent mounting of zebrafish embryos. This permits observation of neurodevelopment in the dorso-ventral and anterior-posterior axes, as it preserves the three-dimensionality of the tissue.

Electroablation: a method for neurectomy and localized tissue injury.

BACKGROUND: Tissue injury has been employed to study diverse biological processes such as regeneration and inflammation. In addition to physical or surgical based methods for tissue injury, current protocols for localized tissue damage include laser and two-photon wounding, which allow a high degree of accuracy, but are expensive and difficult to apply. In contrast, electrical injury is a simple and inexpensive technique, which allows reproducible and localized cell or tissue damage in a variety of contexts. RESULTS: We describe a novel technique that combines the advantages of zebrafish for in vivo visualization of cells with those of electrical injury methods in a simple and versatile protocol which allows the study of regeneration and inflammation. The source of the electrical pulse is a microelectrode that can be placed with precision adjacent to specific cells expressing fluorescent proteins. We demonstrate the use of this technique in zebrafish larvae by damaging different cell types and structures. Neurectomy can be carried out in peripheral nerves or in the spinal cord allowing the study of degeneration and regeneration of nerve fibers. We also apply this method for the ablation of single lateral line mechanosensory neuromasts, showing the utility of this approach as a tool for the study of organ regeneration. In addition, we show that electrical injury induces immune cell recruitment to damaged tissues, allowing in vivo studies of leukocyte dynamics during inflammation within a confined and localized injury. Finally, we show that it is possible to apply electroablation as a method of tissue injury and inflammation induction in adult fish. CONCLUSIONS: Electrical injury using a fine microelectrode can be used for axotomy of neurons, as a general tissue ablation tool and as a method to induce a powerful inflammatory response. We demonstrate its utility to studies in both larvae and in adult zebrafish but we expect that this technique can be readily applied to other organisms as well. We have called this method of electrical based tissue ablation, electroablation.

[A surgery protocol to construct zebrafish heart damage and regeneration model].

In recent years, zebrafish has been found to be strikingly capable of heart regeneration at adult stage, which sheds lights on cardiac regenerative medicine, and has also become an important direction for the study of vertebrate genetic and developmental mechanisms. Dissecting the regeneration process and unraveling the underlying molecular and cellular mechanisms might help recover the regeneration capacity of the mammalian heart and further provide instructions to potential therapies for certain cardiac diseases such as myocardial infarction. Here, we introduced a simple surgical method to construct heart injury and regeneration model in zebrafish through amputation of ~20% ventricle. The main procedure includes anaesthetization of adult zebrafish, exposure of the heart by dissecting the nearby abdominal skin and opening the pericardium, and amputation of certain fraction of the ventricle at the cardiac apex under a stereo microscope. Over 90% success rate and easy handling with high reproducibility enable this method to be the most commonly used one for the study of zebrafish heart regeneration.

A surgery protocol for adult zebrafish spinal cord injury.

Adult zebrafish has a remarkable capability to recover from spinal cord injury, providing an excellent model for studying neuroregeneration. Here we list equipment and reagents, and give a detailed protocol for complete transection of the adult zebrafish spinal cord. In this protocol, potential problems and their solutions are described so that the zebrafish spinal cord injury model can be more easily and reproducibly performed. In addition, two assessments are introduced to monitor the success of the surgery and functional recovery: one test to assess free swimming capability and the other test to assess extent of neuroregeneration by in vivo anterograde axonal tracing. In the swimming behavior test, successful complete spinal cord transection is monitored by the inability of zebrafish to swim freely for 1 week after spinal cord injury, followed by the gradual reacquisition of full locomotor ability within 6 weeks after injury. As a morphometric correlate, anterograde axonal tracing allows the investigator to monitor the ability of regenerated axons to cross the lesion site and increasingly extend into the gray and white matter with time after injury, confirming functional recovery. This zebrafish model provides a paradigm for recovery from spinal cord injury, enabling the identification of pathways and components of neuroregeneration.

Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development, however, work examining post-embryonic through adult cardiac development has been limited. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5 mm standard length (SL) with hearts of 100 µm ventricle length (VL), to adults, with SL of 30 mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques. We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)(twu34), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Characterization of immune-matched hematopoietic transplantation in zebrafish.

Evaluating hematopoietic stem cell (HSC) function in vivo requires a long-term transplantation assay. Although zebrafish are a powerful model for discovering the genetics of hematopoiesis, hematopoietic transplantation approaches have been underdeveloped. Here we established a long-term reconstitution assay in adult zebrafish. Primary and secondary recipients showed multilineage engraftment at 3 months after transplantation. Limiting dilution data suggest that at least 1 in 65 000 zebrafish marrow cells contain repopulating activity, consistent with mammalian HSC frequencies. We defined zebrafish haplotypes at the proposed major histocompatibility complex locus on chromosome 19 and tested functional significance through hematopoietic transplantation. Matching donors and recipients dramatically increased engraftment and percentage donor chimerism compared with unmatched fish. These data constitute the first functional test of zebrafish histocompatibility genes, enabling the development of matched hematopoietic transplantations. This lays the foundation for competitive transplantation experiments with mutant zebrafish HSCs and chemicals to test for effects on engraftment, thereby providing a model for human hematopoietic diseases and treatments not previously available.

Leukemic cell xenograft in zebrafish embryo for investigating drug efficacy.

Zebrafish were proposed as an alternative to mammalian models to assess the efficacy and toxicity of antileukemic drugs. Due to the limited number of transgenic zebrafish leukemia models, we explored human leukemic cell xenograft in zebrafish embryos. Human leukemic cell lines and blast cells sorted from patients with acute myelogenous leukemia were injected 48 hours post-fertilization and remained in the circulation of zebrafish embryos for several days without affecting their development. Imatinib and oxaphorines did not demonstrate any toxicity on normal zebrafish embryos and decreased the leukemic burden in animals xenografted with sensitive leukemic cell lines. Two other molecules, all-trans retinoic acid and the translation inhibitor 4EGI-1, demonstrated teratogenic effects at concentrations shown to be efficient in vitro, which precluded investigation of their antileukemic activity in such models. Altogether, xenografted leukemic cells in zebrafish embryos are a pharmacologically relevant model for screening non-teratogenic drugs.

Microdissection of zebrafish embryonic eye tissues.

Zebrafish is a popular animal model for research on eye development because of its rapid ex utero development and good fecundity. By 3 days post fertilization (dpf), the larvae will show the first visual response. Many genes have been identified to control a proper eye development, but we are far from a complete understanding of the underlying genetic architecture. Whole genome gene expression profiling is a useful tool to elucidate genetic regulatory network for eye development. However, the small size of the embryonic eye in zebrafish makes it challenging to obtain intact and pure eye tissues for expression analysis. For example, the anterior-posterior length of the eye between day 2 and 3 is only approximately 200-300 microm, while the diameter of the lens is less 100 microm. Also, the retinal pigment epithelium (RPE) underlying the retina is just a single-layer epithelium. While gene expression profiles can be obtained from the whole embryo, they do not accurately represent the expression of these tissues. Therefore pure tissue must be obtained for a successful gene expression profiling of eye development. To address this issue, we have developed an approach to microdissect intact retina and retina with RPE attached from 1-3 dpf, which cover major stages of eye morphogenesis. All procedures can be done with fine forceps and general laboratory supplies under standard stereomicroscopes. For retinal dissection, the single-layer RPE is removed and peeled off by brushing action and the preferential adherence of the RPE remnants to the surface of the culture plate for dissection. For RPE-attached retinal dissection, the adherence of RPE to the dissection plate is removed before the dissection so that the RPE can be completely preserved with the retina. A careful lifting action of this tissue can efficiently separate the presumptive choroid and sclera. The lens can be removed in both cases by a chemically etched tungsten needle. In short, our approach can obtain intact eye tissues and has been successfully utilized to study tissue-specific expression profiles of zebrafish retina and retinal pigment epithelium.

Dissection of organs from the adult zebrafish.

Over the last 20 years, the zebrafish has become a powerful model organism for understanding vertebrate development and disease. Although experimental analysis of the embryo and larva is extensive and the morphology has been well documented, descriptions of adult zebrafish anatomy and studies of development of the adult structures and organs, together with techniques for working with adults are lacking. The organs of the larva undergo significant changes in their overall structure, morphology, and anatomical location during the larval to adult transition. Externally, the transparent larva develops its characteristic adult striped pigment pattern and paired pelvic fins, while internally, the organs undergo massive growth and remodeling. In addition, the bipotential gonad primordium develops into either testis or ovary. This protocol identifies many of the organs of the adult and demonstrates methods for dissection of the brain, gonads, gastrointestinal system, heart, and kidney of the adult zebrafish. The dissected organs can be used for in situ hybridization, immunohistochemistry, histology, RNA extraction, protein analysis, and other molecular techniques. This protocol will assist in the broadening of studies in the zebrafish to include the remodeling of larval organs, the morphogenesis of organs specific to the adult and other investigations of the adult organ systems.

Gross and fine dissection of inner ear sensory epithelia in adult zebrafish (Danio rerio).

Neurosensory epithelia in the inner ear are the crucial structures for hearing and balance functions. Therefore, it is important to understand the cellular and molecular features of the epithelia, which are mainly composed of two types of cells: hair cells (HCs) and supporting cells (SCs). Here we choose to study the inner ear sensory epithelia in adult zebrafish not only because the epithelial structures are highly conserved in all vertebrates studied, but also because the adult zebrafish is able to regenerate HCs, an ability that mammals lose shortly after birth. We use the inner ear of adult zebrafish as a model system to study the mechanisms of inner ear HC regeneration in adult vertebrates that could be helpful for clinical therapy of hearing/balance deficits in human as a result of HC loss. Here we demonstrate how to do gross and fine dissections of inner ear sensory epithelia in adult zebrafish. The gross dissection removes the tissues surrounding the inner ear and is helpful for preparing tissue sections, which allows us to examine the detailed structure of the sensory epithelia. The fine dissection cleans up the non-sensory-epithelial tissues of each individual epithelium and enables us to examine the heterogeneity of the whole epithelium easily in whole-mount epithelial samples.