Isolation of Tissue Macrophages in Adult Zebrafish.

Tissue macrophages are essential components of the immune system that also play key roles in vertebrate development and homeostasis, including in zebrafish, which has gained popularity over the years as a translational model for human disease. Commonly, zebrafish macrophages are identified based on expression of fluorescent transgenic reporters, allowing for real-time imaging in living animals. Several of these lines have also proven instrumental to isolate pure populations of macrophages in the developing embryo and larvae using fluorescence-activated cell sorting (FACS). However, the identification of tissue macrophages in adult fish is not as clear, and robust protocols are needed that would take into account changes in reporter specificity as well as the heterogeneity of mononuclear phagocytes as fish reach adulthood. In this chapter, we describe the methodology for analyzing macrophages in various tissues in the adult zebrafish by flow cytometry. Coupled with FACS, these protocols further allow for the prospective isolation of enriched populations of tissue-specific mononuclear phagocytes that can be used in downstream transcriptomic and/or epigenomic analyses. Overall, we aim at providing a guide for the zebrafish community based on our expertise investigating the adult mononuclear phagocyte system.

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-oncogene biology, and whether any analyzed pathways converge for potential clinically actionable targets.

Delivering Traumatic Brain Injury to Larval Zebrafish.

We describe a straightforward, scalable method for administering traumatic brain injury (TBI) to zebrafish larvae. The pathological outcomes appear generalizable for all TBI types, but perhaps most closely model closed-skull, diffuse lesion (blast injury) neurotrauma. The injury is delivered by dropping a weight onto the plunger of a fluid-filled syringe containing zebrafish larvae. This model is easy to implement, cost-effective, and provides a high-throughput system that induces brain injury in many larvae at once. Unique to vertebrate TBI models, this method can be used to deliver TBI without anesthetic or other metabolic agents. The methods simulate the main aspects of traumatic brain injury in humans, providing a preclinical model to study the consequences of this prevalent injury type and a way to explore early interventions that may ameliorate subsequent neurodegeneration. We also describe a convenient method for executing pressure measurements to calibrate and validate this method. When used in concert with the genetic tools readily available in zebrafish, this model of traumatic brain injury offers opportunities to examine many mechanisms and outcomes induced by traumatic brain injury. For example, genetically encoded fluorescent reporters have been implemented with this system to measure protein misfolding and neural activity via optogenetics.

Cancer Modeling by Transgene Electroporation in Adult Zebrafish (TEAZ).

Transgenic expression of genes is a mainstay of cancer modeling in zebrafish. Traditional transgenic techniques rely upon injection into one-cell embryos, but ideally these transgenes would be expressed only in adult somatic tissues. We provide a method to model cancer in adult zebrafish in which transgenes can be expressed via electroporation. Using melanoma as an example, we demonstrate the feasibility of expressing oncogenes such as BRAFV600E as well as CRISPR/Cas9 inactivation of tumor suppressors such as PTEN. These approaches can be performed in any genetic background such as existing fluorophore reporter lines or the casper line. These methods can readily be extended to other cell types allowing for rapid adult modeling of cancer in zebrafish.

Genetic Identification of Neural Circuits Essential for Active Avoidance Fear Conditioning in Adult Zebrafish.

Inhibition or ablation of neuronal activity combined with behavioral assessment is crucial in identifying neural circuits or populations essential for specific behaviors and to understand brain function. In the model vertebrate zebrafish, the development of genetic methods has allowed not only visualization but also targeted manipulation of neuronal activity, and quantitative behavioral assays allow precise measurement of animal behavior. Here, we describe a method to inhibit a specific neuronal population in adult zebrafish brain and assess their role in a learning behavior. We employed the Gal4-UAS system, gene trap and enhancer trap methods, and isolated transgenic zebrafish lines expressing Gal4FF transactivator in specific populations of neurons in the adult zebrafish brain. In these lines, a genetically engineered neurotoxin, botulinum toxin B light chain, was expressed and the fish were assessed in the active avoidance fear conditioning paradigm. The transgenic lines that showed impaired avoidance response were isolated and, in these fish, the Gal4-expressing neurons were analyzed to identify the neuronal circuits involved in avoidance learning.

Optimized Primary Culture of Neuronal Populations for Subcellular Omics Applications.

Primary cell culture is an invaluable method frequently used to overcome challenges associated with in vivo experiments. In zebrafish research, in vivo live imaging experiments are routine owing to the high optical transparency of embryos, and, as a result, primary cell culture has been less utilized. However, the approach still boasts powerful advantages, emphasizing the importance of sophisticated zebrafish cell culture protocols. Here, we present an enhanced protocol for the generation of primary cell cultures by dissociation of 24 hpf zebrafish embryos. We include a novel cell culture medium recipe specifically favoring neuronal growth and survival, enabling relatively long-term culture. We outline primary zebrafish neuronal culture on glass coverslips, as well as in transwell inserts which allow isolation of neurite tissue for experiments such as investigating subcellular transcriptomes.

Selective Cell Ablation Using an Improved Prodrug-Converting Nitroreductase.

Selective cell ablation is an invaluable tool to investigate the function of cell types, the regeneration of cells, and the modeling of diseases associated with cell loss. The nitroreductase (NTR)-mediated cell ablation system is a simple method enabling the elimination of targeted cells through the expression of a nitroreductase enzyme and the application of a prodrug (such as metronidazole). The prodrug is reduced to a cytotoxic product by nitroreductase, thereby leading to DNA damage-induced cell death. In species with elevated regenerative capacity such as zebrafish, removing the prodrug allows endogenous tissue to replace the lost cells. Herein, we describe a method for the use of a markedly improved nitroreductase enzyme for spatially and temporally controlled targeted cell ablation in the zebrafish. Recently, we identified an NTR variant (NTR 2.0) that achieves effective targeted cell ablation at concentrations of metronidazole well below those causing toxic side effects. NTR 2.0 thereby enables the ablation of "resistant" cell types and novel cell ablation paradigms. These advances simplify investigations of cell function, enable interrogations of the effects of chronic inflammation on regenerative processes and facilitate modeling of degenerative diseases associated with chronic cell loss. Techniques for transgenic nitroreductase expression and prodrug application are discussed.

Lineage Tracing of Bone Cells in the Regenerating Fin and During Repair of Bone Lesions.

Small teleost fishes such as zebrafish and medaka show remarkable regeneration capabilities upon tissue injury or amputation. To elucidate cellular mechanisms of teleost tissue repair and regeneration processes, the Cre/LoxP recombination system for cell lineage tracing is a widely used technique. In this chapter, we describe protocols used for inducible Cre/LoxP recombination-mediated lineage tracing of osteoblast progenitors during medaka fin regeneration as well as during the repair of osteoporosis-like bone lesions in the medaka vertebral column. Our approach can be adapted for lineage tracing of other cell populations in the regenerating teleost fin or in other tissues undergoing repair.

Generation of Conditional Knockout Zebrafish Using an Invertible Gene-Trap Cassette.

Conditional knockout (cKO) is a genetic technique to inactivate gene expression in specific tissues or cell types in a temporally regulated manner. cKO analysis is essential to investigate gene function while avoiding the confounding effects of global gene deletion. Genetic techniques enabling cKO analysis were developed in mice based on culturable embryonic stem cells that were not generally available in zebrafish, which hampered precise analysis of genetic mechanisms of organ development and regeneration. However, recent advances in genome editing technologies have resolved this limitation, providing a platform for the generation of cKO models in any organism. Here we describe a detailed protocol for the generation of cKO zebrafish using a Cre-dependent genetic switch.

Holographic Optogenetic Activation of Neurons Eliciting Locomotion in Head-Embedded Larval Zebrafish.

Understanding how motor circuits are organized and recruited in order to perform complex behavior is an essential question of neuroscience. Here we present an optogenetic protocol on larval zebrafish that allows spatial selective control of neuronal activity within a genetically defined population. We combine holographic illumination with the use of effective opsin transgenic lines, alongside high-speed behavioral monitoring to dissect the motor circuits of the larval zebrafish.

Spinal Cord Injury and Assays for Regeneration.

Due to their renowned regenerative capacity, adult zebrafish are a premier vertebrate model to interrogate mechanisms of innate spinal cord regeneration. Following complete transection to their spinal cord, zebrafish extend glial and axonal bridges across severed tissue, regenerate neurons proximal to the lesion, and regain swim capacity within 8 weeks of injury. Here, we describe methods to perform complete spinal cord transections and to assess functional and cellular recovery during regeneration. For spinal cord injury, a complete transection is performed 4 mm caudal to the brainstem. Swim endurance is quantified as a central readout of functional spinal cord repair. For swim endurance, zebrafish are subjected to a constantly increasing water current velocity until exhaustion, and time at exhaustion is reported. To assess cellular regeneration, histological examination is performed to analyze the extents of glial and axonal bridging across the lesion.

Simultaneous Behavioral and Neuronal Imaging by Tracking Microscopy.

Tracking microscopy enables whole-brain cellular resolution imaging in freely swimming animals. This technique enables both structural and functional imaging without immobilizing the animal, and greatly expands the range of the behaviors accessible to neuroscientists. We use infrared imaging to track the target animal in a behavioral arena. Based on the predicted trajectory of the brain, we apply optimal control theory to a motorized stage system to cancel brain motion in three dimensions. We have combined this motion cancellation system with Differential Illumination Focal Filtering (DIFF), a form of structured illumination microscopy, which enables us to image the brain of a freely swimming larval zebrafish for over an hour. Here we describe the typical experimental procedure for data acquisition and processing using the tracking microscope.

Methods to Study Liver Disease Using Zebrafish Larvae.

Liver disease affects millions of people worldwide, and the high morbidity and mortality is attributed in part to the paucity of treatment options. In many cases, liver injury self-resolves due to the remarkable regenerative capacity of the liver, but in cases when regeneration cannot compensate for the injury, inflammation and fibrosis occur, creating a setting for the emergence of liver cancer. Whole animal models are crucial for deciphering the basic biological underpinnings of liver biology and pathology and, importantly, for developing and testing new treatments for liver disease before it progresses to a terminal state. The cellular components and functions of the zebrafish liver are highly similar to mammals, and zebrafish develop many diseases that are observed in humans, including toxicant-induced liver injury, fatty liver, fibrosis, and cancer. Therefore, the widespread use of zebrafish larvae for studying the mechanisms of these pathologies and for developing potential treatments necessitates the optimization of experimental approaches to assess liver disease in this model. Here, we describe protocols using staining methods, imaging, and gene expression analysis to assess liver injury, fibrosis, and preneoplastic changes in the liver of larval zebrafish.

Brain Imaging and Registration in Larval Zebrafish.

Registration of larval zebrafish brain scans to a common reference brain enables comparison of transgene and gene expression patterns, neuroanatomy, and morphometry. Here we describe methods for staining and mounting larval zebrafish to facilitate whole-brain fluorescence imaging. Following image acquisition, we provide a template for aligning brain images to a reference atlas using nonlinear registration with the ANTs software package.

Quantitative Live Imaging of Zebrafish Scale Regeneration: From Adult Fish to Signaling Patterns and Tissue Flows.

In regeneration, a damaged body part grows back to its original form. Understanding the mechanisms and physical principles underlying this process has been limited by the difficulties of visualizing cell signals and behaviors in regeneration. Zebrafish scales are emerging as a model system to investigate morphogenesis during vertebrate regeneration using quantitative live imaging. Scales are millimeter-sized dermal bone disks forming a skeletal armor on the body of the fish. The scale bone is deposited by an adjacent monolayer of osteoblasts that, after scale loss, regenerates in about 2 weeks. This intriguing regenerative process is accessible to live confocal microscopy, quantifications, and mathematical modeling. Here, I describe methods to image scale regeneration live, tissue-wide and at sub-cellular resolution. Furthermore, I describe methods to process the resulting images and quantify cell, tissue, and signal dynamics.

Section Immunostaining for Protein Expression and Cell Proliferation Studies of Regenerating Fins.

Adult zebrafish fins fully regenerate after resection, providing a highly accessible and remarkable vertebrate model of organ regeneration. Fin injury triggers wound epidermis formation and the dedifferentiation of injury-adjacent mature cells to establish an organized blastema of progenitor cells. Balanced cell proliferation and redifferentiation along with cell movements then progressively reestablish patterned tissues and restore the fin to its original size and shape. A mechanistic understanding of these coordinated cell behaviors and transitions requires direct knowledge of proteins in their physiological context, including expression, subcellular localization, and activity. Antibody-based staining of sectioned fins facilitates such high-resolution analyses of specific, native proteins. Therefore, such methods are mainstays of comprehensive, hypothesis-driven fin regeneration studies. However, section immunostaining requires labor-intensive, empirical optimization. Here, we present detailed, multistep procedures for antibody staining and co-detecting proliferating cells using paraffin and frozen fin sections. We include suggestions to avoid common pitfalls and to streamline the development of optimized, validated protocols for new and challenging antibodies.

Scalable CRISPR Screens in Zebrafish Using MIC-Drop.

CRISPR-Cas9 is a powerful tool to interrogate gene function in a targeted and systematic manner. Although the technology has been scaled up for large-scale genetic screens in cell culture, similar scale screens in vivo have been extremely challenging due to the cost, labor, and time required to generate and keep track of thousands of mutant animals. We reported the development of Multiplexed Intermixed CRISPR Droplets (MIC-Drop), a platform that makes large-scale reverse genetic screens possible in zebrafish. In this chapter, we provide a detailed protocol to conduct large-scale genetic screens using this novel platform.

The Goldfish Genome and Its Utility for Understanding Gene Regulation and Vertebrate Body Morphology.

Goldfish, widely viewed as an ornamental fish, is a member of Cyprinidae family and has a very long history in research for both genetics and physiology studies. Among Cyprinidae, the chromosomal locations of orthologs and the amino acid sequences are usually highly conserved. Adult goldfish are 1000 times larger than adult zebrafish (who are in the same family of fishes), which can make it easier to perform several types of experiments compared to their zebrafish cousins. Comparing mutant phenotypes in orthologous genes between goldfish and zebrafish can often be very informative and provide a deeper insight into the gene function than studying the gene in either species alone. Comparative genomics and phenotypic comparisons between goldfish and zebrafish will provide new opportunities for understanding the development and evolution of body forms in the vertebrate lineage.

Colorimetric Barcoding to Track, Isolate, and Analyze Hematopoietic Stem Cell Clones.

In zebrafish, hematopoietic stem cells (HSCs) are born in the developing aorta during embryogenesis. From the definitive wave of hematopoiesis onward, blood homeostasis relies on self-renewal and differentiation of progeny of existing HSCs, or clones, rather than de novo generation. Here, we describe an approach to quantify the number and size of HSC clones at various times throughout the lifespan of the animal using a fluorescent, multicolor labeling strategy. The system is based on combining the multicolor Zebrabow system with an inducible, early lateral plate mesoderm and hematopoietic lineage specific cre driver (draculin (drl)). The cre driver can be temporally controlled and activated in early hematopoiesis to introduce a color barcoding unique to each HSC and subsequently inherited by their daughter cells. Clonal diversity and dominance can be investigated in normal development and blood disease progression, such as blood cancers. This adoptable method allows researchers to obtain quantitative insight into clonality-defining events and their contribution to adult hematopoiesis.

Developmental Toxicity Assessment Using Zebrafish-Based High-Throughput Screening.

Zebrafish-based high-throughput screening has been extensively used to study toxicological profiles of individual chemicals and mixtures, identify novel toxicants, and study modes of action to prioritize chemicals for further testing and policy decisions. Within this chapter, we describe a protocol for automated zebrafish developmental high-throughput screening in our laboratory, with emphasis on exposure setups, morphological and behavioral readouts, and quality control.