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Key Points Phenotypic screening has been one of the most effective approaches to drug discovery, but only a subset of disease-associated phenotypes can be modelled in cultured cells. Zebrafish exhibit a much broader range of disease-associated phenotypes, including disorders of physiology, metabolism and behaviour, while retaining the ability to be used for high-throughput applications. Zebrafish are phylogenetically more distant from humans than rodents are, but they possess orthologues of 82% of human disease-associated genes. In many cases, they exhibit physiological and pharmacological conservation that approaches (and occasionally surpasses) that of rodents. Zebrafish screens have discovered a few compounds that have made it to advanced preclinical and clinical trials, including new compound classes and repurposed drugs. Zebrafish have been used in several toxicology applications, including the screening of large compound collections for potential liabilities. Although target identification remains a challenge for phenotype-based small-molecule discovery, rich databases of zebrafish phenotypes have facilitated the identification of targets for several small molecules. Rapid advances in genome editing and high-throughput phenotyping point to promising new applications for zebrafish in drug discovery over the coming years, including the discovery of compounds that suppress disease phenotypes associated with specific human mutations. The zebrafish, a well-established model organism in basic biomedical research, has also become tractable for numerous applications in drug discovery, from phenotypic screens to testing for toxicity. In this Review, MacRae and Peterson discuss how zebrafish have been used in such applications, advances that could make it an increasingly attractive tool — particularly for phenotypic screening — and the challenges that need to be addressed. The zebrafish has become a prominent vertebrate model for disease and has already contributed to several examples of successful phenotype-based drug discovery. For the zebrafish to become useful in drug development more broadly, key hurdles must be overcome, including a more comprehensive elucidation of the similarities and differences between human and zebrafish biology. Recent studies have begun to establish the capabilities and limitations of zebrafish for disease modelling, drug screening, target identification, pharmacology, and toxicology. As our understanding increases and as the technologies for manipulating zebrafish improve, it is hoped that the zebrafish will have a key role in accelerating the emergence of precision medicine.

We report the evolution of mScarlet3, a cysteine-free monomeric red fluorescent protein with fast and complete maturation, as well as record brightness, quantum yield (75%) and fluorescence lifetime (4.0 ns). The mScarlet3 crystal structure reveals a barrel rigidified at one of its heads by a large hydrophobic patch of internal residues. mScarlet3 behaves well as a fusion tag, displays no apparent cytotoxicity and it surpasses existing red fluorescent proteins as a Förster resonance energy transfer acceptor and as a reporter in transient expression systems. Two mScarlet variants with high brightness and fast maturation times have been evolved. These variants behave favorably as fusion tags and Förster resonance energy transfer acceptors.

Zebrafish have several advantages compared to other vertebrate models used in modeling human diseases, particularly for large-scale genetic mutant and therapeutic compound screenings, and other biomedical research applications. With the impactful developments of CRISPR and next-generation sequencing technology, disease modeling in zebrafish is accelerating the understanding of the molecular mechanisms of human genetic diseases. These efforts are fundamental for the future of precision medicine because they provide new diagnostic and therapeutic solutions. This review focuses on zebrafish disease models for biomedical research, mainly in developmental disorders, mental disorders, and metabolic diseases. Animal models: Zebrafish help unlock clues to human disease With their see-through bodies, low maintenance costs and genetic similarity to humans, zebrafish provide a powerful animal model for studying mental disorders and metabolic diseases in the laboratory. Tae-Young Choi from Wonkwang University, Iksan, South Korea, and coworkers review the many physiological advantages and logistical benefits of rearing these small tropical fish for biomedical research. These include the ease of tissue imaging, the large number of offspring in each generation and the increasing number of genetic techniques available. The researchers highlight the various ways in which zebrafish have contributed to scientists’ understanding of mental disorders and the communication pathways between brain and other organs in the body. They also discuss the potential of zebrafish for tracking metabolism and how it can go awry in various disease settings.

Visualizing dynamic processes over large, three-dimensional fields of view at high speed is essential for many applications in the life sciences. Light-field microscopy (LFM) has emerged as a tool for fast volumetric image acquisition, but its effective throughput and widespread use in biology has been hampered by a computationally demanding and artifact-prone image reconstruction process. Here, we present a framework for artificial intelligence–enhanced microscopy, integrating a hybrid light-field light-sheet microscope and deep learning–based volume reconstruction. In our approach, concomitantly acquired, high-resolution two-dimensional light-sheet images continuously serve as training data and validation for the convolutional neural network reconstructing the raw LFM data during extended volumetric time-lapse imaging experiments. Our network delivers high-quality three-dimensional reconstructions at video-rate throughput, which can be further refined based on the high-resolution light-sheet images. We demonstrate the capabilities of our approach by imaging medaka heart dynamics and zebrafish neural activity with volumetric imaging rates up to 100 Hz.A deep learning–based algorithm enables efficient reconstruction of light-field microscopy data at video rate. In addition, concurrently acquired light-sheet microscopy data provide ground truth data for training, validation and refinement of the algorithm.

The mechanical properties of tissues can be measured by deforming magnetically responsive microdroplets that are implanted in the tissue. Serwane et al . apply this method to study the mechanical properties of tissues in the living zebrafish embryo. The mechanical properties of the cellular microenvironment and their spatiotemporal variations are thought to play a central role in sculpting embryonic tissues, maintaining organ architecture and controlling cell behavior, including cell differentiation. However, no direct in vivo and in situ measurement of mechanical properties within developing 3D tissues and organs has yet been performed. Here we introduce a technique that employs biocompatible, magnetically responsive ferrofluid microdroplets as local mechanical actuators and allows quantitative spatiotemporal measurements of mechanical properties in vivo . Using this technique, we show that vertebrate body elongation entails spatially varying tissue mechanics along the anteroposterior axis. Specifically, we find that the zebrafish tailbud is viscoelastic (elastic below a few seconds and fluid after just 1 min) and displays decreasing stiffness and increasing fluidity toward its posterior elongating region. This method opens new avenues to study mechanobiology in vivo , both in embryogenesis and in disease processes, including cancer.

Dense reconstruction of synaptic connectivity requires high-resolution electron microscopy images of entire brains and tools to efficiently trace neuronal wires across the volume. To generate such a resource, we sectioned and imaged a larval zebrafish brain by serial block-face electron microscopy at a voxel size of 14 × 14 × 25 nm 3 . We segmented the resulting dataset with the flood-filling network algorithm, automated the detection of chemical synapses and validated the results by comparisons to transmission electron microscopic images and light-microscopic reconstructions. Neurons and their connections are stored in the form of a queryable and expandable digital address book. We reconstructed a network of 208 neurons involved in visual motion processing, most of them located in the pretectum, which had been functionally characterized in the same specimen by two-photon calcium imaging. Moreover, we mapped all 407 presynaptic and postsynaptic partners of two superficial interneurons in the tectum. The resource developed here serves as a foundation for synaptic-resolution circuit analyses in the zebrafish nervous system. This Resource presents a serial block-face EM dataset of the whole larval zebrafish brain, including automated segmentation of neurons, detection of synapses and reconstruction of circuitry for visual motion processing.

Z-Brain is an atlas of the larval zebrafish brain. It can be combined with pERK-based neural-activity measurements from freely behaving zebrafish to identify brain regions involved in generating behavior. In order to localize the neural circuits involved in generating behaviors, it is necessary to assign activity onto anatomical maps of the nervous system. Using brain registration across hundreds of larval zebrafish, we have built an expandable open-source atlas containing molecular labels and definitions of anatomical regions, the Z-Brain. Using this platform and immunohistochemical detection of phosphorylated extracellular signal–regulated kinase (ERK) as a readout of neural activity, we have developed a system to create and contextualize whole-brain maps of stimulus- and behavior-dependent neural activity. This mitogen-activated protein kinase (MAP)-mapping assay is technically simple, and data analysis is completely automated. Because MAP-mapping is performed on freely swimming fish, it is applicable to studies of nearly any stimulus or behavior. Here we demonstrate our high-throughput approach using pharmacological, visual and noxious stimuli, as well as hunting and feeding. The resultant maps outline hundreds of areas associated with behaviors.

Understanding complex biological systems requires visualizing structures and processes deep within living organisms. We developed a compact adaptive optics module and incorporated it into two- and three-photon fluorescence microscopes, to measure and correct tissue-induced aberrations. We resolved synaptic structures in deep cortical and subcortical areas of the mouse brain, and demonstrated high-resolution imaging of neuronal structures and somatosensory-evoked calcium responses in the mouse spinal cord at great depths in vivo.A compact adaptive optics module corrects aberrations in two-photon and three-photon microscopy, enabling structural and functional imaging deep in the mouse brain, the mouse spinal cord and the zebrafish larva.

Tissue morphogenesis is driven by mechanical forces that elicit changes in cell size, shape and motion. The extent by which forces deform tissues critically depends on the rheological properties of the recipient tissue. Yet, whether and how dynamic changes in tissue rheology affect tissue morphogenesis and how they are regulated within the developing organism remain unclear. Here, we show that blastoderm spreading at the onset of zebrafish morphogenesis relies on a rapid, pronounced and spatially patterned tissue fluidization. Blastoderm fluidization is temporally controlled by mitotic cell rounding-dependent cell–cell contact disassembly during the last rounds of cell cleavages. Moreover, fluidization is spatially restricted to the central blastoderm by local activation of non-canonical Wnt signalling within the blastoderm margin, increasing cell cohesion and thereby counteracting the effect of mitotic rounding on contact disassembly. Overall, our results identify a fluidity transition mediated by loss of cell cohesion as a critical regulator of embryo morphogenesis. Studying blastoderm spreading in zebrafish, Petridou et al. discover that this process is facilitated by tissue fluidization, mediated by a local loss of cell–cell adhesion during mitotic rounding and spatially restricted by Wnt.

Behaviors emerge from activity throughout the brain, but noninvasive optical access in adult vertebrate brains is limited. We show that three-photon (3P) imaging through the head of intact adult zebrafish allows structural and functional imaging at cellular resolution throughout the telencephalon and deep into the cerebellum and optic tectum. With 3P imaging, considerable portions of the brain become noninvasively accessible from embryo to sexually mature adult in a vertebrate model. Three-photon microscopy provides access to most of the adult zebrafish brain for both structural and functional imaging, as well as to the Danionella dracula brain.