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Danionella translucida (DT) are cyprinid fish with the smallest known vertebrate brain, a small transparent body, and a partially open cranium. They are also very closely related to the more popular genetic model zebrafish. This makes the adaptability of established genetic tools for transgenesis and mutagenesis easier. These features, along with a diverse behavioral repertoire, make them an interesting model organism in neuroscience to investigate complex behaviors. In addition to neuroscience, they can also be a valuable model organism in developmental and evolutionary biology, and for modeling adult-onset diseases. Here, we describe the biology and ecology of this fish with an emphasis on the research questions that are made more accessible with this new fish model. We also report the husbandry and breeding practices used in our laboratory, which can make it easier for other laboratories to house this fish species.

Studying neuronal circuits at cellular resolution is very challenging in vertebrates due to the size and optical turbidity of their brains. Danionella translucida, a close relative of zebrafish, was recently introduced as a model organism for investigating neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of this novel model organism., Design Type(s)sequence assembly objective • sequence annotation objective • transcription profiling designMeasurement Type(s)transcription profiling assay • genomeTechnology Type(s)RNA sequencing • DNA sequencingFactor Type(s)ageSample Characteristic(s)Danionella translucida • whole body Machine-accessible metadata file describing the reported data (ISA-Tab format)

Genetic engineering techniques have rapidly developed since the first large-scale forward genetics screen was developed in 1996, which generated a spectrum of phenotypic zebrafish lines with mutations in many essential genes for normal development. Management of this original collection was relatively straightforward and embryonic analysis was the focus. Adults were heterozygous in their genotype, and phenotypes were primarily within homozygous embryos. Homozygous fish were rarely viable and reporter lines were in their molecular infancy. Genotyping methods were not available since extensive molecular investigation was required to piece together the pathways involved. Mapping and genetic characterization of mutations was propelled forward in the early 2000s by the full sequencing of the Danio rerio genome. This chapter aims to provide a comprehensive overview of the ethical issues in zebrafish (Danio rerio) handling and the most recent practices for the generation and management of zebrafish transgenic, mutant, and mosaic lines. The discussion will take into account recent legislation, international publications, and standard guidelines applied in world reference centers such as the Zebrafish International Resource Center and the European Zebrafish Resource Center, as well as our personal experience in the management of national-level facilities. A set of schemes will be included to provide the reader with an immediate overview of the main operational phases in the different workflows.

Studying the activity of distributed neuronal circuits at a cellular resolution in vertebrates is very challenging due to the size and optical turbidity of their brains. We recently presented Danionella translucida, a close relative of zebrafish, as a model organism suited for studying large-scale neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of the smallest known vertebrate brain.

The rapid adoption of the larval zebrafish as a systems neuroscience model has been driven largely by its small size and optical transparency, which enable the use of imaging and optogenetics techniques to record and manipulate activity throughout the brain. Unfortunately, larval fish lack the mature behavioral repertoire of adults and so a number of learning phenomena and social behaviors cannot be investigated in these animals. Here we establish the pedomorphic fish speciesDanionella translucidaas a laboratory model that overcomes this limitation. AdultDanionellapossess the size and optical transparency of late-larval zebrafish, which we exploit to image deep within the brains of a behaviorally mature animals using two-photon microscopy. The close phylogenetic relationship betweenDanionellaand zebrafish enabled us to use existing reagents and techniques for transgenesis, and zebrafish-derived enhancer elements drove transgene expression inDanionellawith their intended specificities. In a behavioral assay for socially-reinforced place preference, interactions between fish were found to be positively reinforcing and dependent on gender, demonstrating the power of this species for studies of learning and memory as well as social behavior. The establishment ofDanionella’sbehavioral, molecular, and optical tractability provides a unique opportunity for researchers seeking to understand the relationship between circuits and adult-onset behaviors at a whole-brain level.

Understanding how distributed neuronal circuits integrate sensory information and generate behavior is a central goal of neuroscience. However, it has been difficult to study neuronal networks at single-cell resolution across the entire adult brain in vertebrates because of their size and opacity. We address this challenge here by introducing the fish Danionella translucida to neuroscience as a potential model organism. This teleost remains small and transparent even in adulthood, when neural circuits and behavior have matured. Despite having the smallest known adult vertebrate brain, D. translucida displays a rich set of complex behaviors, including courtship, shoaling, schooling, and acoustic communication. In order to carry out optical measurements and perturbations of neural activity with genetically encoded tools, we established CRISPR-Cas9 genome editing and Tol2 transgenesis techniques. These features make D. translucida a promising model organism for the study of adult vertebrate brain function at single-cell resolution.

The version of this paper originally published contained errors in reference citations: in the first paragraph of the Results section, the text "This extent of optical clarity probably results from the absence of skull above the brain22. In our specimens, Nissl-stained coronal sections through the head showed that the skull surrounds the brain only laterally and ventrally” should have read “This extent of optical clarity probably results from the absence of skull above the brain21. In our specimens, Nissl-stained coronal sections through the head22 showed that the skull surrounds the brain only laterally and ventrally.” In addition, the unit abbreviation “μm” was incorrectly divided at a line break in the third paragraph of the Discussion, which might have led to some confusion. These errors have been corrected in the PDF and HTML versions of the article.

Studying neuronal circuits at cellular resolution is very challenging in vertebrates due to the size and optical turbidity of their brains. Danionella translucida, a close relative of zebrafish, was recently introduced as a model organism for investigating neural network interactions in adult individuals. Danionella remains transparent throughout its life, has the smallest known vertebrate brain and possesses a rich repertoire of complex behaviours. Here we sequenced, assembled and annotated the Danionella translucida genome employing a hybrid Illumina/Nanopore read library as well as RNA-seq of embryonic, larval and adult mRNA. We achieved high assembly continuity using low-coverage long-read data and annotated a large fraction of the transcriptome. This dataset will pave the way for molecular research and targeted genetic manipulation of this novel model organism.

Locomotion exists in diverse forms in nature and is adapted to the environmental constraints of each species1. However, little is known about how closely related species with similar neuronal circuitry can evolve different navigational strategies to explore their environments. We established a powerful approach in comparative neuroethology to investigate evolution of neuronal circuits in vertebrates by comparing divergent swimming pattern of two closely related larval fish species,Danionella translucida(DT) andDanio rerioor zebrafish (ZF)2,3. During swimming, we demonstrate that DT utilizes lower half tail-beat frequency and amplitude to generate a slower and continuous swimming pattern when compared to the burst-and-glide swimming pattern in ZF. We found a high degree of conservation in the brain anatomy between the two species. However, we revealed that the activity of a higher motor region, referred here as the Mesencephalic Locomotion Maintenance Neurons (MLMN) correlates with the duration of swim events and differs strikingly between DT and ZF. Using holographic stimulation, we show that the activation of the MLMN is sufficient to increase the frequency and duration of swim events in ZF. Moreover, we propose two characteristics, availability of dissolved oxygen and timing of swim bladder inflation, which drive the observed differences in the swim pattern. Our findings uncover the neuronal circuit substrate underlying the evolutionary divergence of navigational strategies and how they are adapted to their respective environmental constraints.