Genome structures resolve the early diversification of teleost fishes.

Accurate species phylogenies are a prerequisite for all evolutionary research. Teleosts are the largest and most diversified group of extant vertebrates, but relationships among their three oldest extant lineages remain unresolved. On the basis of seven high-quality new genome assemblies in Elopomorpha (tarpons, eels), we revisited the topology of the deepest branches of the teleost phylogeny using independent gene sequence and chromosomal rearrangement phylogenomic approaches. These analyses converged to a single scenario that unambiguously places the Elopomorpha and Osteoglossomorpha (arapaima, elephantnose fish) in a monophyletic sister group to all other teleosts, i.e., the Clupeocephala lineage (zebrafish, medaka). This finding resolves more than 50 years of controversy on the evolutionary relationships of these lineages and highlights the power of combining different levels of genome-wide information to solve complex phylogenies.

Automated Zebrafish Phenotype Pattern Recognition: 6 Years Ago, and Now.

The article assesses the developments in automated phenotype pattern recognition: Potential spikes in classification performance, even when facing the common small-scale biomedical data set, and as a reader, you will find out about changes in the development effort and complexity for researchers and practitioners. After reading, you will be aware of the benefits and unreasonable effectiveness and ease of use of an automated end-to-end deep learning pipeline for classification tasks of biomedical perception systems.

Behavioral effects of acute ethanol in larval zebrafish (D. rerio) depend on genotype and volume of experimental well.

Ethanol consumption is a worldwide problem. Sensitivity to acute effects of ethanol influences the development of chronic ethanol abuse and ethanol dependence. Environmental and genetic factors have been found to contribute to differential effects of acute ethanol. Animal models have been employed to investigate these factors. An increasingly frequently utilized animal model in ethanol research is the zebrafish. A large proportion of ethanol studies with zebrafish have been conducted with adult zebrafish. However, high throughput drug and mutation screens are particularly well adapted to larval zebrafish. These studies are often carried out using the 96-well-plate that allows monitoring large numbers of fish efficiently. Here, we investigate the effects of acute (30 min long) ethanol exposure in 8-day post-fertilization (dpf) old zebrafish. We compare four genetically distinct populations (strains) of zebrafish, measuring numerous parameters of their swim path in two well sizes, i.e., in the 96-well-plate (small volume wells) and in the 6-well-plate (large volume wells). In general, we found that the highest dose of ethanol (1% vol/vol) reduced swim speed, increased duration of immobility, increased turn angle, and increased intra-individual variance of turn angle, while the intermediate dose (0.5%) had a less strong effect, compared to control. However, we also found that these ethanol effects were strain dependent and, in general, were better detected in the larger volume well. We conclude that larval zebrafish are appropriate for quantification of acute ethanol effects and also for the analysis of environmental and genetic factors that influence these effects. We also speculate that using larger wells will likely increase sensitivity of detection and precision in screening applications.

Screening by deep sequencing reveals mediators of microRNA tailing in C. elegans.

microRNAs are frequently modified by addition of untemplated nucleotides to the 3' end, but the role of this tailing is often unclear. Here we characterize the prevalence and functional consequences of microRNA tailing in vivo, using Caenorhabditis elegans. MicroRNA tailing in C. elegans consists mostly of mono-uridylation of mature microRNA species, with rarer mono-adenylation which is likely added to microRNA precursors. Through a targeted RNAi screen, we discover that the TUT4/TUT7 gene family member CID-1/CDE-1/PUP-1 is required for uridylation, whereas the GLD2 gene family member F31C3.2-here named GLD-2-related 2 (GLDR-2)-is required for adenylation. Thus, the TUT4/TUT7 and GLD2 gene families have broadly conserved roles in miRNA modification. We specifically examine the role of tailing in microRNA turnover. We determine half-lives of microRNAs after acute inactivation of microRNA biogenesis, revealing that half-lives are generally long (median = 20.7 h), as observed in other systems. Although we observe that the proportion of tailed species increases over time after biogenesis, disrupting tailing does not alter microRNA decay. Thus, tailing is not a global regulator of decay in C. elegans. Nonetheless, by identifying the responsible enzymes, this study lays the groundwork to explore whether tailing plays more specialized context- or miRNA-specific regulatory roles.

Characterization of zebrafish GABAA receptor subunits.

γ-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system, exerts its effect through the activation of GABA receptors. GABAA receptors are ligand-gated chloride channels composed of five subunit proteins. Mammals have 19 different GABAA receptor subunits (α1-6, ß1-3, γ1-3, δ, ε, π, θ, and ρ1-3), the physiological properties of which have been assayed by electrophysiology. However, the evolutionary conservation of the physiological characteristics of diverged GABAA receptor subunits remains unclear. Zebrafish have 23 subunits (α1, α2a, α2b, α3-5, α6a, α6b, ß1-4, γ1-3, δ, π, ζ, ρ1, ρ2a, ρ2b, ρ3a, and ρ3b), but the electrophysiological properties of these subunits have not been explored. In this study, we cloned the coding sequences for zebrafish GABAA receptor subunits and investigated their expression patterns in larval zebrafish by whole-mount in situ hybridization. We also performed electrophysiological recordings of GABA-evoked currents from Xenopus oocytes injected with one or multiple zebrafish GABAA receptor subunit cRNAs and calculated the half-maximal effective concentrations (EC50s) for each. Our results revealed the spatial expressions and electrophysiological GABA sensitivities of zebrafish GABAA receptors, suggesting that the properties of GABAA receptor subunits are conserved among vertebrates.

Paracellular and Transcellular Leukocytes Diapedesis Are Divergent but Interconnected Evolutionary Events.

Infiltration of the endothelial layer of the blood-brain barrier by leukocytes plays a critical role in health and disease. When passing through the endothelial layer during the diapedesis process lymphocytes can either follow a paracellular route or a transcellular one. There is a debate whether these two processes constitute one mechanism, or they form two evolutionary distinct migration pathways. We used artificial intelligence, phylogenetic analysis, HH search, ancestor sequence reconstruction to investigate further this intriguing question. We found that the two systems share several ancient components, such as RhoA protein that plays a critical role in controlling actin movement in both mechanisms. However, some of the key components differ between these two transmigration processes. CAV1 genes emerged during Trichoplax adhaerens, and it was only reported in transcellular process. Paracellular process is dependent on PECAM1. PECAM1 emerged from FASL5 during Zebrafish divergence. Lastly, both systems employ late divergent genes such as ICAM1 and VECAM1. Taken together, our results suggest that these two systems constitute two different mechanical sensing mechanisms of immune cell infiltrations of the brain, yet these two systems are connected. We postulate that the mechanical properties of the cellular polarity is the main driving force determining the migration pathway. Our analysis indicates that both systems coevolved with immune cells, evolving to a higher level of complexity in association with the evolution of the immune system.

Evolution of the potassium channel gene Kcnj13 underlies colour pattern diversification in Danio fish.

The genetic basis of morphological variation provides a major topic in evolutionary developmental biology. Fish of the genus Danio display colour patterns ranging from horizontal stripes, to vertical bars or spots. Stripe formation in zebrafish, Danio rerio, is a self-organizing process based on cell-contact mediated interactions between three types of chromatophores with a leading role of iridophores. Here we investigate genes known to regulate chromatophore interactions in zebrafish that might have evolved to produce a pattern of vertical bars in its sibling species, Danio aesculapii. Mutant D. aesculapii indicate a lower complexity in chromatophore interactions and a minor role of iridophores in patterning. Reciprocal hemizygosity tests identify the potassium channel gene obelix/Kcnj13 as evolved between the two species. Complementation tests suggest evolutionary change through divergence in Kcnj13 function in two additional Danio species. Thus, our results point towards repeated and independent evolution of this gene during colour pattern diversification.

Defining endogenous barcoding sites for CRISPR/Cas9-based cell lineage tracing in zebrafish.

There is a growing interest in developing experimental methods for tracking the developmental cell lineages of a complex organism. The recently developed CRISPR/Cas9-based barcoding method is, although highly promising, difficult to scale up because it relies on exogenous barcoding sequences that are engineered into the genome. In this study, we characterized 78 high-quality endogenous sites in the zebrafish genome that can be used as CRISPR/Cas9-based barcoding sites. The 78 sites are all highly expressed in most of the cell types according to single-cell RNA sequencing (scRNA-seq) data. Hence, the barcoding information of the 78 endogenous sites is recovered by the available scRNA-seq platforms, enabling simultaneous characterization of cell type and cell lineage information.

Understanding neurobehavioral genetics of zebrafish.

Due to its fully sequenced genome, high genetic homology to humans, external fertilization, fast development, transparency of embryos, low cost and active reproduction, the zebrafish (Danio rerio) has become a novel promising model organism in biomedicine. Zebrafish are a useful tool in genetic and neuroscience research, including linking various genetic mutations to brain mechanisms using forward and reverse genetics. These approaches have produced novel models of rare genetic CNS disorders and common brain illnesses, such as addiction, aggression, anxiety and depression. Genetically modified zebrafish also foster neuroanatomical studies, manipulating neural circuits and linking them to different behaviors. Here, we discuss recent advances in neurogenetics of zebrafish, and evaluate their unique strengths, inherent limitations and the rapidly growing potential for elucidating the conserved roles of genes in neuropsychiatric disorders.

Whole genome experimental maps of DNA G-quadruplexes in multiple species.

Genomic maps of DNA G-quadruplexes (G4s) can help elucidate the roles that these secondary structures play in various organisms. Herein, we employ an improved version of a G-quadruplex sequencing method (G4-seq) to generate whole genome G4 maps for 12 species that include widely studied model organisms and also pathogens of clinical relevance. We identify G4 structures that form under physiological K+ conditions and also G4s that are stabilized by the G4-targeting small molecule pyridostatin (PDS). We discuss the various structural features of the experimentally observed G-quadruplexes (OQs), highlighting differences in their prevalence and enrichment across species. Our study describes diversity in sequence composition and genomic location for the OQs in the different species and reveals that the enrichment of OQs in gene promoters is particular to mammals such as mouse and human, among the species studied. The multi-species maps have been made publicly available as a resource to the research community. The maps can serve as blueprints for biological experiments in those model organisms, where G4 structures may play a role.

Bacterial Cohesion Predicts Spatial Distribution in the Larval Zebrafish Intestine.

Are there general biophysical relationships governing the spatial organization of the gut microbiome? Despite growing realization that spatial structure is important for population stability, interbacterial competition, and host functions, it is unclear in any animal gut whether such structure is subject to predictive, unifying rules or if it results from contextual, species-specific behaviors. To explore this, we used light sheet fluorescence microscopy to conduct a high-resolution comparative study of bacterial distribution patterns throughout the entire intestinal volume of live, larval zebrafish. Fluorescently tagged strains of seven bacterial symbionts, representing six different species native to zebrafish, were each separately monoassociated with animals that had been raised initially germ-free. The strains showed large differences in both cohesion-the degree to which they auto-aggregate-and spatial distribution. We uncovered a striking correlation between each strain's mean position and its cohesion, whether quantified as the fraction of cells existing as planktonic individuals, the average aggregate size, or the total number of aggregates. Moreover, these correlations held within species as well; aggregates of different sizes localized as predicted from the pan-species observations. Together, our findings indicate that bacteria within the zebrafish intestine are subject to generic processes that organize populations by their cohesive properties. The likely drivers of this relationship-peristaltic fluid flow, tubular anatomy, and bacterial growth and aggregation kinetics-are common throughout animals. We therefore suggest that the framework introduced here of biophysical links between bacterial cohesion and spatial organization should be useful for directing explorations in other host-microbe systems, formulating detailed models that can quantitatively map onto experimental data, and developing new tools that manipulate cohesion to engineer microbiome function.

Expression patterns of dscam and sdk gene paralogs in developing zebrafish retina.

Purpose: The differential adhesion hypothesis states that a cell adhesion code provides cues that direct the specificity of nervous system development. The Down syndrome cell adhesion molecule (DSCAM) and sidekick (SDK) proteins belong to the immunoglobulin superfamily of cell adhesion molecules (CAMs) and provide both attractive and repulsive cues that help to organize the nervous system during development, according to the differential adhesion hypothesis. The zebrafish genome is enriched in dscam and sdk genes, making the zebrafish an excellent model system to further test this hypothesis. The goal of this study is to describe the phylogenetic relationships of the paralogous CAM genes and their spatial expression and co-expression patterns in the embryonic zebrafish retina. Methods: Exon-intron structures, karyotypic locations, genomic context, and amino acid sequences of the zebrafish CAM genes (dscama, dscamb, dscaml1, sdk1a, sdk1b, sdk2a, and sdk2b) were obtained from the Ensembl genome database. The Prosite and SMART programs were used to determine the number and identity of protein domains for each CAM gene. The randomized axelerated maximum likelihood (RaxML) program was used to perform a phylogenetic analysis of the zebrafish CAM genes and orthologs in other vertebrates. A synteny analysis of regions surrounding zebrafish CAM paralogs was performed. Digoxigenin (dig)-labeled cRNA probes for each CAM gene were generated to perform in situ hybridization of retinal cryosections from zebrafish embryos and larvae. Dual in situ hybridization of retinal cryosections from zebrafish larvae was performed with dig- and fluorescein-labeled cRNA probes. Results: We found the studied zebrafish CAM genes encode similar protein domain structures as their corresponding orthologs in mammals and possess similar intron-exon organizations. CAM paralogs were located on different chromosomes. Phylogenetic and synteny analyses provided support for zebrafish dscam and sdk2 paralogs having originated during the teleost genome duplication. We found that dscama and dscamb are co-expressed in the ganglion cell layer (GCL) and the basal portion of the inner nuclear layer (INL), with weak expression in the photoreceptor-containing outer nuclear layer (ONL). Of the dscam genes, only dscamb was strongly expressed in ONL. Sdk1a and sdk1b were co-expressed in the GCL and the basal portion of the INL. Sdk2a and sdk2b also showed co-expression in the GCL and basal portion of the INL. All Sdk genes were expressed in the ciliary marginal zone (CMZ). Dual in situ hybridizations revealed alternating patterns of co-expression and exclusive expression for the dscam and sdk1 paralogs in cells of the GCL and the INL. The same alternating pattern was observed between dscam and sdk2 paralogs and between sdk1 and sdk2 paralogs. The expression of dscaml1 was observed in the INL and the GCL, with some cells in the basal portion of the INL showing co-expression of dscaml1 and dscama. Conclusions: These findings suggest that zebrafish dscam and sdk2 paralogs were likely the result of the teleost whole genome duplication and that all CAM duplicates show some differential expression patterns. We also demonstrate that the comparative expression patterns of CAM genes in the zebrafish are distinct from the exclusive expression patterns observed in chick retina, in which retinal ganglion cells express one of the four chick Dscam or Sdk genes only. The patterns in zebrafish are more similar to those of mice, in which co-expression of Dscam and Sdk genes is observed. These findings provide the groundwork for future functional analysis of the roles of the CAM paralogs in zebrafish.

Protection from UV light is an evolutionarily conserved feature of the haematopoietic niche.

Haematopoietic stem and progenitor cells (HSPCs) require a specific microenvironment, the haematopoietic niche, which regulates HSPC behaviour1,2. The location of this niche varies across species, but the evolutionary pressures that drive HSPCs to different microenvironments remain unknown. The niche is located in the bone marrow in adult mammals, whereas it is found in other locations in non-mammalian vertebrates, for example, in the kidney marrow in teleost fish. Here we show that a melanocyte umbrella above the kidney marrow protects HSPCs against ultraviolet light in zebrafish. Because mutants that lack melanocytes have normal steady-state haematopoiesis under standard laboratory conditions, we hypothesized that melanocytes above the stem cell niche protect HSPCs against ultraviolet-light-induced DNA damage. Indeed, after ultraviolet-light irradiation, unpigmented larvae show higher levels of DNA damage in HSPCs, as indicated by staining of cyclobutane pyrimidine dimers and have reduced numbers of HSPCs, as shown by cmyb (also known as myb) expression. The umbrella of melanocytes associated with the haematopoietic niche is highly evolutionarily conserved in aquatic animals, including the sea lamprey, a basal vertebrate. During the transition from an aquatic to a terrestrial environment, HSPCs relocated into the bone marrow, which is protected from ultraviolet light by the cortical bone around the marrow. Our studies reveal that melanocytes above the haematopoietic niche protect HSPCs from ultraviolet-light-induced DNA damage in aquatic vertebrates and suggest that during the transition to terrestrial life, ultraviolet light was an evolutionary pressure affecting the location of the haematopoietic niche.

Fully Automated Pipetting Sorting System for Different Morphological Phenotypes of Zebrafish Embryos.

Systems biology methods, such as transcriptomics and metabolomics, require large numbers of small model organisms, such as zebrafish embryos. Manual separation of mutant embryos from wild-type embryos is a tedious and time-consuming task that is prone to errors, especially if there are variable phenotypes of a mutant. Here we describe a zebrafish embryo sorting system with two cameras and image processing based on template-matching algorithms. In order to evaluate the system, zebrafish rx3 mutants that lack eyes due to a patterning defect in brain development were separated from their wild-type siblings. These mutants show glucocorticoid deficiency due to pituitary defects and serve as a model for human secondary adrenal insufficiencies. We show that the variable phenotypes of the mutant embryos can be safely distinguished from phenotypic wild-type zebrafish embryos and sorted from one petri dish into another petri dish or into a 96-well microtiter plate. On average, classification of a zebrafish embryo takes approximately 1 s, with a sensitivity and specificity of 87% to 95%, respectively. Other morphological phenotypes may be classified and sorted using similar techniques.

Tdrd12 Is Essential for Germ Cell Development and Maintenance in Zebrafish.

The regularity of Piwi-interacting RNA (piRNA) biogenesis is crucial to germline development. Functioning as Piwi-interacting proteins, Tudor domain-related proteins (Tdrds) have been demonstrated to be involved in spermatogenesis and the piRNA pathway. In this study, zebrafish tdrd12 was identified, and the maternal and germ cell-specific expression patterns of zebrafish tdrd12 were observed. Utilizing TALEN (transcription activator-like effector nuclease) techniques, two independent tdrd12 mutant zebrafish lines were generated. Although no defects were found during the generation of the primordial germ cells (PGCs) in the tdrd12-null fish progenies obtained from the heterozygous tdrd12 mutant parents, all Tdrd12-deficient fish developed into infertile males. The reduced numbers and eventually loss of the germ cells by 35 days post fertilization (dpf) led to masculinization and infertility of the Tdrd12-deficient fish. Meiosis defects of the germ cells in the tdrd12 mutants during the gonad-transitioning period were observed, revealing the indispensable functions of Tdrd12 in gametogenesis. Our studies demonstrated that zebrafish Tdrd12 is essential for germ cell development and maintenance.

Nuclear receptor research in zebrafish.

Nuclear receptors (NRs) form a superfamily of transcription factors that can be activated by ligands and are involved in a wide range of physiological processes. NRs are well conserved between vertebrate species. The zebrafish, an increasingly popular animal model system, contains a total of 73 NR genes, and orthologues of almost all human NRs are present. In this review article, an overview is presented of NR research in which the zebrafish has been used as a model. Research is described on the three most studied zebrafish NRs: the estrogen receptors (ERs), retinoic acid receptors (RARs) and peroxisome proliferator-activated receptors (PPARs). The studies on these receptors illustrate the versatility of the zebrafish as a model for ecotoxicological, developmental and biomedical research. Although the use of the zebrafish in NR research is still relatively limited, it is expected that in the next decade the full potential of this animal model will be exploited.

Further characterisation of differences between TL and AB zebrafish (Danio rerio): Gene expression, physiology and behaviour at day 5 of the larval stage.

Zebrafish (Danio rerio) have become popular as model organism in research. Many strains are readily available, which not only differ morphologically, but also genetically, physiologically and behaviourally. Here, we focus on the AB and Tupfel long-fin (TL) strain for which we have previously shown that adults differ in baseline hypothalamus-pituitary-interrenal (HPI)-axis activity (AB higher than TL) affecting inhibitory avoidance behaviour (absent in AB). To assess whether strain differences are already present in early life stages, we compared baseline HPI-axis related gene expression as well as cortisol levels, (neuro)development related as well as (innate) immune system related gene expression, and light-dark as well as startle behaviour in larvae 5 days post fertilisation. The data show that AB and TL larvae differ in baseline HPI-axis activity (AB higher than TL), expression of (neuro)development and immune system related genes (AB higher than TL), habituation to acoustic/vibrational stimuli (AB habituate faster than TL) and light-dark induced changes in motor behaviour (AB stronger than TL). Our data show that already in larval stages differences exist between zebrafish of the AB and TL strain confirming and extending data of earlier studies. To what extent the mutation in connexin 41.8, leading to spots rather than stripes in TL, but also (possibly) affecting eye, heart and brain function, is involved in the expression of (some of) these differences needs to be studied. These results emphasise that differences between strains need to be taken into account to enhance reproducibility both within, and between, laboratories.

Expression pattern analysis of IRF4 and its related genes revealed the functional differentiation of IRF4 paralogues in teleost.

In mammals, interferon regulatory factor 4 (IRF4) plays an important role in the process of development and differentiation of B cells, T cells and dendritic cells. It can regulate immune pathway through IRF5, MyD88, IL21, PGC1α, and NOD2. In the present study, we investigated the expression pattern of IRF4 paralogues and these related genes for the first time in teleosts. The results showed that these genes were all expressed predominantly in known immune tissues while IRF5 was also relatively highly expressed in muscle. IRF4b, IL21, MyD88, IRF5 and NOD2 showed maternal expression in the oocyte and the higher expression of IRF4a, Mx and PGC1α before hatching might be involved in the embryonic innate defense system. Zebrafish embryonic fibroblast (ZF4) cells were infected with GCRV and SVCV. During GCRV infection, the expression of Mx was significantly up-regulated from 3 h to 24 h, reaching the highest level at 12 h (101.5-fold over the controls, P < 0.001). And the expression of IRF4a was significantly up-regulated from 3 h to 48 h, reaching the highest level at 12 h (13.75-fold over the controls, P < 0.001). While the expression of IRF4b was only slightly up-regulated at 12 h and 24 h (3.39-fold, 1.93-fold) above control levels, respectively. Whereas the expression of Mx was significantly up-regulated during SVCV infection from 1 h to 48 h, reaching the highest level at 24 h (11.49-fold over the controls, P < 0.001). IRF4a transcripts were significantly up-regulated from 6 h to 24 h, reaching the highest level at 24 h (41-fold over the controls, P < 0.01). IRF4b only showed a slightly up-regulation by SVCV at 24 h (3.2-fold over the controls, P < 0.01). IRF4a and IRF4b displayed a distinct tissue expression pattern, embryonic stages expression and inducible expression in vivo and in vitro, suggesting that IRF4 paralogues might play different roles in immune system.

Genome Wide Identification, Phylogeny, and Expression of Aquaporin Genes in Common Carp (Cyprinus carpio).

BACKGROUND: Aquaporins (Aqps) are integral membrane proteins that facilitate the transport of water and small solutes across cell membranes. Among vertebrate species, Aqps are highly conserved in both gene structure and amino acid sequence. These proteins are vital for maintaining water homeostasis in living organisms, especially for aquatic animals such as teleost fish. Studies on teleost Aqps are mainly limited to several model species with diploid genomes. Common carp, which has a tetraploidized genome, is one of the most common aquaculture species being adapted to a wide range of aquatic environments. The complete common carp genome has recently been released, providing us the possibility for gene evolution of aqp gene family after whole genome duplication. RESULTS: In this study, we identified a total of 37 aqp genes from common carp genome. Phylogenetic analysis revealed that most of aqps are highly conserved. Comparative analysis was performed across five typical vertebrate genomes. We found that almost all of the aqp genes in common carp were duplicated in the evolution of the gene family. We postulated that the expansion of the aqp gene family in common carp was the result of an additional whole genome duplication event and that the aqp gene family in other teleosts has been lost in their evolution history with the reason that the functions of genes are redundant and conservation. Expression patterns were assessed in various tissues, including brain, heart, spleen, liver, intestine, gill, muscle, and skin, which demonstrated the comprehensive expression profiles of aqp genes in the tetraploidized genome. Significant gene expression divergences have been observed, revealing substantial expression divergences or functional divergences in those duplicated aqp genes post the latest WGD event. CONCLUSIONS: To some extent, the gene families are also considered as a unique source for evolutionary studies. Moreover, the whole set of common carp aqp gene family provides an essential genomic resource for future biochemical, toxicological, physiological, and evolutionary studies in common carp.