Single Zebrafish Research Articles

Determining Photoreceptor Cell Identity: Rod Versus Cone Fate Governed by tbx2b Opposing nrl.

NRL is an influential transcription factor and central to animal modeling in ophthalmology. Disrupting NRL abrogates rod development and produces an excess of S-cones (also known as "UV cones" or "short-wavelength-sensitive1 [SWS1] cones"). Strikingly, mutations in zebrafish tbx2b produce the exact opposite phenotypes (excess rods and loss of SWS1 cones). We sought to define what genetic relationship exists, if any, between these transcription factors. We also infer whether these two phenotypes (altered rod abundance and altered SWS1 cone abundance) are independent versus inter-related. Zebrafish mutants were bred to disrupt nrl and tbx2b in concert. Rods and SWS1 cones were quantified and characterized at ultrastructural and transcriptional levels. Considering single mutant zebrafish, we confirmed previously established phenotypes and noted that the number of rods lost in nrl-/- mutants is reflected by a concomitant increase in SWS1 cone abundance. The tbx2b-/- mutants present the opposite phenotype(s) but exhibit a similar trade-off in cell abundances, with lots of rods and a concomitant decrease in SWS1 cones. Double mutant nrl-/-;tbx2b-/- zebrafish recapitulate the nrl-/- mutant phenotype(s). The tbx2b is thought to be required for producing SWS1 cones in zebrafish, but this can be over-ridden when nrl is absent. Regarding the altered cell abundances observed in either tbx2b-/- or nrl-/- mutants, the alterations in rod and SWS1 cones appear to not be two separate phenotypes but are instead a single intertwined outcome. The tbx2b and nrl are in an epistatic relationship, with nrl phenotypes dominating, implying that tbx2b is upstream of nrl in photoreceptor cell fate determination.

Xenophagy receptors Optn and p62 and autophagy modulator Dram1 independently promote the zebrafish host defense against Mycobacterium marinum.

Anti-bacterial autophagy, also known as xenophagy, is a crucial innate immune process that helps maintain cellular homeostasis by targeting invading microbes. This defense pathway is widely studied in the context of infections with mycobacteria, the causative agents of human tuberculosis and tuberculosis-like disease in animal models. Our previous work in a zebrafish tuberculosis model showed that host defense against Mycobacterium marinum (Mm) is impaired by deficiencies in xenophagy receptors, optineurin (Optn) or sequestome 1 (p62), and Damage-regulated autophagy modulator 1 (Dram1). However, the interdependency of these receptors and their interaction with Dram1 remained unknown. In the present study, we used single and double knockout zebrafish lines in combination with overexpression experiments. We show that Optn and p62 can compensate for the loss of each other's function, as their overexpression restores the infection susceptibility of the mutant phenotypes. Similarly, Dram1 can compensate for deficiencies in Optn and p62, and, vice versa, Optn and p62 compensate for the loss of Dram1, indicating that these xenophagy receptors and Dram1 do not rely on each other for host defense against Mm. In agreement, Dram1 overexpression in optn/p62 double mutants restored the interaction of autophagosome marker Lc3 with Mm. Finally, optn/p62 double mutants displayed more severe infection susceptibility than the single mutants. Taken together, these results suggest that Optn and p62 do not function downstream of each other in the anti-mycobacterial xenophagy pathway, and that the Dram1-mediated defense against Mm infection does not rely on specific xenophagy receptors.

Adult‐ and childhood onset dementias: related causes and related solutions?

AbstractBackgroundThe endo‐lysosomal pathway (ELP) of cells is central to their importation of nutrients and for recycling of cellular components. Much genetic evidence supports the importance of dysfunction of the ELP in Alzheimer’s disease (AD). However, AD is a complex disorder and how AD‐associated genetic variation affects ELP function is unclear. Sanfilippo syndrome childhood dementia is a consequence of extreme ELP dysfunction, where inherited recessive mutations result in the ELP’s inability to properly catabolise a particular polysaccharide. Unlike AD, Sanfilippo is genetically simple. All the causative genes have been identified and the biochemical consequences of their mutations are understood. Animal models of Sanfilippo are considered excellent models of the human disease. Using zebrafish as a model organism, we are dissecting shared disease mechanisms between these two dementia subtypes.MethodZebrafish models of both (early‐onset familial) AD and Sanfilippo syndrome were generated using CRISPR‐Cas9‐based approaches. The brain transcriptomes (RNA‐seq) and metabolomes (metabolomics/fluxomics) of both models are compared within single zebrafish families. Spatial working memory in both models is assessed using a Y‐maze.ResultPreliminary brain transcriptome analyses have identified changes‐in‐common to both oxidative phosphorylation and ribosome gene expression in both AD and Sanfilippo model zebrafish. Metabolomics and behavioural analyses of these models are ongoing.ConclusionNeurodegeneration in Sanfilippo syndrome and Alzheimer’s disease may share common mechanisms that are amenable to common therapeutic approaches.

Pulsed stimulated Brillouin microscopy enables high-sensitivity mechanical imaging of live and fragile biological specimens

Brillouin microscopy is an emerging optical elastography technique capable of assessing mechanical properties of biological samples in a three-dimensional, all-optical and noncontact fashion. The typically weak Brillouin scattering signal can be substantially enhanced via a stimulated Brillouin scattering (SBS) process; however, current implementations require high pump powers, which prohibit applications to photosensitive or live imaging of biological samples. Here we present a pulsed SBS scheme that takes advantage of the nonlinearity of the pump–probe interaction. In particular, we show that the required pump laser power can be decreased ~20-fold without affecting the signal levels or spectral precision. We demonstrate the low phototoxicity and high specificity of our pulsed SBS approach by imaging, with subcellular detail, sensitive single cells, zebrafish larvae, mouse embryos and adult Caenorhabditis elegans. Furthermore, our method permits observing the mechanics of organoids and C. elegans embryos over time, opening up further possibilities for the field of mechanobiology.

Simultaneous extraction and detection of peptides, steroids, and proteins in small tissue samples.

The detection and quantification of hormones are important to assess the reproductive and stress status of experimental models and for the diagnosis of diseases in human and veterinary clinics. Traditionally, steroid, peptide, and protein hormones are analyzed in individual experiments using different extraction methodologies. With the new advancement on HPLC sorbents, the simultaneous measurement of hormones from different categories becomes possible. In this study, we present a novel sample processing strategy for the simultaneous extraction and detection of peptides, steroids, and proteins using high-resolution liquid chromatography tandem mass spectrometry. We demonstrate the sensitivity of our method for small tissues by acquiring data from brain, pituitary gland, and gonads of single zebrafish samples. This approach promises to shed light on the hormonal pathways and their interrelationships, providing knowledge on the integration of hormone systems.

Effects of the major formaldehyde catalyzer ADH5 on phenotypes of fanconi anemia zebrafish model.

Fanconi anemia (FA) is a devastating hereditary disorder for which we desperately need a novel therapeutic strategy. It is caused by mutations in one of at least 22 genes in the FA pathway and is characterized by developmental abnormalities, bone marrow failure, and cancer predisposition. The FA pathway is required for the efficient repair of damaged DNA, including interstrand cross-links (ICL). Recent studies indicate formaldehyde as an ultimate endogenous cause of DNA damage in FA pathophysiology. Formaldehyde can form DNA adducts as well as ICLs by inducing covalent linkages between opposite strands of double-stranded DNA. In this study, we generated a disease model of FA in zebrafish by disrupting the ube2t or fancd2 gene, which resulted in a striking phenotype of female-to-male sex reversal. Since formaldehyde is detoxified from the body by alcohol dehydrogenase 5 (ADH5), we generated fancd2-/-/adh5-/- zebrafish. We observed a body size reduction and a lower number of mature spermatozoa than wild-type or single knockout zebrafish. To evaluate if increased activity in ADH5 can affect the FA phenotype, we overexpressed human ADH5 in fancd2-/- zebrafish. The progress of spermatogenesis seemed to be partially recovered due to ADH5 overexpression. Our results suggest potential utility of an ADH5 enzyme activator as a therapeutic measure for the clearance of formaldehyde and treatment of FA.

Mitochondria-targeting two-photon fluorescent probe for sequential recognition of Cu2+ and ATP in neurons and zebrafish

Highly active mitochondria play a significant role in neuron function. Cu2+ and ATP levels in mitochondria regulate neuronal mitochondrial activity. However, mitochondrial activity was often evaluated by mitochondrial membrane potential. Less is known about the dynamics of Cu2+ and ATP in mitochondria. Herein, we developed a two-photon fluorescence probe (MP), which provided a determination of mitochondrial ATP and Cu2+. The fluorescence of MP showed remarkable quenching in the presence of Cu2+ and then gradually recovered in the presence of ATP, which can be used for sequential recognition. MP has high sensitivity to Cu2+ and ATP, with limits of detection (LOD) close to 0.31 nM and 13.6 nM, respectively. Using this useful probe, we monitor the fluctuation of concentrations of Cu2+ and ATP by fluorescence imaging at single neuron and zebrafish.

Single-cell m6A mapping in vivo using picoMeRIP–seq

Current N6-methyladenosine (m6A) mapping methods need large amounts of RNA or are limited to cultured cells. Through optimized sample recovery and signal-to-noise ratio, we developed picogram-scale m6A RNA immunoprecipitation and sequencing (picoMeRIP–seq) for studying m6A in vivo in single cells and scarce cell types using standard laboratory equipment. We benchmark m6A mapping on titrations of poly(A) RNA and embryonic stem cells and in single zebrafish zygotes, mouse oocytes and embryos.

A fluidic platform for mobility evaluation of zebrafish with gene deficiency.

Zebrafish is a suitable animal model for molecular genetic tests and drug discovery due to its characteristics including optical transparency, genetic manipulability, genetic similarity to humans, and cost-effectiveness. Mobility of the zebrafish reflects pathological conditions leading to brain disorders, disrupted motor functions, and sensitivity to environmental challenges. However, it remains technologically challenging to quantitively assess zebrafish's mobility in a flowing environment and simultaneously monitor cellular behavior in vivo. We herein developed a facile fluidic device using mechanical vibration to controllably generate various flow patterns in a droplet housing single zebrafish, which mimics its dynamically flowing habitats. We observe that in the four recirculating flow patterns, there are two equilibrium stagnation positions for zebrafish constrained in the droplet, i.e., the "source" with the outward flow and the "sink" with the inward flow. Wild-type zebrafish, whose mobility remains intact, tend to swim against the flow and fight to stay at the source point. A slight deviation from streamline leads to an increased torque pushing the zebrafish further away, whereas zebrafish with motor neuron dysfunction caused by lipin-1 deficiency are forced to stay in the "sink," where both their head and tail align with the flow direction. Deviation angle from the source point can, therefore, be used to quantify the mobility of zebrafish under flowing environmental conditions. Moreover, in a droplet of comparable size, single zebrafish can be effectively restrained for high-resolution imaging. Using the proposed methodology, zebrafish mobility reflecting pathological symptoms can be quantitively investigated and directly linked to cellular behavior in vivo.

A minimally invasive fin scratching protocol for fast genotyping and early selection of zebrafish embryos

Current genetic modification and phenotyping methods in teleost fish allow detailed investigation of vertebrate mechanisms of development, modeling of specific aspects of human diseases and efficient testing of drugs at an organ/organismal level in an unparalleled fast and large-scale mode. Fish-based experimental approaches have boosted the in vivo verification and implementation of scientific advances, offering the quality guaranteed by animal models that ultimately benefit human health, and are not yet fully replaceable by even the most sophisticated in vitro alternatives. Thanks to highly efficient and constantly advancing genetic engineering as well as non-invasive phenotyping methods, the small zebrafish is quickly becoming a popular alternative to large animals’ experimentation. This approach is commonly associated to invasive procedures and increased burden. Here, we present a rapid and minimally invasive method to obtain sufficient genomic material from single zebrafish embryos by simple and precise tail fin scratching that can be robustly used for at least two rounds of genotyping already from embryos within 48 h of development. The described protocol betters currently available methods (such as fin clipping), by minimizing the relative animal distress associated with biopsy at later or adult stages. It allows early selection of embryos with desired genotypes for strategizing culturing or genotype–phenotype correlation experiments, resulting in a net reduction of “surplus” animals used for mutant line generation.

Regulation of Iron Transport Is Required for Terminal Erythroid Differentiation Even Under Iron-Replete Conditions Sufficient for Hemoglobinization

Developing erythroid cells are the main consumer of iron in vertebrates, as their main function is to oxygenate tissues via hemoglobin. Iron metabolism is a key driver of erythroid development and hemoglobin synthesis. When iron deficient, or when iron metabolism is dysregulated, erythroid cells are both lacking in hemoglobin and exhibit differentiation defects. Currently, the efficacy of iron supplementation in patients is monitored by quantitating hemoglobin levels. However, the effect of iron supplementation on erythroid differentiation is less clear. As hemoglobinization and erythroid differentiation are inextricably intertwined with erythroid function, we sought to understand whether and how iron supplementation functions to restore erythroid differentiation during iron deficiency. Tackling this problem in vivo has been fraught with technical challenges as vertebrates with mutations in iron metabolism genes often die from anemia in utero. To circumvent this problem, we used zebrafish with genetic iron metabolism defects to determine if iron supplementation could rescue erythropoietic defects at both the cellular and systemic level. Zebrafish are tractable in vivo models for such studies as they develop externally and can easily be treated by exogenous iron in their water. To carry out this study, we designed a system to sort Tg(globin lcr:eGFP) erythrocytes from single zebrafish embryos onto slides for imaging, while keeping sufficient numbers of cells for genotyping of the same embryo. We used two zebrafish lines which carry loss of function mutations in the mfrn1 (carrying a mitochondrial iron trafficking defect) and fpn1 (carrying a defect in iron export from intestinal and yolk syncytial epithelium-a model of dietary iron deficiency). Both lines had decreased erythroid cell numbers. In addition, the erythroid cells were hemoglobin deficient and had erythroid differentiation defects which included enlarge nuclei. We showed that exogenous iron supplementation (labeled as "rescue) in mfrn1 mutant zebrafish restored hemoglobinization (benzidine staining) but not erythroid cell number (% lcr:eGFP) or terminal differentiation. However, iron supplementation in fpn1 mutant zebrafish, restored erythroid cell number but not terminal differentiation defects. The main difference in the 2 mutants is that the mfrn1 mutant has a defect in intracellular iron trafficking leading to defective iron metabolism, while the fpn1 mutant has defective iron export to erythroid cells but their erythroid cells have intact intracellular iron trafficking. mfrn1 mutants had defects in cell cycle progression and increased apoptosis in their erythroid cells which likely is a cause for decreased erythroid cell numbers. Our data suggests that in addition to adequate iron levels, correct regulation of iron trafficking is required for optimal erythroid iron utilization, terminal erythropoiesis and potentially, proliferation and differentiation of erythroid progenitors. This may indicate a role for iron transporters in directing iron fate, as well as signaling to developmental pathways. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Toxicometabolomics and Biotransformation Product Elucidation in Single Zebrafish Embryos Exposed to Carbamazepine from Environmentally-Relevant to Morphologically Altering Doses.

Toxicometabolomics and biotransformation product (bioTP) elucidation were carried out in single zebrafish (ZF) embryos exposed to carbamazepine (CBZ). Exposures were conducted in 96-well plates containing six CBZ concentrations ranging from 0.5 μg/L to 50 mg/L (n = 12 embryos per dose). In the 50 mg/L dose group, 33% of embryos developed edema during the exposure (120 hpf), while hatching was significantly delayed in three of the lower-dose groups (0.46, 3.85, and 445 μg/L) compared to the control at 48 hpf. Toxicometabolomic analysis together with random forest modeling revealed a total of 80 significantly affected metabolites (22 identified via targeted lipidomics and 58 via nontarget analysis). The wide range of doses enabled the observation of both monotonic and nonmonotonic dose responses in the metabolome, which ultimately produced a unique and comprehensive biochemical picture that aligns with existing knowledge on the mode of action of CBZ. The combination of high dose exposures and apical endpoint assessment in single embryos also enabled hypothesis generation regarding the target organ for the morphologically altering insult. In addition, two CBZ bioTPs were identified without additional exposure experiments. Overall, this work showcases the potential of toxicometabolomics and bioTP determination in single ZF embryos for rapid and comprehensive chemical hazard assessment.

Pluripotency factors determine gene expression repertoire at zygotic genome activation

Awakening of zygotic transcription in animal embryos relies on maternal pioneer transcription factors. The interplay of global and specific functions of these proteins remains poorly understood. Here, we analyze chromatin accessibility and time-resolved transcription in single and double mutant zebrafish embryos lacking pluripotency factors Pou5f3 and Sox19b. We show that two factors modify chromatin in a largely independent manner. We distinguish four types of direct enhancers by differential requirements for Pou5f3 or Sox19b. We demonstrate that changes in chromatin accessibility of enhancers underlie the changes in zygotic expression repertoire in the double mutants. Pou5f3 or Sox19b promote chromatin accessibility of enhancers linked to the genes involved in gastrulation and ventral fate specification. The genes regulating mesendodermal and dorsal fates are primed for activation independently of Pou5f3 and Sox19b. Strikingly, simultaneous loss of Pou5f3 and Sox19b leads to premature expression of genes, involved in regulation of organogenesis and differentiation.

Advances in alternative methods upon the vision for toxicity testing in the 21st century

With the increase of global chemical production and the aggravation of population exposure and health risks, higher requirements are put forward for chemical toxicity testing and safety evaluation.'Toxicity testing in the 21st century: a vision and a strategy' has greatly promoted the reform of toxicity testing. Toxicity testing in the new era has made great progress by using new models, new methods and new strategies, combined with interdisciplinary and high-tech advantages. While improving the efficiency of chemical toxicity testing, it also realizes more comprehensive, multi-level and high-quality data acquisition and toxicity evaluation, which provides strong support for the exploration of toxicity mode, toxicity mechanism and toxicity pathway. Focusing on the current alternative new methods of toxicity testing, this issue invites many scholars to introduce and summarize high-content analysis, three-dimensional (3D) cell culture technology, Ex vivo test, single cell sequencing and zebrafish experimental methods, in order to promote the leapfrog development of chemical toxicity testing and evaluation in China.

Total Nucleic Acid Extraction from Single Zebrafish Embryos for Genotyping and RNA-seq.

RNA sequencing allows for the quantification of the transcriptome of embryos to investigate transcriptional responses to various perturbations (e.g., mutations, infections, drug treatments). Previous protocols either lack the option to genotype individual samples, or are laborious and therefore difficult to do at a large scale. We have developed a protocol to extract total nucleic acid from individual zebrafish embryos. Individual embryos are lysed in 96-well plates and nucleic acid is extracted using SPRI beads. The total nucleic acid can be genotyped and then DNase I treated to produce RNA samples for sequencing. This protocol allows for processing large numbers of individual samples, with the ability to genotype each sample, which makes it possible to undertake transcriptomic analysis on mutants at timepoints before the phenotype is visible. Graphic abstract: Extraction of total nucleic acid from individual zebrafish embryos for genotyping and RNA-seq.

A Simple Method to Establish Metaphase Chromosomes from Individual Zebrafish Embryos.

Cytogenetic approach based on metaphase chromosomes established from dividing cells enables direct microscopic visualization of individual chromosomes, a powerful technique to investigate aneuploidy, chromosome aberrations, and genomic instability. In this study, we describe a simple method based on direct blocking of metaphases in individual zebrafish embryo and dropping slides with temperature changes, water vapor, and acetic acid treatment to increase the metaphase diameters. We demonstrate that well-separated metaphases could be established from single zebrafish embryos using this method. Our method could be further adapted for the analyses of DNA damage, chromosome aberrations, and genomic instability using zebrafish and other teleost models.

Tracking leukocytes in intravital time lapse images using 3D cell association learning network

Leukocytes are key cellular elements of the innate immune system in all vertebrates, which play a crucial role in defending organisms against invading pathogens. Tracking these highly migratory and amorphous cells in in vivo models such as zebrafish embryos is a challenging task in cellular immunology. As temporal and special analysis of these imaging datasets by a human operator is quite laborious, developing an automated cell tracking method is highly in demand. Despite the remarkable advances in cell detection, this field still lacks powerful algorithms to accurately associate the detected cell across time frames. The cell association challenge is mostly related to the amorphous nature of cells, and their complicated motion profile through their migratory paths. To tackle the cell association challenge, we proposed a novel deep-learning-based object linkage method. For this aim, we trained the 3D cell association learning network (3D-CALN) with enough manually labelled paired 3D images of single fluorescent zebrafish's neutrophils from two consecutive frames. Our experiment results prove that deep learning is significantly applicable in cell linkage and particularly for tracking highly mobile and amorphous leukocytes. A comparison of our tracking accuracy with other available tracking algorithms shows that our approach performs well in relation to addressing cell tracking problems.

Persistent Ventricle Partitioning in the Adult Zebrafish Heart.

The vertebrate heart integrates cells from the early-differentiating first heart field (FHF) and the later-differentiating second heart field (SHF), both emerging from the lateral plate mesoderm. In mammals, this process forms the basis for the development of the left and right ventricle chambers and subsequent chamber septation. The single ventricle-forming zebrafish heart also integrates FHF and SHF lineages during embryogenesis, yet the contributions of these two myocardial lineages to the adult zebrafish heart remain incompletely understood. Here, we characterize the myocardial labeling of FHF descendants in both the developing and adult zebrafish ventricle. Expanding previous findings, late gastrulation-stage labeling using drl-driven CreERT2 recombinase with a myocardium-specific, myl7-controlled, loxP reporter results in the predominant labeling of FHF-derived outer curvature and the right side of the embryonic ventricle. Raised to adulthood, such lineage-labeled hearts retain broad areas of FHF cardiomyocytes in a region of the ventricle that is positioned at the opposite side to the atrium and encompasses the apex. Our data add to the increasing evidence for a persisting cell-based compartmentalization of the adult zebrafish ventricle even in the absence of any physical boundary.

Rapid in-plate screening of biotransformation products in single zebrafish embryos.

A procedure was developed for rapid screening of xenobiotic biotransformation products (bioTPs) in single zebrafish (ZF; Danio rerio) embryos. Exposure was carried out from 0–120 hours post fertilization (hpf) to 6 different concentrations of the model compound propranolol (PPL). Following in-plate extraction and non-target instrumental analysis by high resolution mass spectrometry, suspected bioTPs were identified using custom data filtration scripts and matching to in silico structural predictions. A total of eight PPL bioTPs were identified (five at a level 1 confidence and one at a level 2–3 confidence). These findings supplement previously generated toxicometabolomic models derived from the same dataset, and were obtained without conducting additional exposure experiments. In addition to facilitating assessments of inter-individual variability in bioTP production in ZF embryos, we demonstrate that bioTPs can be elucidated using extremely small quantities of biomass (i.e. ∼200 μg). To the best of our knowledge, this is the first time bioTP elucidation has been carried out in single ZF embryos.

Disruption of foxc1 genes in zebrafish results in dosage-dependent phenotypes overlapping Axenfeld-Rieger syndrome

The Forkhead Box C1 (FOXC1) gene encodes a forkhead/winged helix transcription factor involved in embryonic development. Mutations in this gene cause dysgenesis of the anterior segment of the eye, most commonly Axenfeld-Rieger syndrome (ARS), often with other systemic features. The developmental mechanisms and pathways regulated by FOXC1 remain largely unknown. There are two conserved orthologs of FOXC1 in zebrafish, foxc1a and foxc1b. To further examine the role of FOXC1 in vertebrates, we generated foxc1a and foxc1b single knockout zebrafish lines and bred them to obtain various allelic combinations. Three genotypes demonstrated visible phenotypes: foxc1a-/- single homozygous and foxc1-/- double knockout homozygous embryos presented with similar characteristics comprised of severe global vascular defects and early lethality, as well as microphthalmia, periocular edema and absence of the anterior chamber of the eye; additionally, fish with heterozygous loss of foxc1a combined with homozygosity for foxc1b (foxc1a+/-;foxc1b-/-) demonstrated craniofacial defects, heart anomalies and scoliosis. All other single and combined genotypes appeared normal. Analysis of foxc1 expression detected a significant increase in foxc1a levels in homozygous and heterozygous mutant eyes, suggesting a mechanism for foxc1a upregulation when its function is compromised; interestingly, the expression of another ARS-associated gene, pitx2, was responsive to the estimated level of wild-type Foxc1a, indicating a possible role for this protein in the regulation of pitx2 expression. Altogether, our results support a conserved role for foxc1 in the formation of many organs, consistent with the features observed in human patients, and highlight the importance of correct FOXC1/foxc1 dosage for vertebrate development.