Zebrafish are a valuable model organism in biomedical research. Their rapid development, ability to model human diseases, utility for testing genetic variants identified from next-generation ...sequencing, amenity to CRISPR mutagenesis, and potential for therapeutic compound screening, has led to their wide-spread adoption in diverse fields of study. However, their power for large-scale screens is limited by the absence of automated genotyping tools for live animals. This constrains potential drug screen options, limits analysis of embryonic and larval phenotypes, and requires raising additional animals to adulthood to ensure obtaining an animal of the desired genotype. Our objective was to develop an automated system that would rapidly obtain cells and DNA from zebrafish embryos and larvae for genotyping, and that would keep the animals alive. We describe the development, testing, and validation of a zebrafish embryonic genotyping device, termed "ZEG" (Zebrafish Embryo Genotyper). Using microfluidic harmonic oscillation of the animal on a roughened glass surface, the ZEG is able to obtain genetic material (cells and DNA) for use in genotyping, from 24 embryos or larvae simultaneously in less than 10 minutes. Loading and unloading of the ZEG is performed manually with a standard pipette tip or transfer pipette. The obtained genetic material is amplified by PCR and can be used for subsequent analysis including sequencing, gel electrophoresis, or high-resolution melt-analysis. Sensitivity of genotyping and survival of animals are both greater than 90%. There are no apparent effects on body morphology, development, or motor behavior tests. In summary, the ZEG device enables rapid genotyping of live zebrafish embryos and larvae, and animals are available for downstream applications, testing, or raising.
X-linked adrenoleukodystrophy (ALD) is a progressive neurodegenerative disease caused by mutations in ABCD1, the peroxisomal very long-chain fatty acid (VLCFA) transporter. ABCD1 deficiency results ...in accumulation of saturated VLCFAs. A drug screen using a phenotypic motor assay in a zebrafish ALD model identified chloroquine as the top hit. Chloroquine increased expression of stearoyl-CoA desaturase-1 (scd1), the enzyme mediating fatty acid saturation status, suggesting that a shift toward monounsaturated fatty acids relieved toxicity. In human ALD fibroblasts, chloroquine also increased SCD1 levels and reduced saturated VLCFAs. Conversely, pharmacological inhibition of SCD1 expression led to an increase in saturated VLCFAs, and CRISPR knockout of scd1 in zebrafish mimicked the motor phenotype of ALD zebrafish. Importantly, saturated VLCFAs caused ER stress in ALD fibroblasts, whereas monounsaturated VLCFA did not. In parallel, we used liver X receptor (LXR) agonists to increase SCD1 expression, causing a shift from saturated toward monounsaturated VLCFA and normalizing phospholipid profiles. Finally, Abcd1-/y mice receiving LXR agonist in their diet had VLCFA reductions in ALD-relevant tissues. These results suggest that metabolic rerouting of saturated to monounsaturated VLCFAs may alleviate lipid toxicity, a strategy that may be beneficial in ALD and other peroxisomal diseases in which VLCFAs play a key role.
Zebrafish have emerged as a useful model for biomedical research and have been used in environmental toxicology studies. However, the presence of the chorion during the embryo stage limits cellular ...exposure to toxic elements and creates the possibility of a false-negative or reduced sensitivity in fish embryo toxicity testing (FET). This paper presents the use of electroporation as a technique to improve the delivery of toxic elements inside the chorion, increasing the exposure level of the toxins at an early embryo stage (<3 h post-fertilization). A custom-made electroporation device with the required electrical circuitry has been developed to position embryos between electrodes that provide electrical pulses to expedite the entry of molecules inside the chorion. The optimized parameters facilitate material entering into the chorion without affecting the survival rate of the embryos. The effectiveness of the electroporation system is demonstrated using Trypan blue dye and gold nanoparticles (AuNPs, 20-40 nm). Our results demonstrate the feasibility of controlling the concentration of dye and nanoparticles delivered inside the chorion by optimizing the electrical parameters, including pulse width, pulse number, and amplitude. Next, we tested silver nanoparticles (AgNPs, 10 nm), a commonly used toxin that can lower mortality, affect heart rate, and cause phenotypic defects. We found that electroporation of AgNPs reduces the exposure time required for toxicity testing from 4 days to hours. Electroporation for FET can provide rapid entry of potential toxins into zebrafish embryos, reducing the time required for toxicity testing and drug delivery experiments.
The autosomal dominant mutation in the human αB-crystallin gene inducing a R120G amino acid exchange causes a multisystem, protein aggregation disease including cardiomyopathy. The pathogenesis of ...cardiomyopathy in this mutant (hR120GCryAB) is poorly understood. Here, we show that transgenic mice overexpressing cardiac-specific hR120GCryAB recapitulate the cardiomyopathy in humans and find that the mice are under reductive stress. The myopathic hearts show an increased recycling of oxidized glutathione (GSSG) to reduced glutathione (GSH), which is due to the augmented expression and enzymatic activities of glucose-6-phosphate dehydrogenase (G6PD), glutathione reductase, and glutathione peroxidase. The intercross of hR120GCryAB cardiomyopathic animals with mice with reduced G6PD levels rescues the progeny from cardiac hypertrophy and protein aggregation. These findings demonstrate that dysregulation of G6PD activity is necessary and sufficient for maladaptive reductive stress and suggest a novel therapeutic target for abrogating R120GCryAB cardiomyopathy and heart failure in humans.
•Implemented a novel method for transgenesis of zebrafish embryos combining microinjection and electroporation and developed a high-throughput electroporation system.•Electroporation for multiple ...zebrafish embryos without removing the chorion is possible by injecting genetic material inside the chorion before application of pulses.•Optimized electrical parameters (Electric pulse amplitude, number of pulses, pulse duration and pulse gap).•Optimized volume and concentration of genetic material injected inside the chorion.•The system can be converted to a high throughput automated transgenesis system for zebrafish embryos.
Electroporation is regularly used to deliver agents into cells, including transgenic materials, but it is not used for mutating zebrafish embryos due to the lack of suitable systems, information on appropriate operating parameters, and the challenges posed by the protective chorion. Here, a novel method for gene delivery in zebrafish embryos was developed by combining microinjection into the space between the chorion and the embryo followed by electroporation. This method eliminates the need for chorion removal and injecting into the space between the chorion and embryo eliminates the need for finding and identifying key cell locations before performing an injection, making the process much simpler and more automatable. We also developed a microfluidic electroporation system and optimized electric pulse parameters for transgenesis of embryos. The study provided a novel method for gene delivery in zebrafish embryos that can be potentially implemented in a high throughput transgenesis or mutagenesis system.
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Vanishing white matter disease (VWM) is a severe leukodystrophy of the central nervous system caused by mutations in subunits of the eukaryotic initiation factor 2B complex (eIF2B). Current models ...only partially recapitulate key disease features, and pathophysiology is poorly understood. Through development and validation of zebrafish (
) models of VWM, we demonstrate that zebrafish
mutants phenocopy VWM, including impaired somatic growth, early lethality, effects on myelination, loss of oligodendrocyte precursor cells, increased apoptosis in the CNS, and impaired motor swimming behavior. Expression of human
in the zebrafish
mutant rescues lethality and CNS apoptosis, demonstrating conservation of function between zebrafish and human. In the mutants, intron 12 retention leads to expression of a truncated
transcript. Expression of the truncated
in wild-type larva impairs motor behavior and activates the ISR, suggesting that a feed-forward mechanism in VWM is a significant component of disease pathophysiology.
Vanishing white matter (VWM) is a devastating autosomal recessive leukodystrophy, resulting in neurological deterioration and premature death, and without curative treatment. Pathogenic hypomorphic ...variants in subunits of the eukaryotic initiation factor 2B (eIF2B) cause VWM. eIF2B is required for regulating the integrated stress response (ISR), a physiological response to cellular stress. In patients' central nervous system, reduced eIF2B activity causes deregulation of the ISR. In VWM mouse models, the extent of ISR deregulation correlates with disease severity. One approach to restoring eIF2B activity is by inhibition of GSK3β, a kinase that phosphorylates eIF2B and reduces its activity. Lithium, an inhibitor of GSK3β, is thus expected to stimulate eIF2B activity and ameliorate VWM symptoms. The effects of lithium were tested in zebrafish and mouse VWM models. Lithium improved motor behavior in homozygous
mutant zebrafish. In lithium-treated
mutant mice, a paradoxical increase in some ISR transcripts was found. Furthermore, at the dosage tested, lithium induced significant polydipsia in both healthy controls and
mutant mice and did not increase the expression of other markers of lithium efficacy. In conclusion, lithium is not a drug of choice for further development in VWM based on the limited or lack of efficacy and significant side-effect profile.
X-linked adrenoleukodystrophy (ALD) is a devastating inherited neurodegenerative disease caused by defects in the ABCD1 gene and affecting peripheral and central nervous system myelin. ABCD1 encodes ...a peroxisomal transmembrane protein required for very long chain fatty acid (VLCFA) metabolism. We show that zebrafish (Danio rerio) Abcd1 is highly conserved at the amino acid level with human ABCD1, and during development is expressed in homologous regions including the central nervous system and adrenal glands. We used TALENs to generate five zebrafish abcd1 mutant allele lines introducing premature stop codons in exon 1, as well as obtained an abcd1 allele from the Zebrafish Mutation Project carrying a point mutation in a splice donor site. Similar to patients with ALD, zebrafish abcd1 mutants have elevated VLCFA levels. Interestingly, we found that CNS development of the abcd1 mutants is disrupted, with hypomyelination in the spinal cord, abnormal patterning and decreased numbers of oligodendrocytes, and increased cell death. By day of life five abcd1 mutants demonstrate impaired motor function, and overall survival to adulthood of heterozygous and homozygous mutants is decreased. Expression of human ABCD1 in oligodendrocytes rescued apoptosis in the abcd1 mutant. In summary, we have established a zebrafish model of ALD that recapitulates key features of human disease pathology and which reveals novel features of underlying disease pathogenesis.
Leukoencephalopathy with vanishing white matter (VWM) is a progressive incurable white matter disease that most commonly occurs in childhood and presents with ataxia, spasticity, neurological ...degeneration, seizures, and premature death. A distinctive feature is episodes of rapid neurological deterioration provoked by stressors such as infection, seizures, or trauma. VWM is caused by autosomal recessive mutations in one of five genes that encode the eukaryotic initiation factor 2B complex, which is necessary for protein translation and regulation of the integrated stress response. The majority of mutations are in EIF2B5. Astrocytic dysfunction is central to pathophysiology, thereby constituting a potential therapeutic target. Herein we characterize two VWM murine models and investigate astrocyte-targeted adeno-associated virus serotype 9 (AAV9)-mediated EIF2B5 gene supplementation therapy as a therapeutic option for VWM. Our results demonstrate significant rescue in body weight, motor function, gait normalization, life extension, and finally, evidence that gene supplementation attenuates demyelination. Last, the greatest rescue results from a vector using a modified glial fibrillary acidic protein (GFAP) promoter—AAV9-gfaABC(1)D-EIF2B5—thereby supporting that astrocytic targeting is critical for disease correction. In conclusion, we demonstrate safety and early efficacy through treatment with a translatable astrocyte-targeted gene supplementation therapy for a disease that has no cure.
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Herstine and colleagues detail the first-ever adeno-associated virus gene supplementation therapy for the rare leukodystrophy, vanishing white matter disease. Through the characterization of two mouse models utilizing translatable readouts such as MRI, they were able to show promising signs of efficacy and safety in treating this stress-sensitive disease.
The mechanisms of hypoxic injury to the developing human brain are poorly understood, despite being a major cause of chronic neurodevelopmental impairments. Recent work in the invertebrate ...Caenorhabditis elegans has shown that hypoxia causes discrete axon pathfinding errors in certain interneurons and motorneurons. However, it is unknown whether developmental hypoxia would have similar effects in a vertebrate nervous system. We have found that developmental hypoxic injury disrupts pathfinding of forebrain neurons in zebrafish (Danio rerio), leading to errors in which commissural axons fail to cross the midline. The pathfinding defects result from activation of the hypoxia-inducible transcription factor (hif1) pathway and are mimicked by chemical inducers of the hif1 pathway or by expression of constitutively active hif1α. Further, we found that blocking transcriptional activation by hif1α helped prevent the guidance defects. We identified ephrinB2a as a target of hif1 pathway activation, showed that knock-down of ephrinB2a rescued the guidance errors, and showed that the receptor ephA4a is expressed in a pattern complementary to the misrouting axons. By targeting a constitutively active form of ephrinB2a to specific neurons, we found that ephrinB2a mediates the pathfinding errors via a reverse-signaling mechanism. Finally, magnesium sulfate, used to improve neurodevelopmental outcomes in preterm births, protects against pathfinding errors by preventing upregulation of ephrinB2a. These results demonstrate that evolutionarily conserved genetic pathways regulate connectivity changes in the CNS in response to hypoxia, and they support a potential neuroprotective role for magnesium.
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