Zebrafish Model Organism Research Articles

Experimental approaches to modeling delirium in zebrafish (<i>Danio rerio</i>)

Delirium is an acute psychiatric syndrome characterized by impaired attention, perception and orientation in space. Albeit known for millennia, the mechanisms and risk factors of delirium remain poorly understood. Experimental animal models, especially rodents, become widely used to understand these pathogenetic aspects. However, a new model organism zebrafish (Danio rerio) also has promise in this field. Discussed here, recent behavioral tests and protocols for zebrafish enable assessing their behavioral and cognitive function, and its experimental modulation by deliriant drugs and other factors.

In vivo anti-inflammatory and antioxidant effects of microbial polysaccharides extracted from Euganean therapeutic muds

Therapeutic thermal mud produced by spas of the Euganean Thermal District (Italy) is used as a treatment for arthro-rheumatic diseases. Its production involves the growth of a specific microbiota embedded in a polysaccharidic matrix. Polysaccharides (Microbial-PolySaccharides, M-PS) released in the mud by the resident microorganisms were extracted and analyzed. The monosaccharidic composition analysis showed the presence of galacturonic acid, mannose, xylose, ribose and glucose and a high percentage of sulfated groups in the polymers. To assess their involvement in the therapeutic efficacy of the mud, the M-PS were tested using the model organism zebrafish (Danio rerio). The anti-inflammatory and antioxidant activities were evaluated after confirming the lack of toxic effects during development. Inflammatory state was induced chemically with copper sulfate, or through tail fin amputation procedure and UVB exposure. Recovery from inflammatory condition after exposure to M-PS was always observed with specific morphometric analyses, and further supported by qPCR. Genes linked with the inflammatory and oxidative stress response were investigated confirming the M-PS treatment's efficacy.

Application of Model Organism Zebrafish in Constructing Animal Models of Human Diseases

As one of the most important model organisms in biological research, due to its unique advantages, zebrafish have been widely used in developmental biology, genetics, oncology, pharmacology, toxicology, environmental protection, and many other fields since the 1970s making many new important contributions. It may be expected that with the enrichment of research methods and the development of science, zebrafish will play a greater role in biomedicine as an ideal model organism and help us to understand the basic biological and human mechanisms greatly.

Investigating the toxically homogenous effects of three lanthanides on zebrafish

The adverse effects of rare earth elements (REEs) have been increasingly reported in the past decades and have raised concern about their environmental toxicities. However, the available data is insufficient to elucidate the toxic effects, mechanisms, and whether the toxicity across all REEs is uniform. In this study, zebrafish were exposed to 0, 0.8, 1.6, 3.2, 6.4, 12.8 and 25.6 mg/L Ln(NO3)3•6H2O to test the acute toxicity of La(III), Ce(III), and Nd(III). LC50 of the three lanthanides was compared to the extent of the impact on gene expression. We carried out the functionally grouped network-based transcriptome analysis using gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) to explore the molecular mechanisms. The acute toxicity test showed that LC50 of La(III), Ce(III), and Nd(III) were 2.53, 2.03, and 2.76 mg/L, respectively. Consistent with acute toxicity, Ce(III) caused a little more DEGs than La(III) and Nd(III). Some biological processes such as metabolism of xenobiotics, oocyte meiosis, steroid biosynthesis, DNA replication, and p53 signaling pathway were affected following exposure of all the three lanthanides. Ce(III) also induced changes in the chemokine-mediated signaling pathway. The results indicated that the lethality is comparable, and the toxic patterns are similar across the three lanthanides. This study gives comparative research on the toxicities of three lanthanides to model organism zebrafish.

Plastic nanoparticles cause mild inflammation, disrupt metabolic pathways, change the gut microbiota and affect reproduction in zebrafish: A full generation multi-omics study

Plastic pollution has become a major concern on a global scale. The plastic is broken down into minuscule particles, which have an impact on the biosystems, however long-term impacts through an entire generation is largely unknown. Here, we present the first whole generation study exposing fish to a 500 nm polystyrene plastic particle at environmentally relevant concentrations. Short- and long-term adverse effects were investigated in the zebrafish model organism using a holistic multi-omics approach. The particles accumulated in the yolk sac of young larvae and short-term biological impacts included immune-relevant gene regulation related to inflammation and tolerance as well as disruption of metabolic processes, such as the fatty acid and lipid pathways. The long-term effects comprised gene regulations pointing towards skin and/or gill inflammation, dysbiosis of the gut microbiota, a tendency towards decreased condition factor in adult males as well as a lowered reproductive capability. From this study, it can be concluded that exposures to plastic nanoparticles have an impact on population as well as ecosystem level in fish and likely also in other vertebrates.

Molecular and Cellular Mechanisms of Vascular Development in Zebrafish.

The establishment of a functional cardiovascular system is crucial for the development of all vertebrates. Defects in the development of the cardiovascular system lead to cardiovascular diseases, which are among the top 10 causes of death worldwide. However, we are just beginning to understand which signaling pathways guide blood vessel growth in different tissues and organs. The advantages of the model organism zebrafish (Danio rerio) helped to identify novel cellular and molecular mechanisms of vascular growth. In this review we will discuss the current knowledge of vasculogenesis and angiogenesis in the zebrafish embryo. In particular, we describe the molecular mechanisms that contribute to the formation of blood vessels in different vascular beds within the embryo.

Upregulation of GBP1 in thyroid primordium is required for developmental thyroid morphogenesis

PurposeCongenital hypothyroidism (CH) is a common congenital endocrine disorder in humans. CH-related diseases such as athyreosis, thyroid ectopy, and hypoplasia are primarily caused by dysgenic thyroid development. However, the underlying molecular mechanisms remain unknown. MethodsTo identify novel CH candidate genes, 192 CH patients were enrolled, and target sequencing of 21 known CH-related genes was performed. The remaining 98 CH patients carrying no known genes were subjected to exome sequencing (ES). The functions of the identified variants were confirmed using thyroid epithelial cells in vitro and in zebrafish model organisms in vivo. ResultsFour pathogenic GBP1 variations from three patients were identified. In zebrafish embryos, gbp1 knockdown caused defective thyroid primordium morphogenesis and hypothyroidism. The thyroid cells were stuck together and failed to dissociate from each other to form individual follicles in gbp1-deficient embryos. Furthermore, defects were restored with wild-type human GBP1 (hGBP1) messenger RNA (mRNA) except for mutated hGBP1 (p.H150Y, p.L187P) overexpression. GBP1 promoted β-catenin translocation into the cytosol and suppressed the formation of cellular adhesion complexes. Suppression of cell–cell adhesion restored the thyroid primordium growth defect observed in gbp1-deficient zebrafish embryos. ConclusionThis study provides further understanding regarding thyroid development and shows that defective cellular remodeling could cause congenital hypothyroidism.

Making Blood from the Vessel: Extrinsic and Environmental Cues Guiding the Endothelial-to-Hematopoietic Transition.

It is increasingly recognized that specialized subsets of endothelial cells carry out unique functions in specific organs and regions of the vascular tree. Perhaps the most striking example of this specialization is the ability to contribute to the generation of the blood system, in which a distinct population of “hemogenic” endothelial cells in the embryo transforms irreversibly into hematopoietic stem and progenitor cells that produce circulating erythroid, myeloid and lymphoid cells for the lifetime of an animal. This review will focus on recent advances made in the zebrafish model organism uncovering the extrinsic and environmental factors that facilitate hemogenic commitment and the process of endothelial-to-hematopoietic transition that produces blood stem cells. We highlight in particular biomechanical influences of hemodynamic forces and the extracellular matrix, metabolic and sterile inflammatory cues present during this developmental stage, and outline new avenues opened by transcriptomic-based approaches to decipher cell–cell communication mechanisms as examples of key signals in the embryonic niche that regulate hematopoiesis.

From start-up to scale-up: a journey through AAV process development

Kevin Mouillesseaux, PhD, joined StrideBio in October 2016, when the entire company could take conference calls around a single Bluetooth speakerphone. Dr Mouillesseaux leads StrideBio’s upstream and downstream process development efforts and engineered the company’s proprietary suspension producer cell line, HEKMo. Before coming to StrideBio, Dr Mouillesseaux worked in Biogen’s small-scale upstream process development group. As an academic scientist, Dr Mouillesseaux earned his doctorate at the University of California, Los Angeles, using molecular and cellular biology and genetics to study endothelial cell biology in the zebrafish model organism. During his postdoctoral fellowship at the University of North Carolina at Chapel Hill, Dr Mouillesseaux leveraged those same skills to study intercellular communication during cell fate determination.

2-Amino-3-methylimidazo[4,5-f]quinoline induced oxidative stress and inflammation via TLR4/MAPK and TLR4/NF-κB signaling pathway in zebrafish (Danio rerio) livers

2-Amino-3-methylimidazole[4,5-f]quinoline (IQ) is a harmful substance, mainly existing in protein-abundant thermally processed foods and polluted environments. This study investigated the hepatotoxicity of IQ by exposing zebrafish model organisms at 0, 8, 80, and 800 ng/mL concentrations for 35 days and was supposed to reveal the mechanism of IQ-induced oxidative stress and inflammation in the liver. The results showed that, after IQ exposure, alanine aminotransferase (ALT), aspartate aminotransferase (AST), reactive oxygen species (ROS), and malondialdehyde (MDA) levels in zebrafish liver increased significantly; meanwhile, significantly increased tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), interleukin-6 (IL-6), and interleukin-12 (IL-12) levels induced severe oxidative stress and inflammation; however, glutathione (GSH), superoxide dismutase (SOD), catalase (CAT), glutathione s-transferase (GST) and glutathione peroxidase (GSH-Px) levels significantly decreased. The results indicated that the increased IQ exposure gradually aggravated pathological changes of zebrafish liver tissue (irregular cell morphology, cytoplasmic vacuolation, and inflammatory cell infiltration) and induced significant liver damage at last. Alterations in the expressions of genes and proteins involved in the IQ-induced TLR4/MAPK and TLR4/NF-κB pathways can elucidate the mechanism of its hepatotoxicity. The study provides evidence of IQ-induced hepatotoxicity and helps to draw attention to the health risks of dietary and environmental exposure to IQ.

Short-term dietary restriction maintains synaptic plasticity whereas short-term overfeeding alters cellular dynamics in the aged brain: evidence from the zebrafish model organism

Increased caloric intake (OF) impairs quality of life causing comorbidities with other diseases and cognitive deficits, whereas dietary restriction (DR) increases healthspan by preventing age-related deteriorations. To understand the effects of these opposing dietary regimens on the cellular and synaptic dynamics during brain aging, the zebrafish model, which shows gradual aging like mammals, was utilized. Global changes in cellular and synaptic markers with respect to age and a 12 week dietary regimen of OF and DR demonstrated that aging reduces the levels of the glutamate receptor subunits, GLUR2/3, inhibitory synaptic clustering protein, GEP, synaptic vesicle protein, SYP, and early-differentiated neuronal marker, HuC. DR significantly elevates levels of glutamate receptor subunits, GLUR2/3, and NMDA clustering protein, PSD95, levels, while OF subtly increases the level of the neuronal protein, DCAMKL1. These data suggest that decreased caloric intake within the context of aging has more robust effects on synapses than cellular proteins, whereas OF alters cellular dynamics. Thus, patterns like these should be taken into account for possible translation to human subjects.

Environmental enrichment applied with sensory components prevents age-related decline in synaptic dynamics: Evidence from the zebrafish model organism.

Progression of cognitive decline with or without neurodegeneration varies among elderly subjects. The main aim of the current study was to illuminate the molecular mechanisms that promote and retain successful aging in the context of factors such as environment and gender, both of which alter the resilience of the aging brain. Environmental enrichment (EE) is one intervention that may lead to the maintenance of cognitive processing at older ages in both humans and animal subjects. EE is easily applied to different model organisms, including zebrafish, which show similar age-related molecular and behavioral changes as humans. Global changes in cellular and synaptic markers with respect to age, gender and 4-weeks of EE applied with sensory stimulation were investigated using the zebrafish model organism. Results indicated that EE increases brain weight in an age-dependent manner without affecting general body parameters like body mass index (BMI). Age-related declines in the presynaptic protein synaptophysin, AMPA-type glutamate receptor subunits and a post-mitotic neuronal marker were observed and short-term EE prevents these changes in aged animals, as well as elevates levels of the inhibitory scaffolding protein, gephyrin. Gender-driven alterations were observed in the levels of the glutamate receptor subunits. Oxidative stress markers were significantly increased in the old animals, while exposure to EE did not alter this pattern. These data suggest that EE with sensory stimulation exerts its effects mainly on age-related changes in synaptic dynamics, which likely increase brain resilience through specific cellular mechanisms.

Zebrafish Tools for Deciphering Habenular Network-Linked Mental Disorders.

Simple SummaryEverything that we think, feel or do depends on the function of neural networks in the brain. These are highly complex structures made of cells (neurons) and their interconnections (axons), which develop dependent on precisely coordinated interactions of genes. Any gene mutation can result in unwanted alterations in neural network formation and concomitant brain disorders. The habenula neural network is one of these important circuits, which has been linked to autism, schizophrenia, depression and bipolar disorder. Studies using the zebrafish have uncovered genes involved in the development of this network. Intriguingly, some of these genes have also been identified as risk genes of human brain disorders highlighting the power of this animal model to link risk genes and the affected network to human disease. But can we use the advantages of this model to identify new targets and compounds with ameliorating effects on brain dysfunction? In this review, we summarise the current knowledge on techniques to manipulate the habenula neural network to study the consequences on behavior. Moreover, we give an overview of existing behavioral test to mimic aspects of mental disorders and critically discuss the applicability of the zebrafish model in this field of research.The prevalence of patients suffering from mental disorders is substantially increasing in recent years and represents a major burden to society. The underlying causes and neuronal circuits affected are complex and difficult to unravel. Frequent disorders such as depression, schizophrenia, autism, and bipolar disorder share links to the habenular neural circuit. This conserved neurotransmitter system relays cognitive information between different brain areas steering behaviors ranging from fear and anxiety to reward, sleep, and social behaviors. Advances in the field using the zebrafish model organism have uncovered major genetic mechanisms underlying the formation of the habenular neural circuit. Some of the identified genes involved in regulating Wnt/beta-catenin signaling have previously been suggested as risk genes of human mental disorders. Hence, these studies on habenular genetics contribute to a better understanding of brain diseases. We are here summarizing how the gained knowledge on the mechanisms underlying habenular neural circuit development can be used to introduce defined manipulations into the system to study the functional behavioral consequences. We further give an overview of existing behavior assays to address phenotypes related to mental disorders and critically discuss the power but also the limits of the zebrafish model for identifying suitable targets to develop therapies.

The evolving cardiac lymphatic vasculature in development, repair and regeneration.

The lymphatic vasculature has an essential role in maintaining normal fluid balance in tissues and modulating the inflammatory response to injury or pathogens. Disruption of normal development or function of lymphatic vessels can have severe consequences. In the heart, reduced lymphatic function can lead to myocardial oedema and persistent inflammation. Macrophages, which are phagocytic cells of the innate immune system, contribute to cardiac development and to fibrotic repair and regeneration of cardiac tissue after myocardial infarction. In this Review, we discuss the cardiac lymphatic vasculature with a focus on developments over the past 5 years arising from the study of mammalian and zebrafish model organisms. In addition, we examine the interplay between the cardiac lymphatics and macrophages during fibrotic repair and regeneration after myocardial infarction. Finally, we discuss the therapeutic potential of targeting the cardiac lymphatic network to regulate immune cell content and alleviate inflammation in patients with ischaemic heart disease.

Insights into structure and dynamics of extracellular domain of Toll-like receptor 5 in Cirrhinus mrigala (mrigala): A molecular dynamics simulation approach.

The toll-like receptor 5 (TLR5) is the most conserved important pattern recognition receptors (PRRs) often stimulated by bacterial flagellins and plays a major role in the first-line defense against invading pathogenic bacteria and in immune homeostasis. Experimental crystallographic studies have shown that the extracellular domain (ECD) of TLR5 recognizes flagellin of bacteria and functions as a homodimer in model organism zebrafish. However, no structural information is available on TLR5 functionality in the major carp Cirrhinus mrigala (mrigala) and its interaction with bacterial flagellins. Therefore, the present study was undertaken to unravel the structural basis of TLR5-flagellin recognition in mrigala using structural homodimeric TLR5-flagellin complex of zebrafish as reference. Integrative structural modeling and molecular dynamics simulations were employed to explore the structural and mechanistic details of TLR5 recognition. Results from structural snapshots of MD simulation revealed that TLR5 consistently formed close interactions with the three helices of the D1 domain in flagellin on its lateral side mediated by several conserved amino acids. Results from the intermolecular contact analysis perfectly substantiate with the findings of per residue-free energy decomposition analysis. The differential recognition mediated by flagellin to TLR5 in mrigala involves charged residues at the interface of binding as compared to the zebrafish complex. Overall our results shows TLR5 of mrigala involved in innate immunity specifically recognized a conserved site on flagellin which advocates the scientific community to explore host-specific differences in receptor activation.

Light-sheet fluorescence imaging charts the gastrula origin of vascular endothelial cells in early zebrafish embryos

It remains challenging to construct a complete cell lineage map of the origin of vascular endothelial cells in any vertebrate embryo. Here, we report the application of in toto light-sheet fluorescence imaging of embryos to trace the origin of vascular endothelial cells (ECs) at single-cell resolution in zebrafish. We first adapted a previously reported method to embryo mounting and light-sheet imaging, created an alignment, fusion, and extraction all-in-one software (AFEIO) for processing big data, and performed quantitative analysis of cell lineage relationships using commercially available Imaris software. Our data revealed that vascular ECs originated from broad regions of the gastrula along the dorsal–ventral and anterior–posterior axes, of which the dorsal–anterior cells contributed to cerebral ECs, the dorsal–lateral cells to anterior trunk ECs, and the ventral–lateral cells to posterior trunk and tail ECs. Therefore, this work, to our knowledge, charts the first comprehensive map of the gastrula origin of vascular ECs in zebrafish, and has potential applications for studying the origin of any embryonic organs in zebrafish and other model organisms.

Inhibitory Effect of Morin Against Candida albicans Pathogenicity and Virulence Factor Production: An in vitro and in vivo Approaches.

Candida albicans is considered an exclusive etiologic agent of candidiasis, a very common fungal infection in human. The expression of virulence factors contributes highly to the pathogenicity of C. albicans. These factors include biofilm formation, yeast-to-hyphal transition, adhesins, aspartyl proteases, and phospholipases secretion. Moreover, resistance development is a critical issue for the therapeutic failure of antifungal agents against systemic candidiasis. To circumvent resistance development, the present study investigated the virulence targeted therapeutic activity of the phyto-bioactive compound morin against C. albicans. Morin is a natural compound commonly found in medicinal plants and widely used in the pharmaceutical and cosmetic products/industries. The present study explicated the significant inhibitory potential of morin against biofilm formation and other virulence factors’ production, such as yeast-hyphal formation, phospholipase, and exopolymeric substances, in C. albicans. Further, qPCR analysis confirmed the downregulation of biofilm and virlence-related genes in C. albicans upon morin treatment, which is in correspondence with the in vitro bioassays. Further, the docking analysis revealed that morin shows strong affinity with Hwp-1 protein, which regulates the expression of biofilm and hyphal formation in C. albicans and, thereby, abolishes fungal pathogenicity. Moreover, the anti-infective potential of morin against C. albicans-associated systemic candidiasis is confirmed through an in vivo approach using biomedical model organism zebrafish (Danio rerio). The outcomes of the in vivo study demonstrate that the morin treatment effectively rescues animals from C. albicans infections and extends their survival rate by inhibiting the internal colonization of C. albicans. Histopathology analysis revealed extensive candidiasis-related pathognomonic changes in the gills, intestine, and kidney of animals infected with C. albicans, while no extensive abnormalities were observed in morin-treated animals. The results evidenced that morin has the ability to protect against the pathognomonic effect and histopathological lesions caused by C. albicans infection in zebrafish. Thus, the present study suggests that the utilization of morin could act as a potent therapeutic medication for C. albicans instigated candidiasis.

Microcystin-LR exposure results in aberrant spindles and induces apoptosis in porcine oocytes

Microcystin-LR (MC-LR), as a well-known hepatotoxin, was recently found to accumulate in gonads and induce a variety of reproductive damages in zebrafish, mice and other model organisms, however, little information is available on whether MC-LR has toxic effects on the mammalian oocytes, especially in livestock species. In this study, the effects of MC-LR on meiotic maturation of porcine oocytes were investigated, and the potential mechanism of MC-LR toxicity was explored. Germinal vesicle (GV)-stage oocytes were exposed to 0, 20, 40 and 60 μM MC-LR, respectively, during the in vitro maturation for 44 h, and the results showed that the first polar body (PbI) extrusion rate of the oocytes decreased significantly when the MC-LR concentration reached 40 (P < 0.01) or 60 μM (P < 0.001). After treated with 60 μM MC-LR for 44 h, a significant higher percentage of the oocytes arrested at anaphase-telophase I (ATI) stage (P < 0.01). Laser scanning confocal results further confirmed that a significantly larger proportion of the 60 μM MC-LR-treated oocytes exhibited aberrant spindles and misaligned chromosomes, suggesting a failure of spindle assembly and homologous chromosome segregation during the ATI stage. Furthermore, the ROS levels in the 60 μM MC-LR-exposed oocytes were significantly higher than the control group (P < 0.01), while the expression of antioxidant related genes (SOD1, CAT and GPX) were much lower compared with control group, indicating that oxidative stress was induced and the antioxidant capacity of oocytes was depleted by 60 μM MC-LR treatment. Additionally, markedly decreased mitochondrial membrane potential (MMP) (P < 0.01) and significantly higher incidence of early apoptosis (P < 0.01) were observed in the 60 μM MC-LR-treated oocytes, suggesting that MC-LR exposure induced apoptosis in porcine oocytes. Moreover, the protein expression of PP2A was remarkably inhibited, whereas the expression of p53, BAX, Caspase3 and Cleaved-caspase3 were prominently increased in the 60 μM MC-LR-exposed oocytes. Together, these results suggested that 60 μM of MC-LR exposure can induce oxidative stress, and lead to aberrant spindles, impaired MMP, and trigger apoptosis, which eventually result in failure of porcine oocyte maturation.

The neurophysiological effects of iron in early life stages of zebrafish

Trace metal/ion homeostasis, neurophysiological performance, and molecular responses to iron (Fe) exposure were investigated in the model organism zebrafish (Danio rerio). The findings demonstrated that exposure to a sublethal concentration of ferric iron (Fe3+) increased Fe contents in both the whole body and head region of developing zebrafish. Among the various trace metals and major ion examined, a dysregulation in manganese, zinc, nickel, and calcium balance was also observed in Fe-exposed larvae. Further biochemical assay and in-vivo imaging revealed that Fe exposure resulted in possible oxidative stress-induced damage, and an increased generation of reactive oxygen species in specific regions of the larvae. Using a droplet digital PCR (ddPCR) technology, it was found that the expression levels of various oxidative stress-responsive genes were temporally modulated by Fe exposure. Additionally, Fe-exposed larvae exhibited an impairment in escape response and a decrease in swimming activity. These larvae also appeared to exhibit a reduced anxiety-like behaviour. Together, our research suggested that larvae experiencing an increased Fe loading exhibited a dysregulation in metal homeostasis and a decrease in neurophysiological performance. These results suggested that neurophysiological assessments are sensitive methods to evaluate Fe toxicity in developing fish.

Zebrafish: A Suitable Tool for the Study of Cell Signaling in Bone.

In recent decades, many studies using the zebrafish model organism have been performed. Zebrafish, providing genetic mutants and reporter transgenic lines, enable a great number of studies aiming at the investigation of signaling pathways involved in the osteoarticular system and at the identification of therapeutic tools for bone diseases. In this review, we will discuss studies which demonstrate that many signaling pathways are highly conserved between mammals and teleost and that genes involved in mammalian bone differentiation have orthologs in zebrafish. We will also discuss as human diseases, such as osteogenesis imperfecta, osteoarthritis, osteoporosis and Gaucher disease can be investigated in the zebrafish model.