Zebrafish Cell Lines Research Articles

Functional analysis of ESRP1/2 gene variants and CTNND1 isoforms in orofacial cleft pathogenesis

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

Establishment and transcriptome analysis of single blastomere-derived cell lines from zebrafish

Maintaining chromosome euploidy in zebrafish embryonic cells is challenging because of the degradation of genomic integrity during cell passaging. In this study, we report the derivation of zebrafish cell lines from single blastomeres. These cell lines have a stable chromosome status attributed to BMP4 and exhibit continuous proliferation in vitro. Twenty zebrafish cell lines are successfully established from single blastomeres. Single-cell transcriptome sequencing analysis confirms the fidelity of gene expression profiles throughout long-term culturing of at least 45 passages. The long-term cultured cells are specialized into epithelial cells, exhibiting similar expression patterns validated by integrative transcriptomic analysis. Overall, this work provides a protocol for establishing zebrafish cell lines from single blastomeres, which can serve as valuable tools for in vitro investigations of epithelial cell dynamics in terms of life–death balance and cell fate determination during normal homeostasis.

Assessing the impact of Ag-ZnO nanoparticle on the induction of oxidative stress, hematological, and molecular changes in zebrafish (Danio rerio) and McCoy fibroblast cell lines

Assessing the impact of Ag-ZnO nanoparticle on the induction of oxidative stress, hematological, and molecular changes in zebrafish (Danio rerio) and McCoy fibroblast cell lines

Functional analysis of ESRP1/2 gene variants and CTNND1 isoforms in orofacial cleft pathogenesis.

Orofacial cleft (OFC) is a common human congenital anomaly. Epithelial-specific RNA splicing regulators ESRP1 and ESRP2 regulate craniofacial morphogenesis and their disruption result in OFC in zebrafish, mouse and humans. Using esrp1/2 mutant zebrafish and murine Py2T cell line models, we functionally tested the pathogenicity of human ESRP1/2 gene variants. We found that many variants predicted by in silico methods to be pathogenic were functionally benign. Esrp1 also regulates the alternative splicing of Ctnnd1 and these genes are co-expressed in the embryonic and oral epithelium. In fact, over-expression of ctnnd1 is sufficient to rescue morphogenesis of epithelial-derived structures in esrp1/2 zebrafish mutants. Additionally, we identified 13 CTNND1 variants from genome sequencing of OFC cohorts, confirming CTNND1 as a key gene in human OFC. This work highlights the importance of functional assessment of human gene variants and demonstrates the critical requirement of Esrp-Ctnnd1 acting in the embryonic epithelium to regulate palatogenesis.

A ‘molecular chameleon’ mimic ZrIV-consolidated Fluoro-specific coordination complex and arseno-selective supramolecular metallogelator as a phase discriminatory sensing probe: Substantiation of in-vitro and in-silico approaches with the sensing response

ZrIV consolidated solvatochromic and halochromic coordination complex [ZrIV-ABH (C)] has been synthesized which exhibited promising selectivity and sensitivity towards aqueous phase F-. Upon addition of a trace amount of F-, ZrIV-ABH (C) showcased concentration-reliant chromogenic alteration and fluorogenic spectral shift. Sensing phenomena were corroborated through, UV–Vis spectroscopy, photoluminescence and cyclic voltammetry along with an in-silico approach. The equivalence-dependent chromo-fluorogenic alteration upon the addition of F- on ZrIV-ABH (C), further has been validated theoretically by means of diminution of energy of fluoro-adducts. The bio-compatibility of the probe has been validated through in-vitro detection of F- from hepatocytes of zebrafish and HepG2 cell line. The anti-interfering sensing response of F- among its geogenic lethal congeners has been further utilized to formulate a six-input-two-output logic gate circuitry. Furthermore, ZrIV epicentric coordination complex-based metallogelator probe namely, ZrIV-ABH (G) has been synthesized which can detect AsO2- selectively from the aqueous phase. ZrIV-ABH (G) has been utilized for the real-field validation of aquifer-genic AsO2-. The solvent as well as the temperature-specific formation of ZrIV-ABH (G) metallogelator has been substantiated by rheological behavior. The thermosensitive activity of ZrIV-ABH (G) has been professed by means of chromogenic alteration and fluorogenic turn-on response at a particular elevated temperature. Therefore, this phase-selective switching of sensing response from F- to AsO2- validated its endurance as a value-added contender in its category.

An Efficient Vector-Based CRISPR/Cas9 System in Zebrafish Cell Line.

The clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system has been widely applied in animals as an efficient genome editing tool. However, the technique is difficult to implement in fish cell lines partially due to the lack of efficient promoters to drive the expression of both sgRNA and the Cas9 protein within a single vector. In this study, it was indicated that the zebrafish U6 RNA polymerase III (ZFU6) promoter could efficiently induce tyrosinase (tyr) gene editing and lead to loss of retinal pigments when co-injection with Cas9 mRNA in zebrafish embryo. Furthermore, an optimized all-in-one vector for expression of the CRISPR/Cas9 system in the zebrafish fibroblast cell line (PAC2) was constructed by replacing the human U6 promoter with ZFU6 promoter, basing on the lentiCRISPRV2 system that widely applied in mammal cells. This new vector could successfully target the cellular communication network factor 2a (ctgfa) gene and demonstrated its function in the PAC2 cell. Notably, the vector could also be used to edit the endogenous EMX1 gene in the mammal 293T cell line, implying its wide application potential. In conclusion, we established a new gene editing tool for zebrafish cell line, which could be a useful in vitro platform for high-throughput analyzing gene function in fish.

A Review on Unlocking Toxicity: How Zebrafish embryos Help Ensure Safe Chemicals

Aquatic ecotoxicity testing has benefited greatly from zebrafish embryo toxicity testing (ZFET) because of its high throughput, low cost, sensitivity to a variety of toxicants, and ethical issues. This study highlights the benefits of ZFET over conventional fish toxicity testing, such as its capacity to detect teratogenic defects and possible long-term impacts. The limitations of ZFET are also addressed, notably its emphasis on acute dosages and difficulties with interspecies extrapolation. In addition to the generation of in vitro alternatives utilizing zebrafish cell lines, recent developments in ZFET technology are also highlighted, including the use of transgenic zebrafish lines and high-throughput screening techniques. The paper's conclusion includes a discussion of potential prospects for ZFET research. These include improving interspecies extrapolation methods, optimizing chronic toxicity assessment procedures, and integrating ZFET with other ecotoxicity testing strategies. ZFET is a great instrument for environmental protection and a crucial component in avoiding chemical contamination of aquatic ecosystems, so long as it is acknowledged for what it is and is open to future improvement

Prediction of acute fish toxicity (AFT) and fish embryo toxicity (FET) tests by cytotoxicity assays using liver and embryo zebrafish cell lines (ZFL and ZEM2S)

Fish cell-based assays represent potential alternative methods to vertebrates' use in ecotoxicology. In this study, we evaluated the cytotoxicity of thirteen chemicals, chosen from OECD guidelines 236 and 249, in two zebrafish cell lines (ZEM2S and ZFL). We aimed to investigate whether the IC50 values obtained by viability assays (alamar blue, MTT, CFDA-AM, and neutral red) can predict the LC50 values of Acute Fish Toxicity (AFT) test and Fish Embryo Toxicity (FET) test. There was no significant difference between the values obtained by the different viability assays. ZFL strongly correlated with AFT and FET tests (R2AFT = 0.73–0.90; R2FET48h = 0.79–0.90; R2FET96h = 0.76–0.87), while ZEM2S correlated better with the FET test (48h) (R2 = 0.70–0.86) and weakly with AFT and FET tests (96h) (R2AFT = 0.68–0.74 and R2FET96h = 0.62–0.64). The predicted LC50 values allowed the correct categorization of the chemicals in 76.9% (AFT test) – 90.9% (FET test) using ZFL and in 30.7% (AFT test) – 63.6% (FET test) using ZEM2S considering the US EPA criterion for classifying acute aquatic toxicity. ZFL is a promising cell line to be used in alternative methods to adult fish and fish embryos in ecotoxicity assessments, and the method performed in 96-well plates is advantageous in promoting high-throughput cytotoxicity assessment.

Isolation and characterization of the morphology, size and particle number of rainbow trout (Oncorhynchus mykiss) and zebrafish (Danio rerio) cell line derived large and small extracellular vesicles

Extracellular vesicles (EVs) are 50–1,000 nm lipid bilayer-bound vesicles, released into the extracellular environment by various cell types for intercellular communication purposes. The quantitative and qualitative characteristics of EVs can be affected by stress and pathological conditions. The majority of extracellular vesicle (EV) studies have been performed on mammalian cell lines or bodily fluids. EVs have been previously described from bodily fluids like plasma, serum or mucus in different fish species, however the available knowledge of fish cell line derived EVs is limited and in the vast majority of studies, the overall focus is on small EVs (< 200 nm). We isolated large and small extracellular vesicles from zebrafish (Danio rerio) liver (ZFL), rainbow trout (Oncorhynchus mykiss) liver (RTL-W1), gill (RTgill-W1) and intestinal epithelial (RTgutGC) cell lines using stepwise centrifugation and characterized the size and morphology of EVs. Here we demonstrated that large and small extracellular vesicles can be successfully isolated using stepwise centrifugation from the serum-free medium of the selected piscine cell lines after a 24-h incubation period. The size distribution of large and small EVs isolated from the piscine cell lines suggest that large and small EV groups show high diversity in size ranges, containing heterogenous subpopulations in sizes, and the results highly depend on the applied method and whether filtration steps were included following the isolation. The spherical morphology of EVs was verified by transmission electron microscopy.

Functional characterization of melanocortin 2 receptor (Mc2r) from a lobe-finned fish (Protopterus annectens) and insights into the molecular evolution of melanocortin receptors

Recent studies from our group on melanocortin 2 receptors (Mc2r) from basal families of actinopterygians have served to resolve that Mrap1 dependence and ACTH selectivity are features of even the most basal ray-finned fishes. However, there have been no studies on Mc2r function of the basal sarcopterygians, the lobe-finned fishes, represented by the extant members coelacanths and lungfishes. Here, we offer the first molecular and functional characterization of an Mc2r from a lobe-finned fish, the West African lungfish (Protopterus annectens). Plasmids containing cDNA constructs of lungfish (lf) Mc2r and Mrap1 were expressed in mammalian and zebrafish cell lines. Cells were then stimulated by human ACTH(1–24) and melanocyte stimulating hormone (α-MSH), as well as alanine-substituted analogs of hACTH(1–24) targeting residues within the H6F7R8W9 and K15K16R17R18P19 motifs. Activation of lfMc2r was assessed using a cAMP-responsive luciferase reporter gene assay. In these assays, lfMc2r required co-expression with lfMrap1, was selective for ACTH over α-MSH at physiological concentrations of the ligands, and was completely inhibited by multiple-alanine substitutions of the HFRW (A6–9) and KKRRP (A15–19) motifs. Single- and partial-alanine substitutions of the HFRW and KKRRP motifs varied in their impacts on receptor-ligand affinity from having no effect to completely inhibiting lfMc2r activation. This characterization of the Mc2r of a lobe-finned fish fulfills the last major extant vertebrate group for which Mc2r function had yet to be characterized. In doing so, we resolve that all basal bony vertebrate groups exhibit Mc2r function that substantially differs from that of the cartilaginous fishes, indicating that rapid and dramatic shift in Mc2r function occurred between the radiation of cartilaginous fishes and the emergence of bony fishes. We support this interpretation with a molecular clock analysis of the melanocortin receptors, which demonstrates the uniquely high rate of sequence divergence in Mc2r. Much remains to be understood regarding the molecular evolution of Mc2r during the early radiation of vertebrates that resulted in the derived functional characteristics of Mrap1 dependence and exclusive selectivity for ACTH.

Oxygen potentiates antiviral ability of zebrafish in response to SVCV infection

Oxygen (O2) profoundly influences the physiological processes of aerobic organisms through a range of mechanisms. Recently, increasing evidence has revealed the relationship between viral infection and oxygen levels. However, due to a lack of feasible methods and in vivo models, how oxygen directly affects antiviral capability remains largely unknown. In contrast to terrestrial animals, fish live in water for life, where oxygen levels change more frequently than on land in areas with similar altitude. Therefore, fish appear to be ideal organisms for elucidating the effect of oxygen levels on antiviral responses. In this study, we report that zebrafish under low oxygen conditions are more susceptible to SVCV infection. Further assays indicate that low oxygen tension not only suppresses SVCV-induced IFN activation but also promotes SVCV replication in both zebrafish cell lines and zebrafish. This study provides novel insights into the effect of oxygen on antiviral responses and virus replication.

Susceptibility and Permissivity of Zebrafish (Danio rerio) Larvae to Cypriniviruses

The zebrafish (Danio rerio) represents an increasingly important model organism in virology. We evaluated its utility in the study of economically important viruses from the genus Cyprinivirus (anguillid herpesvirus 1, cyprinid herpesvirus 2 and cyprinid herpesvirus 3 (CyHV-3)). This revealed that zebrafish larvae were not susceptible to these viruses after immersion in contaminated water, but that infections could be established using artificial infection models in vitro (zebrafish cell lines) and in vivo (microinjection of larvae). However, infections were transient, with rapid viral clearance associated with apoptosis-like death of infected cells. Transcriptomic analysis of CyHV-3-infected larvae revealed upregulation of interferon-stimulated genes, in particular those encoding nucleic acid sensors, mediators of programmed cell death and related genes. It was notable that uncharacterized non-coding RNA genes and retrotransposons were also among those most upregulated. CRISPR/Cas9 knockout of the zebrafish gene encoding protein kinase R (PKR) and a related gene encoding a protein kinase containing Z-DNA binding domains (PKZ) had no impact on CyHV-3 clearance in larvae. Our study strongly supports the importance of innate immunity-virus interactions in the adaptation of cypriniviruses to their natural hosts. It also highlights the potential of the CyHV-3-zebrafish model, versus the CyHV-3-carp model, for study of these interactions.

The infection of murine gammaherpesvirus-68 delays the early embryonic development of zebrafish

Zebrafish is a model animal for aquaculture as well as bio-medicine. Murine gamma-herpesvirus 68 (MHV68) is widely regarded as a model for the study of human gamma herpesviruses such as Kaposi's sarcoma-associated herpesvirus (KSHV). In this study, the infection of MHV68 on zebrafish was investigated through incubating fertilized zebrafish eggs with MHV68. The development of zebrafish delayed or stopped at early stage separately when the zebrafish eggs were incubated with MHV68 at different dose for 2 h. The lytic gene of MHV68, including ORF65, RTA and ORF57, were detected in the genome of larvae hatched from the infected eggs. The transcription of the latent gene LANA but not of these lytic genes was detected by RT-PCR. The mRNA level of growth factor TGF-β1, but not of FGF3, was obviously decreased in the larvae from the MHV68 treated eggs. The mRNA levels of cytokines, such as IFNφ3, ISG15 and TNFα, varied post MHV68 infection; however, the transcription of the cytosolic DNA sensors, including DDX41, DHX9 and cGAS, did not change significantly. Moreover, the data generated in zebrafish cell line demonstrated that MHV68 could infect ZF4 cells. All the data support the conclusion that MHV68 could infect zebrafish embryo in the egg envelope and go into latent infection, which will delay the development of the embryos through some unknown mechanism.

A 3D Culture Method of Spheroids of Embryonic and Liver Zebrafish Cell Lines.

Fish cell lines are promising in vitro models for ecotoxicity assessment; however, conventional monolayer culture systems (2D culture) have well-known limitations (e.g., culture longevity and maintenance of some in vivo cellular functions). Thus, 3D cultures, such as spheroids, have been proposed, since these models can reproduce tissue-like structures, better recapturing the in vivo conditions. This article describes an effective, easy, and fast 3D culture protocol for the formation of spheroids with two zebrafish (Danio rerio) cell lines: ZEM2S (embryo) and ZFL (normal hepatocyte). The protocol consists of plating the cells in a round-bottom, ultra-low attachment, 96-well plate. After 5 days under orbital shaking (70 rpm), a single spheroid per well is formed. The formed spheroids present stable size and shape, and this method avoids the formation of multiple spheroids in a well; thus, it is not necessary to handpick spheroids of similar sizes. The ease, speed, and reproducibility of this spheroid method make it useful for high-throughput in vitro tests.

In vivo biodistribution of kinetically stable Pt2L4 nanospheres that show anti-cancer activity.

There is an increasing interest in the application of metal-organic cages (MOCs) in a biomedicinal context, as they can offer non-classical distribution in organisms compared to molecular substrates, while revealing novel cytotoxicity mechanisms. Unfortunately, many MOCs are not sufficiently stable under in vivo conditions, making it difficult to study their structure-activity relationships in living cells. As such, it is currently unclear whether MOC cytotoxicity stems from supramolecular features or their decomposition products. Herein, we describe the toxicity and photophysical properties of highly-stable rhodamine functionalized platinum-based Pt2L4 nanospheres as well as their building blocks under in vitro and in vivo conditions. We show that in both zebrafish and human cancer cell lines, the Pt2L4 nanospheres demonstrate reduced cytotoxicity and altered biodistribution within the body of zebrafish embryos compared to the building blocks. We anticipate that the composition-dependent biodistribution of Pt2L4 spheres together with their cytotoxic and photophysical properties provides the fundament for MOC application in cancer therapy.

Thyroid hormone disrupting potentials of benzisothiazolinone in embryo-larval zebrafish and rat pituitary GH3 cell line

Benzisothiazolinone (BIT), one of the most widely used antimicrobial agents in consumer products, has frequently been detected in the water environment. The present study was conducted to determine the adverse effects of BIT on the thyroid neuroendocrine system of zebrafish embryos/larvae. Rat pituitary (GH3) cell line was employed to support the underlying mechanism of thyroid hormone disrupting effects. Significant coagulation and hatching delay were observed in embryos exposed to 30 μg/L of BIT, which in turn remarkably decreased hatchability and larval survival. In BIT-exposed larvae, tshβ, tshr, and trh genes were significantly upregulated along with a decrease in thyroxine and triiodothyronine content, indicating that BIT decreased thyroid hormones and increased thyrotropin-releasing hormone and thyroid stimulating hormone secretion through a feedback circuit. The downregulation of trα and deio2 genes in the zebrafish larvae suggests the inhibition of thyroid hormone receptors and deiodination. Similar to the results in zebrafish, upregulation of tshβ and downregulation of trα, trβ, deio1, and deio2 genes were observed in GH3 cells. Our observations suggest that BIT can decrease the level of thyroid hormones by influencing central regulation, receptor binding, and deiodination.

Vitamin D Mitigates Hyperglycemia-Induced Cognition Decline in Danio rerio (Zebrafish) through the Activation of Antioxidant Mechanisms.

Hyperglycemia contributes to the development of cognition impairment and related disorders, induces oxidative stress in neuronal cells; thereby, impairs normal signaling mechanisms involved in cognition processes. Studies have shown a significant decrease in the vitamin D in individuals with hyperglycemia and cognition impairment. But whether supplementing vitamin D has any beneficiary impact on mitigating hyperglycemia-induced cognition impairment is unknown. We have first tested the impact of hyperglycemia on the induction of cognition deficiency in a zebrafish model. Next, the molecular mechanisms related to oxidative stress, which are deregulated in hyperglycemic zebrafish brains, have been explored. Subsequently, the impact of supplementing the water with vitamin D and a known activator of nuclear factor erythroid-2 related factor 2 (Nrf2) i.e., sulforaphane (SFN) on learning and memory functions were assessed. We showed a significant increase in the oxidative stress in the brain tissue of zebrafish residing in hyperglycemic water (111 mM glucose). Addition of vitamin D and SFN increased Nrf2, but differentially modulated its target genes (NQO1, SOD, GPx etc) activity in zebrafish and neuronal cell lines thereby improved the hyperglycemia-induced decline of cognition impairment. Mechanistically, vitamin D binds to the Keap1 protein; thereby, interfering with its binding to Nrf2, which leads to the activation of antioxidant mechanisms in the cells. In summary, reducing the oxidative stress through vitamin D treatment is a possible option for controlling the cognition impairment in diabetic population, but studies testing this possibility in clinical trials are currently needed.

CMTr mediated 2'-O-ribose methylation status of cap-adjacent nucleotides across animals.

Cap methyltransferases (CMTrs) O methylate the 2′ position of the ribose (cOMe) of cap-adjacent nucleotides of animal, protist, and viral mRNAs. Animals generally have two CMTrs, whereas trypanosomes have three, and many viruses encode one in their genome. In the splice leader of mRNAs in trypanosomes, the first four nucleotides contain cOMe, but little is known about the status of cOMe in animals. Here, we show that cOMe is prominently present on the first two cap-adjacent nucleotides with species- and tissue-specific variations in Caenorhabditis elegans, honeybees, zebrafish, mouse, and human cell lines. In contrast, Drosophila contains cOMe primarily on the first cap-adjacent nucleotide. De novo RoseTTA modeling of CMTrs reveals close similarities of the overall structure and near identity for the catalytic tetrad, and for cap and cofactor binding for human, Drosophila and C. elegans CMTrs. Although viral CMTrs maintain the overall structure and catalytic tetrad, they have diverged in cap and cofactor binding. Consistent with the structural similarity, both CMTrs from Drosophila and humans methylate the first cap-adjacent nucleotide of an AGU consensus start. Because the second nucleotide is also methylated upon heat stress in Drosophila, these findings argue for regulated cOMe important for gene expression regulation.

Aging Cell Culture - Genetic and Metabolic Effects of Passage Number on Zebrafish Z3 Cells.

Since cell lines are cultured and extensively used in a variety of different research disciplines, we determined the effects of passage numbers on a commonly used embryonic zebrafish cell line (Z3). Senescence markers, DNA damage, the redox state, gene expression, and metabolic parameters have been investigated in young (passage 5) up to very old (passage 40 and higher) cells. Besides increasing DNA damage, we also found elevated metabolic capacity and a shift to a more reduced cellular redox state in the cells. Interestingly, several parameters showed a non-linear course regarding the passage number or cell age, so that for example young and mid-aged cells appeared to cluster with very old rather than with old cells. This study illustrates the importance of passage number and suggests pre-testing specific parameters to assure the generation of accurate and reproducible data.

Leptin Gene Protects Against Cold Stress in Antarctic Toothfish.

Leptin is a cytokine-like peptide, predominantly biosynthesized in adipose tissue, which plays an important role in regulating food intake, energy balance and reproduction in mammals. However, how it may have been modified to enable life in the chronic cold is unclear. Here, we identified a leptin-a gene (lepa) in the cold-adapted and neutrally buoyant Antarctic toothfish Dissostichus mawsoni that encodes a polypeptide carrying four α-helices and two cysteine residues forming in-chain disulfide bonds, structures shared by most vertebrate leptins. Quantitative RT-PCR confirmed that mRNA levels of the leptin-a gene of D. mawsoni (DM-lepa) were highest in muscle, followed by kidney and liver; detection levels were low in the gill, brain, intestine, and ovary tissues. Compared with leptin-a genes of fishes living in warmer waters, DM-lepa underwent rapid evolution and was subjected to positive selection. Over-expression of DM-lepa in the zebrafish cell line ZFL resulted in signal accumulation in the cytoplasm and significantly increased cell proliferation both at the normal culture temperature and under cold treatment. DM-lepa over-expression also reduced apoptosis under low-temperature stress and activated the STAT3 signaling pathway, in turn upregulating the anti-apoptotic proteins bcl2l1, bcl2a, myca and mdm2 while downregulating the pro-apoptotic baxa, p53 and caspase-3. These results demonstrate that DM-lepa, through STAT3 signaling, plays a protective role in cold stress by preventing apoptotic damage. Our study reveals a new role of lepa in polar fish.