In order to develop a transgenic zebrafish line with green fluorescent protein (enhanced green fluorescent protein, EGFP) expressed specifically in muscle and heart, the recombinant expression vector constructed using the zebrafish ttn.2 gene promoter fragment and EGFP gene coding sequence and the capped mRNA of Tol2 transposase were co-injected into the zebrafish 1-cell stage embryos. The stable genetic Tg (ttn.2: EGFP) transgenic zebrafish line was successfully developed by fluorescence detection, followed by genetic hybridization screening and molecular identification. Fluorescence signals and whole-mount in situ hybridization showed that EGFP expression was located in muscle and heart, the specificity of which was consistent with the expression of ttn.2 mRNA. Inverse PCR showed that EGFP was integrated into chromosomes 4 and 11 of zebrafish in No. 33 transgenic line, while integrated into chromosome 1 in No. 34 transgenic line. The successful construction of this fluorescent transgenic zebrafish line, Tg (ttn.2: EGFP), laid a foundation for the research of muscle and heart development and related diseases. In addition, the transgenic zebrafish lines with strong green fluorescence can also be used as a new ornamental fish.
Animals ; Zebrafish/genetics* ; Animals, Genetically Modified/genetics* ; Green Fluorescent Proteins/metabolism* ; Zebrafish Proteins/genetics* ; Promoter Regions, Genetic

The immunomodulatory effect of Saposhnikoviae Radix polysaccharide(SRP) was evaluated based on the zebrafish mo-del, and its mechanism was explored by transcriptome sequencing and real-time fluorescence-based quantitative PCR(RT-qPCR). The immune-compromised model was induced by navelbine in the immunofluorescence-labeled transgenic zebrafish Tg(lyz: DsRed), and the effect of SRP on the density and distribution of macrophages in zebrafish was evaluated. The effect of SRP on the numbers of macrophages and neutrophils in wild-type AB zebrafish was detected by neutral red and Sudan black B staining. The content of NO in zebrafish was detected by DAF-FM DA fluorescence probe. The content of IL-1β and IL-6 in zebrafish was detected by ELISA. The differentially expressed genes(DEGs) of zebrafish in the blank control group, the model group, and the SRP treatment group were analyzed by transcriptome sequencing. The immune regulation mechanism was analyzed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment, and the expression levels of key genes were verified by RT-qPCR. The results showed that SRP could significantly increase the density of immune cells in zebrafish, increase the number of macrophages and neutrophils, and reduce the content of NO, IL-1β, and IL-6 in immune-compromised zebrafish. The results of transcriptome sequencing analysis showed that SRP could affect the expression level of immune-related genes on Toll-like receptor pathway and herpes simplex infection pathway to affect the release of downstream cytokines and interferon, thereby completing the activation process of T cells and playing a role in regulating the immune activity of the body.
Animals ; Zebrafish/genetics* ; Interleukin-6/genetics* ; Gene Expression Profiling ; Cytokines/genetics* ; Macrophages ; Transcriptome

OBJECTIVE: To evaluate toxicity of raw extract of Panax notoginseng (rPN) and decocted extract of PN (dPN) by a toxicological assay using zebrafish larvae, and explore the mechanism by RNA sequencing assay. METHODS: Zebrafish larvae was used to evaluate acute toxicity of PN in two forms: rPN and dPN. Three doses (0.5, 1.5, and 5.0 µ g/mL) of dPN were used to treat zebrafishes for evaluating the developmental toxicity. Behavior abnormalities, body weight, body length and number of vertebral roots were used as specific phenotypic endpoints. RNA sequencing (RNA-seq) assay was applied to clarify the mechanism of acute toxicity, followed by real time PCR (qPCR) for verification. High performance liquid chromatography analysis was performed to determine the chemoprofile of this herb. RESULTS: The acute toxicity result showed that rPN exerted higher acute toxicity than dPN in inducing death of larval zebrafishes (P<0.01). After daily oral intake for 21 days, dPN at doses of 0.5, 1.5 and 5.0 µ g/mL decreased the body weight, body length, and vertebral number of larval zebrafishes, indicating developmental toxicity of dPN. No other adverse outcome was observed during the experimental period. RNA-seq data revealed 38 genes differentially expressed in dPN-treated zebrafishes, of which carboxypeptidase A1 (cpa1) and opioid growth factor receptor-like 2 (ogfrl2) were identified as functional genes in regulating body development of zebrafishes. qPCR data showed that dPN significantly down-regulated the mRNA expressions of cpa1 and ogfrl2 (both P<0.01), verifying cpa1 and ogfrl2 as target genes for dPN. CONCLUSION This report uncovers the developmental toxicity of dPN, suggesting potential risk of its clinical application in children.
Animals ; Zebrafish/genetics* ; Saponins/pharmacology* ; Panax notoginseng/chemistry* ; Larva ; Sequence Analysis, RNA

This study used the zebrafish model to explore the hepatotoxicity of Rhododendri Mollis Flos(RMF). The mortality was calculated according to the number of the survival of zebrafish larvae 4 days after fertilization under different concentration of RMF, and the dose-toxicity curve was fitted to preliminarily evaluate the toxicity of RMF. The liver phenotypes under the sublethal concentration of RMF in the treatment group and the blank control group were observed by hematoxylin-eosin(HE) staining and acridine orange(AO) staining. Meanwhile, the activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined to confirm the hepatotoxicity of RMF. Real-time quantitative polymerase chain reaction(real-time PCR) and Western blot were used to determine the expressions of genes and proteins in zebrafish larvae. Gas chromatography time-of-flight mass spectrometry(GC-TOF-MS) was used to conduct untargeted metabolomics testing to explore the mechanism. The results showed that the toxicity of RMF to zebrafish larvae was dose-dependent, with 1 100 μg·mL~(-1) of the absolute lethal concentration and 448 μg·mL~(-1) of sublethal concentration. The hepatocyte apoptosis and degeneration appeared in the zebrafish larvae under the sublethal concentration of RMF. The content of ALT and AST in zebrafish larvae at the end of the experiment was significantly increased in a dose-dependent manner. Under the sublethal concentration, the expressions of genes and proteins related to apoptosis in zebrafish larvae were significantly increased as compared with the blank control group. The results of untargeted metabolomics showed that the important metabolites related to the he-patotoxicity of RMF were mainly enriched in alanine, aspartic acid, glutamic acid, and other pathways. In conclusion, it is inferred that RMF has certain hepatotoxicity to zebrafish larvae, and its mechanism may be related to apoptosis.
Animals ; Zebrafish/genetics* ; Apoptosis ; Larva ; Chemical and Drug Induced Liver Injury

OBJECTIVE: To investigate the mechanism by which Chinese medicine Shengmai Yin (SMY) reverses epithelial-mesenchymal transition (EMT) through lipocalin-2 (LCN2) in nasopharyngeal carcinoma (NPC) cells CNE-2R. METHODS: Morphological changes in EMT in CNE-2R cells were observed under a microscope, and the expressions of EMT markers were detected using quantitative real-time PCR (RT-qPCR) and Western blot assays. Through the Gene Expression Omnibus dataset and text mining, LCN2 was found to be highly related to radiation resistance and EMT in NPC. The expressions of LCN2 and EMT markers following SMY treatment (50 and 100 µ g/mL) were detected by RT-qPCR and Western blot assays in vitro. Cell proliferation, migration, and invasion abilities were measured using colony formation, wound healing, and transwell invasion assays, respectively. The inhibitory effect of SMY in vivo was determined by observing a zebrafish xenograft model with a fluorescent label. RESULTS: The CNE-2R cells showed EMT transition and high expression of LCN2, and the use of SMY (5, 10 and 20 µ g/mL) reduced the expression of LCN2 and reversed the EMT in the CNE-2R cells. Compared to that of the CNE-2R group, the proliferation, migration, and invasion abilities of SMY high-concentration group were weakened (P<0.05). Moreover, SMY mediated tumor growth and metastasis in a dose-dependent manner in a zebrafish xenograft model, which was consistent with the in vitro results. CONCLUSIONS SMY can reverse the EMT process of CNE-2R cells, which may be related to its inhibition of LCN2 expression. Therefore, LCN2 may be a potential diagnostic marker and therapeutic target in patients with NPC.
Animals ; Humans ; Nasopharyngeal Carcinoma/genetics* ; Epithelial-Mesenchymal Transition ; Zebrafish ; Cell Proliferation ; Cell Line, Tumor ; Nasopharyngeal Neoplasms/radiotherapy* ; Cell Movement ; Gene Expression Regulation, Neoplastic

Circular RNAs (circRNAs),a group of highly conserved small RNAs,are characterized by a closed circular structure from precursor linear RNA through reverse splicing.They are powerful regulators of the physiological and pathological processes in organisms at different development stages.Zebrafish can be used for the high-throughput drug screening with low cost.Thus,the circRNAs associated with development and inflammation can be mined from zebrafish.Recently,a variety of circRNAs in zebrafish have been identified and characterized.Studies have proved that circRNAs play a vital role in the development and inflammation of zebrafish.The paper summarizes the classification,characteristics,and biological functions of circRNAs,and reviews the research progress in zebrafish's circRNAs.
Animals ; Inflammation ; RNA/genetics* ; RNA, Circular ; Zebrafish/genetics*

Motor neurons are an important type of neurons that control movement. The transgenic fluorescent protein (FP)-labeled motor neurons of zebrafish line is disadvantageous for studying the morphogenesis of motor neurons. For example, the individual motor neuron is indistinguishable in this transgenic line due to the high density of the motor neurons and the interlaced synapses. In order to optimize the in vivo imaging methods for the analysis of motor neurons, the present study was aimed to establish a microtubule-fluorescent fusion protein mosaic system that can label motor neurons in zebrafish. Firstly, the promotor of mnx1, which was highly expressed in the spinal cord motor neurons, was subcloned into pDestTol2pA2 construct combined with the GFP-α-Tubulin fusion protein sequence by Gateway cloning technique. Then the recombinant constructs were co-injected with transposase mRNA into the 4-8 cell zebrafish embryos. Confocal imaging analysis was performed at 72 hours post fertilization (hpf). The results showed that the GFP fusion protein was expressed in three different types of motor neurons, and individual motor neurons were mosaically labeled. Further, the present study analyzed the correlation between the injection dose and the number and distribution of the mosaically labeled neurons. Fifteen nanograms of the recombinant constructs were suggested as an appropriate injection dose. Also, the defects of the motor neuron caused by the down-regulation of insm1a and kif15 were verified with this system. These results indicate that our novel microtubule-fluorescent fusion protein mosaic system can efficiently label motor neurons in zebrafish, which provides a more effective model for exploring the development and morphogenesis of motor neurons. It may also help to decipher the mechanisms underlying motor neuron disease and can be potentially utilized in drug screening.
Animals ; Animals, Genetically Modified ; Green Fluorescent Proteins/pharmacology* ; Microtubules/metabolism* ; Motor Neurons ; Zebrafish/genetics* ; Zebrafish Proteins/genetics*

OBJECTIVES: A study was conducted to explore the expression pattern and function of ferritin heavy polypeptide gene (fth1b) in zebrafish pharyngeal teeth development and lay the foundation for subsequent research on teeth development and mineralization. METHODS: The zebrafish embryos were harvested at 56, 72, 96, and 120 h after fertilization. The expression of fth1b in zebrafish pharyngeal teeth development was detected by whole embryo RESULTS: The expression pattern of fth1b gene was very similar to that of the known zebrafish pharyngeal teeth marker dlx2b and was specifically expressed in the zebrafish pharyngeal teeth during development. After the specific knockout of the gene fth1b, the earliest gene that can be detect in zebrafish pharyngeal teeth-pitx2 was expressed normally during early development. The dlx2b expression was not significantly different from that of wild type zebrafish, but the mineralization of pharyngeal teeth in the mutant was weaker than that of wild type zebrafish. CONCLUSIONS The gene fth1b is specifically expressed in zebrafish pharyngeal teeth and acts on their early mineralization.
Animals ; In Situ Hybridization ; Odontogenesis ; Pharynx ; Tooth ; Zebrafish/genetics*

With the increasing incidence of hepatobiliary diseases, it is particularly important to understand the role of molecular, cellular and physiological factors in the clinical diagnosis and treatment with traditional Chinese medicine(TCM) in the development of liver disease. Appropriate animal models can help us identify the possible mechanisms of relevant diseases. Danio rerio(zebrafish) model was traditionally used to study embryonic development, and has been gradually used in screening and evaluation of liver diseases and relevant drug in recent years. Zebrafish embryos develop rapidly and the digestive organs of 5-day-old juvenile fish are all mature. At this stage, they may develop hepatobiliary diseases induced by developmental defects or compounds. Zebrafish liver is similar to human liver in cell composition, function, signal transduction, response to injury and cell process mediating liver disease. Furthermore, due to the high conservation of genes and proteins between humans and zebrafish, zebrafish becomes an alternative system for studying basic mechanisms of liver disease. Therefore, genetic screening could be performed to identify new genes involving specific disease processes, and chemical screening could be made for drugs in specific processes. This paper briefly introduced the experimental properties of zebrafish as model system, emphasized the study progress of zebrafish models for pathological mechanism of liver diseases, especially fatty liver, and drug screening and evaluation, so as to provide ideas and techniques for the future liver toxicity assessment of TCM.
Animals ; Drug Evaluation, Preclinical ; Humans ; Liver ; Liver Diseases/genetics* ; Medicine, Chinese Traditional ; Zebrafish/genetics*

Osteoporosis is one of the common clinical orthopedic diseases, which can lead to a variety of complications. There are many pathogenic factors in this disease. The latest research found that ATP6V1H is a new gene leading to the occurrence of osteoporosis, and it is likely to become a new target for the future drug treatment of osteoporosis.This paper introduces the biological structure and characteristics of H subunit, summed up the human body caused by loss of ATP6V1H and animal models such as zebrafish, mice bone loss and osteoporosis symptom such as related research reports of the loss, from osteoclast, osteoblast and marrow stromal cell level and the connection between the various subunits further expounds the H subunit regulate bone dynamic balance of mechanism, to explore ATP6V1H in bone developmentand bone related diseases has laid a solid foundation, also provide new ideas for clinical treatment of osteoporosis.
Animals ; Bone and Bones ; Mice ; Osteoblasts ; Osteoclasts ; Osteoporosis/genetics* ; Zebrafish