mitfa deficiency promotes immune vigor and potentiates antitumor effects in zebrafish.

The mitfa gene is a well-known transcription factor associated with microphthalmia and is essential for early melanophore development. However, little is known about how mitfa affects the immune system. Here, we generated a novel mitfa knock-out zebrafish line using the CRISPR/Cas9 system. The mitfa-/- zebrafish exhibited reduced melanin levels compared to the nacre mutant. We investigated the impact on the immune system after exposure to Edwardsiella tarda and bifenazate in zebrafish larvae, and observed that the macrophage numbers were reduced in both treated groups. Remarkably, the expression levels of immune-related genes exhibited significant increases after bacterial challenge or bifenazate exposure in the mitfa-/- zebrafish, except for tlr4 and rela. Furthermore, we conducted xenograft experiments using mouse B16 melanoma cells. Notably, the cancer cells didn't show a high cell migration ratio, implying that the immune system was highly activated after the loss of mifta. Taken together, our findings suggest that mitfa-/- zebrafish serve as a valuable model for investigating the relationship between the immune system and melanocytes, providing new insights into the role of mitfa in immune responses.

Loss of mpv17 affected early embryonic development via mitochondria dysfunction in zebrafish.

MVP17 encodes a mitochondrial inner-membrane protein, and mutation of human MVP17 can cause mitochondria DNA depletion syndrome (MDDS). However, the underlying function of mpv17 is still elusive. Here, we developed a new mutant with mpv17 knockout by using the CRISPR/Cas9 system. The mpv17-/- zebrafish showed developmental defects in muscles, liver, and energy supply. The mpv17-/- larvae hardly survived beyond a month, and they showed abnormal growth during the development stage. Abnormal swimming ability was also found in the mpv17-/- zebrafish. The transmission electron microscope (TEM) observation indicated that the mpv17-/- zebrafish underwent severe mitochondria dysfunction and the disorder of mitochondrial cristae. As an energy producer, the defects of mitochondria significantly reduced ATP content in mpv17-/- zebrafish, compared to wild-type zebrafish. We hypothesized that the disorder of mitochondria cristae was contributed to the dysfunction of muscle and liver in the mpv17-/- zebrafish. Moreover, the content of major energy depot triglycerides (TAG) was decreased dramatically. Interestingly, after rescued with normal exogenous mitochondria by microinjection, the genes involved in the TAG metabolism pathway were recovered to a normal level. Taken together, this is the first report of developmental defects in muscles, liver, and energy supply via mitochondria dysfunction, and reveals the functional mechanism of mpv17 in zebrafish.

Knock-In Strategy for Editing Human and Zebrafish Mitochondrial DNA Using Mito-CRISPR/Cas9 System.

The mitochondria DNA (mtDNA) editing tool, zinc finger nucleases (ZFNs), transcription activator-like effector nuclease (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9 (CRISPR/Cas9) system, is a promising approach for the treatment of mtDNA diseases by eliminating mutant mitochondrial genomes. However, there have been no reports of repairing the mutant mtDNA with homologous recombination strategy to date. Here, we show a mito-CRISPR/Cas9 system that mito-Cas9 protein can specifically target mtDNA and reduce mtDNA copy number in both human cells and zebrafish. An exogenous single-stranded DNA with short homologous arm was knocked into the targeting loci accurately, and this mutagenesis could be steadily transmitted to F1 generation of zebrafish. Moreover, we found some major factors involved in nuclear DNA repair were upregulated significantly by the mito-CRISPR/Cas9 system. Taken together, our data suggested that the mito-CRISPR/Cas9 system could be a useful method to edit mtDNA by knock-in strategy, providing a potential therapy for the treatment of inherited mitochondrial diseases.

Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) is a persistent organic pollutant (POP), an unintentional byproduct of various industrial processes, and a human carcinogen. The expression of the cytochrome P450 1A (cyp1a) gene is upregulated in the presence of TCDD through activating the aryl hydrocarbon receptor pathway in a dose-dependent manner. Several essential response elements, including the 8 potential xenobiotic response elements in the cyp1a promoter region, have been identified to be the main functional parts for the response to TCDD. Thus, we aimed to develop a convenient and sensitive biomonitoring tool to examine the level of POPs in the environment and evaluate its potential human health risks by TCDD. Here, we established a transgenic zebrafish model with a red fluorescent reporter gene ( mCherry) using the truncated cyp1a promoter. Under exposure to TCDD, the expression pattern of mCherry in the reporter zebrafish mirrored that of endogenous cyp1a mRNA, and the primary target tissues for TCDD were the brain vessels, liver, gut, cloaca, and skin. Our results indicated that exposure of the embryos to TCDD at concentrations as low as 0.005 nM for 48 h, which did not elicit morphologic abnormalities in the embryos, markedly increased mCherry expression. In addition, the reporter embryos responded to other POPs, and primary liver cell culture of zebrafish revealed that Cyp1a protein was mainly expressed in the cytoplasm of liver cells. Furthermore, our transgenic fish embryos demonstrated that TCDD exposure can regulate the expression levels of several tumor-related factors, including epidermal growth factor, TNF-α, C-myc, proliferating cell nuclear antigen, TGF-ß, serine/threonine kinase (Akt), and phosphorylated Akt, suggesting that our transgenic fish can be used as a sensitive model to evaluate the carcinogenicity induced by TCDD exposure.-Luo, J.-J., Su, D.-S., Xie, S.-L., Liu, Y., Liu, P., Yang, X.-J., Pei D.-S. Hypersensitive assessment of aryl hydrocarbon receptor transcriptional activity using a novel truncated cyp1a promoter in zebrafish.

Deletion of mstna and mstnb impairs the immune system and affects growth performance in zebrafish.

Myostatin (Mstn) is a negative regulator of muscle development in vertebrates. Although its function in muscle growth has been well studied in mammals and fish, it remains unclear whether or how mstn functions in the immune system. In this study, mstna-/- and mstnb-/- homozygous zebrafish were firstly generated using CRISPR/Cas9 (Clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9). Deletion of mstnb but not mstna enhanced growth performance. Although survival rates under normal conditions were slightly decreased in both strains, mortality after dexamethasone-induced stress was increased by ∼30%. Furthermore, transcriptional levels of several critical immune-related genes were decreased, and the ability to withstand exposure to pathogenic E. tarda was decreased, compared with that of controls. In mstnb-/- but not mstna-/- zebrafish, expression of NF-κB subunits and several pro-inflammatory cytokines failed to respond to E. tarda exposure except nfkb1, c-rel and tnfα. Taken together, these results indicate that mstnb but not mstna plays a key role in zebrafish muscle growth. While each paralogue contributes to the response to bacterial insult, mstnb affects the immune system through activation of the NF-κB pathway, and mstna is likely to act upstream of NF-κB at some as yet unidentified target.

Generation and application of a novel transgenic zebrafish line Tg(cyp1a:mCherry) as an in vivo assay to sensitively monitor PAHs and TCDD in the environment.

The polycyclic aromatic hydrocarbons (PAHs) and 2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) are classified as human carcinogens, and can also cause serious health problems. To develop a convenient bio-monitoring tool for the detection of PAHs and TCDD in the environment, we generated a transgenic zebrafish line Tg(cyp1a:mCherry) with cyp1a promoter driving mCherry expression. Here, Tg(cyp1a:mCherry) embryos were treated with different concentrations of TCDD and five US EPA priority PAHs congeners. The results showed that the expressions of mCherry and endogenous cyp1a were consistent with the PAHs exposure concentrations and were largely induced by TCDD and ≥4-ring PAHs. Moreover, the sensitivity of Tg(cyp1a:mCherry) embryos was also evaluated through monitoring of the PAHs contamination in the water and soil samples. The elevated red fluorescent signals and cyp1a expression levels were observed in Tg(cyp1a:mCherry) zebrafish after exposure to water samples and soil organic extracts with higher concentrations of ≥4-ring PAHs. These results further strengthen our findings of concentration- and congener-dependent response of the newly established zebrafish. Taken together, the newly established zebrafish line will prove as a sensitive, efficient and convenient tool for monitoring PAHs and TCDD contamination in the environment.

A novel technique based on in vitro oocyte injection to improve CRISPR/Cas9 gene editing in zebrafish.

Contemporary improvements in the type II clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9) system offer a convenient way for genome editing in zebrafish. However, the low efficiencies of genome editing and germline transmission require a time-intensive and laborious screening work. Here, we reported a method based on in vitro oocyte storage by injecting oocytes in advance and incubating them in oocyte storage medium to significantly improve the efficiencies of genome editing and germline transmission by in vitro fertilization (IVF) in zebrafish. Compared to conventional methods, the prior micro-injection of zebrafish oocytes improved the efficiency of genome editing, especially for the sgRNAs with low targeting efficiency. Due to high throughputs, simplicity and flexible design, this novel strategy will provide an efficient alternative to increase the speed of generating heritable mutants in zebrafish by using CRISPR/Cas9 system.

Toxicological characterization of a novel wastewater treatment process using EDTA-Na2Zn as draw solution (DS) for the efficient treatment of MBR-treated landfill leachate.

Landfill leachate has become an important source of environmental pollution in past decades, due to the increase of waste volume. Acute toxic and genotoxic hazards to organisms can be caused by landfill leachate. Thus, how to efficiently recover water from landfill leachate and effectively eliminate combined toxicity of landfill leachate are the most pressing issues in waste management. In this study, EDTA-Na2Zn as draw solution (DS) was used to remove the toxicity of membrane bioreactor-treated landfill leachate (MBR-treated landfill leachate) in forward osmosis (FO) process, and nanofiltration (NF) was designed for recovering the diluted DS. Zebrafish and human cells were used for toxicity assay after the novel wastewater treatment process using EDTA-Na2Zn as DS. Results showed that the water recovery rate of MBR-treated landfill leachate (M-LL) in FO membrane system could achieve 66.5% and 71.2% in the PRO and FO mode respectively, and the diluted DS could be efficiently recovered by NF. Toxicity tests performed by using zebrafish and human cells showed that M-LL treated by EDTA-Na2Zn had no toxicity effect on zebrafish larvae and human cells, but it had very slight effect on zebrafish embryos. In conclusion, all results indicated that EDTA-Na2Zn as DS can effectively eliminate toxicity of landfill leachate and this method is economical and eco-friendly for treatment of different types of landfill leachate.