Yong Long

Transcriptomic Characterization of Temperature Stress Responses in Larval Zebrafish

Temperature influences nearly all biochemical, physiological and life history activities of fish, but the molecular mechanisms underlying the temperature acclimation remains largely unknown. Previous studies have identified many temperature-regulated genes in adult tissues; however, the transcriptional responses of fish larvae to temperature stress are not well understood. In this study, we characterized the transcriptional responses in larval zebrafish exposed to cold or heat stress using microarray analysis. In comparison with genes expressed in the control at 28 °C, a total of 2680 genes were found to be affected in 96 hpf larvae exposed to cold (16 °C) or heat (34 °C) for 2 and 48h and most of these genes were expressed in a temperature-specific and temporally regulated manner. Bioinformatic analysis identified multiple temperature-regulated biological processes and pathways. Biological processes overrepresented among the earliest genes induced by temperature stress include regulation of transcription, nucleosome assembly, chromatin organization and protein folding. However, processes such as RNA processing, cellular metal ion homeostasis and protein transport and were enriched in genes up-regulated under cold exposure for 48 h. Pathways such as mTOR signalling, p53 signalling and circadian rhythm were enriched among cold-induced genes, while adipocytokine signalling, protein export and arginine and praline metabolism were enriched among heat-induced genes. Although most of these biological processes and pathways were specifically regulated by cold or heat, common responses to both cold and heat stresses were also found. Thus, these findings provide new interesting clues for elucidation of mechanisms underlying the temperature acclimation in fish.

Molecular characterization and functions of zebrafish ABCC2 in cellular efflux of heavy metals.

Multidrug-resistance associated protein 2 (MRP2/ABCC2) plays crucial roles in bile formation and detoxification by transporting a wide variety of endogenous compounds and xenobiotics, but its functions in zebrafish (Danio rerio) remain to be characterized. In this study, we obtained the full-length cDNA of zebrafish abcc2, analyzed its expression in developing embryos and adult tissues, investigated its transcriptional response to heavy metals, and evaluated its roles in efflux of heavy metals including cadmium, mercury and lead. Zebrafish abcc2 gene is located on chromosome 13 and composed of 32 exons. The deduced polypeptide of zebrafish ABCC2 consists of 1567 amino acids and possesses most of functional domains and critical residues defined in human ABCC2. Zebrafish abcc2 gene is not maternally expressed and its earliest expression was detected in embryos at 72hpf. In larval zebrafish, abcc2 gene was found to be exclusively expressed in liver, intestine and pronephric tubules. In adult zebrafish, the highest expression of abcc2 gene was found in intestine followed by those in liver and kidney, while relative low expression was detected in brain and muscle. Expression of abcc2 in excretory organs including kidney, liver and intestine of zebrafish larvae was induced by exposure to 0.5μM mercury or 5μM lead. Moreover, exposure to 0.125-1μM of mercury or lead also significantly induced abcc2 expression in these excretory organs of adult zebrafish. Furthermore, overexpression of zebrafish ABCC2 in ZF4 cells and zebrafish embryos decreased the cellular accumulation of heavy metals including cadmium, mercury and lead as determined by MRE (metal responsive element)- or EPRE (electrophile response element)-driven luciferase reporters and atomic absorption spectrometry. These results suggest that zebrafish ABCC2/MRP2 is capable of effluxing heavy metals from cells and may play important roles in the detoxification of toxic metals.

Molecular analysis, developmental function and heavy metal-induced expression of ABCC5 in zebrafish

ABCC5/MRP5 is an organic anion transporter that participates in tissue defense and cellular signal transduction through efflux of anticancer drugs, toxicants and a second messenger cGMP, but its physiological functions in zebrafish remain to be defined. Herein, we report the characterization, spatiotemporal expression and developmental function of zebrafish ABCC5 and its transcriptional responses to heavy metals. Zebrafish abcc5 gene is located on chromosome 18 and comprised of 28 exons. The deduced polypeptide of zebrafish ABCC5 consists of 1426 amino acids, which shares high sequence identity with those from other species. Zebrafish abcc5 is maternally expressed and its transcripts are mainly distributed in brain, lens, liver and intestine of developing embryos. In adults, zebrafish abcc5 is extensively expressed, at higher levels in testis, brain, eye, ovary, intestine and kidney, but at relatively lower levels in gill, liver, heart and muscle. Blockage of endogenous ABCC5 activity by its dominant-negative led to the developmental retardation of zebrafish embryos in which activity of p21 signaling was markedly stimulated and cellular cGMP content was significantly increased. In addition, expression of abcc5 in ZF4 cells and zebrafish embryos was significantly induced by cadmium (Cd), lead (Pb), mercury (Hg) or arsenic (As). The induced expression of ABCC5 by heavy metals was mainly detected in the liver of embryos at 96-h post-fertilization (hpf). In adult zebrafish, expression of abcc5 in brain, intestine, liver, kidney and ovary was significantly induced by one or more of these heavy metals. Furthermore, overexpression of ABCC5 attenuated the toxicity of Cd to zebrafish embryos, but did not affect the toxicity of Hg or As. Thus, ABCC5 is likely to play an active role in embryonic development and heavy metal detoxification through the export of second messenger molecules and toxicants out of cells in zebrafish.

MRP proteins as potential mediators of heavy metal resistance in zebrafish cells

Acquired resistance of mammalian cells to heavy metals is closely relevant to enhanced expression of several multidrug resistance-associated proteins (MRP), but it remains unclear whether MRP proteins confer resistance to heavy metals in zebrafish. In this study, we obtained zebrafish (Danio rerio) fibroblast-like ZF4 cells with resistance to toxic heavy metals after chronic cadmium exposure and selection for 6months. These cadmium-resistant cells (ZF4-Cd) were maintained in 5μM cadmium and displayed cross-resistance to cadmium, mercury, arsenite and arsenate. ZF4-Cd cells remained the resistance to heavy metals after protracted culture in cadmium-free medium. In comparison with ZF4-WT cells, ZF4-Cd cells exhibited accelerated rate of cadmium excretion, enhanced activity of MRP-like transport, elevated expression of abcc2, abcc4 and mt2 genes, and increased content of cellular GSH. Inhibition of MRP-like transport activity, GSH biosynthesis and GST activity significantly attenuated the resistance of ZF4-Cd cells to heavy metals. The results indicate that some of MRP transporters are involved in the efflux of heavy metals conjugated with cellular GSH and thus play crucial roles in heavy metal detoxification of zebrafish cells.

De Novo Assembly of Mud Loach (Misgurnus anguillicaudatus) Skin Transcriptome to Identify Putative Genes Involved in Immunity and Epidermal Mucus Secretion

Fish skin serves as the first line of defense against a wide variety of chemical, physical and biological stressors. Secretion of mucus is among the most prominent characteristics of fish skin and numerous innate immune factors have been identified in the epidermal mucus. However, molecular mechanisms underlying the mucus secretion and immune activities of fish skin remain largely unclear due to the lack of genomic and transcriptomic data for most economically important fish species. In this study, we characterized the skin transcriptome of mud loach using Illumia paired-end sequencing. A total of 40364 unigenes were assembled from 86.6 million (3.07 gigabases) filtered reads. The mean length, N50 size and maximum length of assembled transcripts were 387, 611 and 8670 bp, respectively. A total of 17336 (43.76%) unigenes were annotated by blast searches against the NCBI non-redundant protein database. Gene ontology mapping assigned a total of 108513 GO terms to 15369 (38.08%) unigenes. KEGG orthology mapping annotated 9337 (23.23%) unigenes. Among the identified KO categories, immune system is the largest category that contains various components of multiple immune pathways such as chemokine signaling, leukocyte transendothelial migration and T cell receptor signaling, suggesting the complexity of immune mechanisms in fish skin. As for mucin biosynthesis, 37 unigenes were mapped to 7 enzymes of the mucin type O-glycan biosynthesis pathway and 8 members of the polypeptide N-acetylgalactosaminyltransferase family were identified. Additionally, 38 unigenes were mapped to 23 factors of the SNARE interactions in vesicular transport pathway, indicating that the activity of this pathway is required for the processes of epidermal mucus storage and release. Moreover, 1754 simple sequence repeats (SSRs) were detected in 1564 unigenes and dinucleotide repeats represented the most abundant type. These findings have laid the foundation for further understanding the secretary processes and immune functions of loach skin mucus.

Transcriptional events co-regulated by hypoxia and cold stresses in Zebrafish larvae

BackgroundHypoxia and temperature stress are two major adverse environmental conditions often encountered by fishes. The interaction between hypoxia and temperature stresses has been well documented and oxygen is considered to be the limiting factor for the thermal tolerance of fish. Although both high and low temperature stresses can impair the cardiovascular function and the cross-resistance between hypoxia and heat stress has been found, it is not clear whether hypoxia acclimation can protect fish from cold injury.ResultsPre-acclimation of 96-hpf zebrafish larvae to mild hypoxia (5% O2) significantly improved their resistance to lethal hypoxia (2.5% O2) and increased the survival rate of zebrafish larvae after lethal cold (10°C) exposure. However, pre-acclimation of 96-hpf larvae to cold (18°C) decreased their tolerance to lethal hypoxia although their ability to endure lethal cold increased. RNA-seq analysis identified 132 up-regulated and 41 down-regulated genes upon mild hypoxia exposure. Gene ontology enrichment analyses revealed that genes up-regulated by hypoxia are primarily involved in oxygen transport, oxidation-reduction process, hemoglobin biosynthetic process, erythrocyte development and cellular iron ion homeostasis. Hypoxia-inhibited genes are enriched in inorganic anion transport, sodium ion transport, very long-chain fatty acid biosynthetic process and cytidine deamination. A comparison with the dataset of cold-regulated gene expression identified 23 genes co-induced by hypoxia and cold and these genes are mainly associated with oxidation-reduction process, oxygen transport, hemopoiesis, hemoglobin biosynthetic process and cellular iron ion homeostasis. The alleviation of lipid peroxidation damage by both cold- and hypoxia-acclimation upon lethal cold stress suggests the association of these genes with cold resistance. Furthermore, the alternative promoter of hmbsb gene specifically activated by hypoxia and cold was identified and confirmed.ConclusionsAcclimation responses to mild hypoxia and cold stress were found in zebrafish larvae and pre-acclimation to hypoxia significantly improved the tolerance of larvae to lethal cold stress. RNA-seq and bioinformatics analyses revealed the biological processes associated with hypoxia acclimation. Transcriptional events co-induced by hypoxia and cold may represent the molecular basis underlying the protection of hypoxia-acclimation against cold injury.

Zebrafish Abcb4 is a potential efflux transporter of microcystin-LR

Microcystin-LR (MC-LR) is one of the most common microcystins (MCs), which are hepatotoxic and released into a water body during a period of cyanobacterial blooms. These toxicants can be accumulated in aquatic animals and transferred along the food chain and thus pose adverse effects on aquatic environment and public health. Zebrafish Abcb4 is reported to mediate the cellular efflux of ecotoxicologically relevant compounds including galaxolide, tonalide and phenanthrene; however, it remains unclear whether Abcb4 functions in the detoxification of MC-LR. Here, we demonstrated the role of zebrafish Abcb4 in cellular efflux of MC-LR. Transcripts of zebrafish abcb4 were detected in all of adult tissues examined. MC-LR was able to induce the expression of abcb4 gene and overexpression of Abcb4 significantly decreased the cytotoxicity and accumulation of MC-LR in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcb4-G1177D mutant abolished its transporter function but not substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to this cyanobacterial toxin. Moreover, ATPase activity in developing embryos can be induced by MC-LR. Thus, zebrafish Abcb4 plays crucial roles in cellular efflux of MC-LR and is a potential molecular marker for the monitoring of cyanobacteria contamination in the aquatic environment.

Characterization of Zebrafish Abcc4 as an Efflux Transporter of Organochlorine Pesticides

DDT and lindane are highly toxic organochlorine pesticides and posing adverse effects on the environment and public health due to their frequent usage in developing countries. ABCC4/MRP4 is an organic anion transporter that mediates cellular efflux of a wide range of exogenous and endogenous compounds such as cyclic nucleotides and anti-cancer drugs; however, it remains unclear whether ABCC4 and its orthologs function in the detoxification of organochlorine pesticides. Here, we demonstrated the roles of zebrafish Abcc4 in cellular efflux of DDT and lindane. Zebrafish abcc4 was maternally expressed in the oocytes and its transcripts were detected in the lens, pancreas, gills, liver, intestine and bladder of developing embryos and in adult tissues examined. DDT and lindane were able to induce the expression of abcc4 gene and overexpression of Abcc4 significantly decreased the cytotoxicity and accumulation of DDT and lindane in LLC-PK1 cells and developing embryos. In contrast, overexpression of an Abcc4-G1188D mutant abolished its transporter function without effects on its substrate binding activity, and sensitized LLC-PK1 cells and developing embryos to toxic pesticides. Moreover, glutathione (GSH) was involved in the efflux of cellular pesticides and ATPase activity in developing embryos can be induced by DDT or lindane. Thus, zebrafish Abcc4 plays crucial roles in cellular efflux of organochlorine pesticides and can be used a potential molecular marker for the monitor of DDT and lindane contamination in the aquatic environment.

Effective Gene Trapping Mediated by Sleeping Beauty Transposon

Gene trapping is a high-throughput approach to elucidate gene functions by disrupting and recapitulating expression of genes in a target genome. A number of transposon-based gene-trapping systems are developed for mutagenesis in cells and model organisms, but there is still much room for the improvement of their efficiency in gene disruption and mutation. Herein, a gene-trapping system mediated by Sleeping Beauty (SB) transposon was developed by inclusion of three functional cassettes. The mutation cassette can abrogate the splice of trapped genes and terminate their translation. Once an endogenous gene is captured, the finding cassette independently drives the translation of reporter gene in HeLa cells and zebrafish embryos. The efficiency cassette controls the remobilization of integrated traps through inducible expression of SB gene. Analysis of transposon-genome junctions indicate that most of trap cassettes are integrated into an intron without an obvious 3′ bias. The transcription of trapped genes was abrogated by alternative splicing of the mutation cassette. In addition, integrated transposons can be induced to excise from their original insertion sites. Furthermore, the Cre/LoxP system was introduced to delete the efficiency cassette for stabilization of gene interruption and bio-safety. Thus, this gene-trap vector is an alternative and effective tool for the capture and disruption of endogenous genes in vitro and in vivo.

Lzts2 Regulates Embryonic Cell Movements and Dorsoventral Patterning through Interaction with and Export of Nuclear β-Catenin in Zebrafish

Background: The activities of leucine zipper tumor suppressor 2 (Lzts2) in vertebrate embryogenesis remain unclear. Results: Lzts2 regulates gastrula CE movements, dorsoventral patterning, and midline convergence and specification of organ precursors in zebrafish. Conclusion: Lzts2 functions through interaction with and the export of nuclear β-catenins. Significance: This work has uncovered the developmental roles of Lzts2 and regulatory mechanisms in zebrafish. Leucine zipper tumor suppressor 2 (Lzts2) functions in the development and progression of various tumors, but its activities in vertebrate embryogenesis remain unclear. Here, we demonstrate that lzts2 transcripts are of maternal origin in zebrafish embryos. Activation of BMP signaling up-regulates zygotic expression of lzts2, whereas canonical Wnt signaling acts upstream of BMP signaling to inhibit lzts2 expression. Abrogation of lzts2 expression by its specific morpholino-enhanced gastrula convergence and extension (CE) movements, dorsalized early embryos, and inhibited specification of midline progenitors for pancreas, liver, and heart. In contrast, ectopic expression of lzts2 led to the delay of CE movements and midline convergence of organ progenitors and resulted in a certain ratio of ventralized embryos. Mechanistically, Lzts2 regulates the migration of embryonic cells and dorsoventral patterning through its limitation of Wnt/β-catenin activity, because it physically interacts with β-catenin-1 and -2 and transports them out of the nucleus. In addition, both β-catenin-1 and -2 exhibit redundant functions in activation of Stat3 signaling and in induction of Wnt5/11 expression through inhibition of BMP signaling and stimulation of Cyclops and Squint expression. Thus, Lzts2 regulates gastrula CE movements, dorsoventral patterning, and midline convergence and specification of organ precursors through interaction with and the export of nuclear β-catenins in zebrafish.