Yan Tong
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dbstongy@nus.edu.sg
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Publication Transcriptome analysis of zebrafish embryogenesis using microarrays(2005) Mathavan, S.; Lee, S.G.P.; Mak, A.; Miller, L.D.; Murthy, K.R.K.; Govindarajan, K.R.; Tong, Y.; Wu, Y.L.; Lam, S.H.; Yang, H.; Ruan, Y.; Korzh, V.; Gong, Z.; Liu, E.T.; Lufkin, T.; BIOLOGICAL SCIENCESZebrafish (Danio rerio) is a well-recognized model for the study of vertebrate developmental genetics, yet at the same time little is known about the transcriptional events that underlie zebrafish embryogenesis. Here we have employed microarray analysis to study the temporal activity of developmentally regulated genes during zebrafish embryogenesis. Transcriptome analysis at 12 different embryonic time points covering five different developmental stages (maternal, blastula, gastrula, segmentation, and pharyngula) revealed a highly dynamic transcriptional profile. Hierarchical clustering, stage-specific clustering, and algorithms to detect onset and peak of gene expression revealed clearly demarcated transcript clusters with maximum gene activity at distinct developmental stages as well as coregulated expression of gene groups involved in dedicated functions such as organogenesis. Our study also revealed a previously unidentified cohort of genes that are transcribed prior to the mid-blastula transition, a time point earlier than when the zygotic genome was traditionally thought to become active. Here we provide, for the first time to our knowledge, a comprehensive list of developmentally regulated zebrafish genes and their expression profiles during embryogenesis, including novel information on the temporal expression of several thousand previously uncharacterized genes. The expression data generated from this study are accessible to all interested scientists from our institute resource database (http://giscompute. gis.a-star.edu.sg/∼govind/zebrafish/data_download.html). © 2005 Mathavan et al.Publication Molecular cloning of zebrafish and medaka vitellogenin genes and comparison of their expression in response to 17β-estradiol(2004-03-17) Tong, Y.; Shan, T.; Poh, Y.K.; Yan, T.; Wang, H.; Lam, S.H.; Gong, Z.; BIOLOGICAL SCIENCESIn the present study, both zebrafish and medaka vitellogenin genes have been isolated and used as a biomarker to compare the two small aquarium fish in response to estrogen treatment and thus to evaluate the two fish models in development of a biomonitoring system for environmental estrogens. The isolated zebrafish vitellogenin gene, zvtg1, is the most abundantly expressed vitellogenin gene in zebrafish and its complete protein sequence of 1360 amino acids was deduced from a genomic and a cDNA clone. The isolated medaka vitellogenin (mvtg1) genomic clone covers 1053 amino acids in the N-terminal. Both zebrafish zvtg1 and medaka mvtg1 are specifically expressed in female liver and their expression can be induced by 17β-estradiol (E2) in male fish both by intramuscular injection and immersion treatment. A real-time reverse transcriptase polymerase chain reaction (RT-PCR) assay was developed for quantification of vitellogenin mRNA level in both control fish and E2-treated fish. The lowest-observed-effect concentrations of E2 for the induction of vitellogenin mRNAs were observed at 1 μg/l for zebrafish and 0. 1 μg/l for medaka in a 2-day exposure experiment. Further kinetics studies of the two fish models indicated that medaka was able to respond much faster to E2 treatment than zebrafish, while the zebrafish can attain a much higher level of vitellogenin mRNAs than medaka after a long-term E2 treatment. The implication of these observations may be that the medaka system is better in monitoring acute treatment while the zebrafish system is better in monitoring chronic exposure. © 2004 Elsevier B.V. All rights reserved.Publication Transcriptome kinetics of arsenic-induced adaptive response in zebrafish liver(2006-11-27) Siew, H.L.; Winata, C.L.; Tong, Y.; Korzh, S.; Wen, S.L.; Korzh, V.; Spitsbergen, J.; Mathavan, S.; Miller, L.D.; Liu, E.T.; Gong, Z.; BIOLOGICAL SCIENCESArsenic is a prominent environmental toxicant and carcinogen; however, its molecular mechanism of toxicity and carcinogenicity remains poorly understood. In this study, we performed microarray-based expression profiling on liver of zebrafish exposed to 15 parts/million (ppm) arsenic [As(V)] for 8-96 h to identify global transcriptional changes and biological networks involved in arsenic-induced adaptive responses in vivo. We found that there was an increase of transcriptional activity associated with metabolism, especially for biosyntheses, membrane transporter activities, cytoplasm, and endoplasmic reticulum in the 96 h of arsenic treatment, while transcriptional programs for proteins in catabolism, energy derivation, and stress response remained active throughout the arsenic treatment. Many differentially expressed genes encoding proteins involved in heat shock proteins, DNA damage/repair, antioxidant activity, hypoxia induction, iron homeostasis, arsenic metabolism, and ubiquitin-dependent protein degradation were identified, suggesting strongly that DNA and protein damage as a result of arsenic metabolism and oxidative stress caused major cellular injury. These findings were comparable with those reported in mammalian systems, suggesting that the zebrafish liver coupled with the available microarray technology present an excellent in vivo toxicogenomic model for investigating arsenic toxicity. We proposed an in vivo, acute arsenic-induced adaptive response model of the zebrafish liver illustrating the relevance of many transcriptional activities that provide both global and specific information of a coordinated adaptive response to arsenic in the liver. Copyright © 2006 the American Physiological Society.Publication PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy(Nature Publishing Group, 2018) Wang L.; Cho Y.-L.; Tang Y.; Wang J.; Park J.-E.; Wu Y.; Wang C.; Tong Y.; Chawla R.; Zhang J.; Shi Y.; Deng S.; Lu G.; Wu Y.; Tan H.W.-S.; Pawijit P.; Lim G.G.-Y.; Chan H.-Y.; Zhang J.; Fang L.; Yu H.; Liou Y.-C.; Karthik M.; Bay B.-H.; Lim K.-L.; Sze S.-K.; Yap C.T.; Shen H.-M.; BIOLOGY; PHYSIOLOGY; ANATOMY; SAW SWEE HOCK SCHOOL OF PUBLIC HEALTH; BIOCHEMISTRYMitophagy is an important type of selective autophagy for specific elimination of damaged mitochondria. PTEN-induced putative kinase protein 1 (PINK1)-catalyzed phosphorylation of ubiquitin (Ub) plays a critical role in the onset of PINK1–Parkin-mediated mitophagy. Phosphatase and tensin homolog (PTEN)-long (PTEN-L) is a newly identified isoform of PTEN, with addition of 173 amino acids to its N-terminus. Here we report that PTEN-L is a novel negative regulator of mitophagy via its protein phosphatase activity against phosphorylated ubiquitin. We found that PTEN-L localizes at the outer mitochondrial membrane (OMM) and overexpression of PTEN-L inhibits, whereas deletion of PTEN-L promotes, mitophagy induced by various mitochondria-damaging agents. Mechanistically, PTEN-L is capable of effectively preventing Parkin mitochondrial translocation, reducing Parkin phosphorylation, maintaining its closed inactive conformation, and inhibiting its E3 ligase activity. More importantly, PTEN-L reduces the level of phosphorylated ubiquitin (pSer65-Ub) in vivo, and in vitro phosphatase assay confirms that PTEN-L dephosphorylates pSer65-Ub via its protein phosphatase activity, independently of its lipid phosphatase function. Taken together, our findings demonstrate a novel function of PTEN-L as a protein phosphatase for ubiquitin, which counteracts PINK1-mediated ubiquitin phosphorylation leading to blockage of the feedforward mechanisms in mitophagy induction and eventual suppression of mitophagy. Thus, understanding this novel function of PTEN-L provides a key missing piece in the molecular puzzle controlling mitophagy, a critical process in many important human diseases including neurodegenerative disorders such as Parkinson’s disease. © 2018 IBCB, SIBS, CASPublication Development of estrogen-responsive transgenic medaka for environmental monitoring of endocrine disrupters(2005-11-15) Zeng, Z.; Shan, T.; Tong, Y.; Lam, S.H.; Gong, Z.; BIOLOGICAL SCIENCESTo develop a transgenic fish system to monitor environmental pollution, we generated a mvtg1.gfp transgenic medaka line, in which the gfp reporter gene was under the control of medaka vitellogenin1 (mvtg1) gene promoter. In this transgenic line, GFP was exclusively expressed in the liver of the mature adult female. Male and juvenile transgenic fish did not express GFP but could be induced to express GFP in the liver after exposure to 17-β-estradiol (E2). Concurrent accumulation of mvtg1 and gfp mRIMAs was observed during both development and estrogen treatment, indicating that the gfp transgene was faithfully expressed under the mvtg1 promoter. Dose- and time-dependent induction of GFP expression by E2 was investigated in male transgenic fish. The lowest-observed-effect concentration (LOEC) of E2 to induce GFP expression was 0.5 μg/L by observation of live fish and 0.05-0.1 μg/L by observation of dissection-exposed liver in a 30 day exposure experiment. GFP expression was observed within 36 h after treatment in high concentrations of E2 (5 μg/L), and it took longer to detect GFP expression under lower concentrations of E2. By removal and readdition of E2, we demonstrated that GFP expression was repeatedly induced. Finally, we also demonstrated that GFP expression could be induced by other estrogenic compounds, including 17-α-ethynylestradiol (EE2, 0.05 μg/L), diethylstibestrol (DES, 5 μg/L), estriol (10 μg/L), and bisphenol A (BPA, 1 mg/L), but not by weak estrogenic chemicals such as nonylphenol (NP, up to 1 mg/L) and methoxychlor (MXC, up to 20 μg/L). Our experiments indicated the broad application of the transgenic line to monitor a wide range of estrogenic chemicals. © 2005 American Chemical Society.Publication Recapitulation of fast skeletal muscle development in zebrafish by transgenic expression of GFP under the mylz2 promoter(2003-05-01) Ju, B.; Chong, S.W.; He, J.; Wang, X.; Xu, Y.; Wan, H.; Tong, Y.; Yan, T.; Korzh, V.; Gong, Z.; INSTITUTE OF MOLECULAR AGROBIOLOGY; CIVIL ENGINEERING; BIOLOGICAL SCIENCESA 1,934-bp muscle-specific promoter from the zebrafish mylz2 gene was isolated and characterized by transgenic analysis. By using a series of 5′ promoter deletions linked to the green fluorescent protein (gfp) reporter gene, transient transgenic analysis indicated that the strength of promoter activity appeared to correlate to the number of muscle cis-elements in the promoter and that a minimal -77-bp region was sufficient for a relatively strong promoter activity in muscle cells. Stable transgenic lines were obtained from several mylz2-gfp constructs. GFP expression in the 1,934-bp promoter transgenic lines mimicked well the expression pattern of endogenous mylz2 mRNA in both somitic muscle and nonsomitic muscles, including fin, eye, jaw, and gill muscles. An identical pattern of GFP expression, although at a much lower level, was observed from a transgenic line with a shorter 871-bp promoter. Our observation indicates that there is no distinct cis-element for activation of mylz2 in different skeletal muscles. Furthermore, RNA encoding a dominant negative form of cAMP-dependent protein kinase A was injected into mylz2-gfp transgenic embryos and GFP expression was significantly reduced due to an expanded slow muscle development at the expense of GFP-expressing fast muscle. The mylz2-gfp transgene was also transferred into two zebrafish mutants, spadetail and chordino, and several novel phenotypes in muscle development in these mutants were discovered. © 2003 Wiley-Liss, Inc.Publication Correction to: PTEN-L is a novel protein phosphatase for ubiquitin dephosphorylation to inhibit PINK1–Parkin-mediated mitophagy (Cell Research, (2018), 10.1038/s41422-018-0056-0)(Nature Publishing Group, 2018) Wang L.; Cho Y.-L.; Tang Y.; Wang J.; Park J.-E.; Wu Y.; Wang C.; Tong Y.; Chawla R.; Zhang J.; Shi Y.; Deng S.; Lu G.; Wu Y.; Tan H.W.-S.; Pawijit P.; Lim G.G.-Y.; Chan H.-Y.; Zhang J.; Fang L.; Yu H.; Liou Y.-C.; Karthik M.; Bay B.-H.; Lim K.-L.; Sze S.-K.; Yap C.T.; Shen H.-M.; BIOLOGY; PHYSIOLOGY; ANATOMY; SAW SWEE HOCK SCHOOL OF PUBLIC HEALTH; BIOCHEMISTRYWe apologize for three errors that we just found in the paper published online on 22 June 2018. © 2018, IBCB, SIBS, CAS.Publication Zebrafish whole-adult-organism chemogenomics for large-scale predictive and discovery chemical biology.(2008-07) Lam, S.H.; Mathavan, S.; Tong, Y.; Li, H.; Karuturi, R.K.; Wu, Y.; Vega, V.B.; Liu, E.T.; Gong, Z.; GENOME INSTITUTE OF SINGAPORE; BIOLOGICAL SCIENCESThe ability to perform large-scale, expression-based chemogenomics on whole adult organisms, as in invertebrate models (worm and fly), is highly desirable for a vertebrate model but its feasibility and potential has not been demonstrated. We performed expression-based chemogenomics on the whole adult organism of a vertebrate model, the zebrafish, and demonstrated its potential for large-scale predictive and discovery chemical biology. Focusing on two classes of compounds with wide implications to human health, polycyclic (halogenated) aromatic hydrocarbons [P(H)AHs] and estrogenic compounds (ECs), we generated robust prediction models that can discriminate compounds of the same class from those of different classes in two large independent experiments. The robust expression signatures led to the identification of biomarkers for potent aryl hydrocarbon receptor (AHR) and estrogen receptor (ER) agonists, respectively, and were validated in multiple targeted tissues. Knowledge-based data mining of human homologs of zebrafish genes revealed highly conserved chemical-induced biological responses/effects, health risks, and novel biological insights associated with AHR and ER that could be inferred to humans. Thus, our study presents an effective, high-throughput strategy of capturing molecular snapshots of chemical-induced biological states of a whole adult vertebrate that provides information on biomarkers of effects, deregulated signaling pathways, and possible affected biological functions, perturbed physiological systems, and increased health risks. These findings place zebrafish in a strategic position to bridge the wide gap between cell-based and rodent models in chemogenomics research and applications, especially in preclinical drug discovery and toxicology.Publication Demonstration of site-directed recombination in transgenic zebrafish using the Cre/loxP system(2005-04) Pan, X.; Wan, H.; Chia, W.; Tong, Y.; Gong, Z.; BIOLOGICAL SCIENCESTo test the Cre/loxP recombination system in zebrafish, a stable transgenic zebrafish line was developed by using a floxed (loxP flanked) gfp(green fluorescent protein) gene construct under the muscle-specific mylz2 promoter. Like our previous non-floxed gfp transgenic line under the same promoter, the new transgenic line expresses GFP reporter faithfully in fast skeletal muscles to the same intensity. To demonstrate the excision of floxed gfp transgene, in vitro synthesized Cre RNA was injected into embryos of floxed gfp transgenic zebrafish and we found a dramatic reduction of GFP expression. To confirm the excision, PCR was performed and a DNA fragment of correct size was amplified as predicted from the Cre/loxP mediated excision. Finally, we cloned the fragment and sequence information confirmed that the excision occurred at the precise site as predicted. Our experiments demonstrated that the Cre/loxP system can function efficiently and accurately in the zebrafish system. © Springer 2005.Publication Conservation of gene expression signatures between zebrafish and human liver tumors and tumor progression(2006-01) Siew, H.L.; Yi, L.W.; Vega, V.B.; Miller, L.D.; Spitsbergen, J.; Tong, Y.; Zhan, H.; Govindarajan, K.R.; Lee, S.; Mathavan, S.; Murthy, K.R.K.; Buhler, D.R.; Liu, E.T.; Gong, Z.; BIOLOGICAL SCIENCESThe zebrafish (Danio rerio) has been long advocated as a model for cancer research, but little is known about the real molecular similarities between zebrafish and human tumors. Comparative analysis of microarray data from zebrafish liver tumors with those from four human tumor types revealed molecular conservation at various levels between fish and human tumors. This approach provides a useful strategy for identifying an expression signature that is strongly associated with a disease phenotype. © 2006 Nature Publishing Group.