Publications by authors named "Sinnakaruppan Mathavan"

43 Publications

Exploring Translational Control of Maternal mRNAs in Zebrafish.

Methods Mol Biol 2021 ;2218:367-380

Skin Research Institute of Singapore, Agency for Science Technology and Research, Singapore, Singapore.

The study of translational regulation requires reliable measurement of both mRNA levels and protein synthesis. Cytoplasmic polyadenylation is a prevalent mode of translational regulation during oogenesis and early embryogenesis. Here the length of the poly(A) tail of an mRNA is coupled to its translatability. We describe a protocol to identify translationally regulated genes and measure their translation rate in the early zebrafish embryo using genome-wide polysome profiling. This protocol relies on the isolation of mRNA by means of an rRNA depletion strategy, which avoids capture bias due to short poly(A) tail that can occur when using conventional oligo(dT)-based methods. We also present a simple PCR-based method to measure the poly(A) tail length of selected mRNAs.
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http://dx.doi.org/10.1007/978-1-0716-0970-5_29DOI Listing
January 2021

CERKL mutation causing retinitis pigmentosa(RP) in Indian population - a genotype and phenotype correlation study.

Ophthalmic Genet 2020 12 31;41(6):570-578. Epub 2020 Aug 31.

Shri Bhagwan Mahavir Vitreoretinal Services, Medical Research Foundation , Chennai, India.

Background: Mutations in gene has been reported to cause Retinitis pigmentosa (RP) and clinically appears discrete from other commonly encountered phenotypes. We report 14 patients who were seen to have CERKL mutation of the 152 patients of RP from Indian population who underwent genetic testing.

Materials And Methods: A retrospective analysis was performed in 28 eyes of the 14 unrelated patients to establish genotype phenotype correlation. Targeted next generation sequencing was performed using the STRAND® NGS v2.5 software. Validation was done using PCR-based bidirectional Sanger sequencing. Clinical data was collected along with imaging such as fundus photo, autofluorescence(AF), Optical coherence tomography and Electroretinogram wherever available.

Results: Three variants c.1045_1046delAT, c.847 C > T and a novel c.899-IG>A were identified. Retinal morphological features were typically bilaterally symmetrical with mild to moderate disc pallor and arteriolar attenuation in all cases, while sparse peripheral pigmentation was noted in seven patients indicating paucipigmentary character. Early macular involvement in all cases was a characteristic finding with central hypo-autofluorescence and surrounding hyper-autofluorescence. Peripheral scalloped chorioretinal atrophic patches were seen in five patients particularly in older patients.

Conclusions: Phenotype associated with CERKL mutation appears clinically discrete from other commonly encountered phenotypes of inherited retinal dystrophies. Recognizing this typical genotype phenotype correlation will help clinicians to identify this form of RP, prognosticate the disease and segregate candidates for futures gene therapy.
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http://dx.doi.org/10.1080/13816810.2020.1814347DOI Listing
December 2020

Cxcl9l and Cxcr3.2 regulate recruitment of osteoclast progenitors to bone matrix in a medaka osteoporosis model.

Proc Natl Acad Sci U S A 2020 08 27;117(32):19276-19286. Epub 2020 Jul 27.

Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore;

Bone homeostasis requires continuous remodeling of bone matrix to maintain structural integrity. This involves extensive communication between bone-forming osteoblasts and bone-resorbing osteoclasts to orchestrate balanced progenitor cell recruitment and activation. Only a few mediators controlling progenitor activation are known to date and have been targeted for intervention of bone disorders such as osteoporosis. To identify druggable pathways, we generated a medaka () osteoporosis model, where inducible expression of receptor-activator of nuclear factor kappa-Β ligand (Rankl) leads to ectopic formation of osteoclasts and excessive bone resorption, which can be assessed by live imaging. Here we show that upon Rankl induction, osteoblast progenitors up-regulate expression of the chemokine ligand Cxcl9l. Ectopic expression of Cxcl9l recruits -positive macrophages to bone matrix and triggers their differentiation into osteoclasts. We also demonstrate that the chemokine receptor Cxcr3.2 is expressed in a distinct subset of macrophages in the aorta-gonad-mesonephros (AGM). Live imaging revealed that upon Rankl induction, Cxcr3.2-positive macrophages get activated, migrate to bone matrix, and differentiate into osteoclasts. Importantly, mutations in prevent macrophage recruitment and osteoclast differentiation. Furthermore, Cxcr3.2 inhibition by the chemical antagonists AMG487 and NBI-74330 also reduced osteoclast recruitment and protected bone integrity against osteoporotic insult. Our data identify a mechanism for progenitor recruitment to bone resorption sites and Cxcl9l and Cxcr3.2 as potential druggable regulators of bone homeostasis and osteoporosis.
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http://dx.doi.org/10.1073/pnas.2006093117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7431079PMC
August 2020

Development of an automated system for the detection of genotype in polypoidal choroidal vasculopathy using retinal image phenotype.

Comput Methods Programs Biomed 2020 Aug 19;192:105460. Epub 2020 Mar 19.

SN ONGC Department of Genetics and Molecular Biology, Vision Research Foundation, Sankara Nethralaya campus, Chennai-600006, India.

Background And Objectives: Polypoidal choroidal vasculopathy (PCV) is a retinal disorder characterized by the presence of aneurismal polypoidal lesions in the choroidal vasculature. A single nucleotide polymorphism (SNP) is a common genetic variant which may be associated with the disease. This study is to investigate the association of HERPUD1 (rs2217332) gene with PCV in the Indian population and develop an automated system for genotype and phenotype correlation using fundus images and machine learning methods.

Methods: A cohort of 54 PCV patients and 120 control subjects were recruited for the study. Genotyping of SNP (HERPUD1, rs2217332) was performed by following polymerase chain reaction and direct sequencing method. Statistical association of SNP to PCV was determined using chi-square analysis. The acquired GG and AG images were preprocessed using an adaptive histogram. 19 and 18 texture features were extracted from the images in the PCV naïve cases and PCV patients on treatment, respectively. Student's independent t-test was then employed for the selection of significant features, which were input to the ensemble tree for automated classification. Leave-one-out validation was used to evaluate the system.

Results: HERPUD1 rs2217332 SNP is significantly associated in PCV patients compared to control (P = 0.0296, odds ratio [OD] = 2.297, 95% confidence interval [CI] = 1.087-4.856) in the Indian population. High F1 and precision values of 85.71%, 86.84% and 85.71%, 93.75% were achieved in the pre and post- treatment phases, respectively.

Conclusion: Our results suggest that the HERPUD1 polymorphism is associated in PCV patients. Based on our analysis, it may be possible to predict the genotype and disease status of PCV patients using fundus images in assistance with a machine learning algorithm.
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http://dx.doi.org/10.1016/j.cmpb.2020.105460DOI Listing
August 2020

A novel hybrid approach for automated detection of retinal detachment using ultrasound images.

Comput Biol Med 2020 05 19;120:103704. Epub 2020 Mar 19.

Department of Electronics and Computer Engineering, Ngee Ann Polytechnic, Clementi, 599489, Singapore; Department of Bioinformatics and Medical Engineering, Asia University, Taichung, Taiwan; International Research Organization for Advanced Science and Technology (IROAST), Kumamoto University, Kumamoto, Japan. Electronic address:

Retinal detachment (RD) is an ocular emergency, which needs quick intervention to preclude permanent vision loss. In general, ocular ultrasound is used by ophthalmologists to enhance their judgment in detecting RD in eyes with media opacities which precludes the retinal evaluation. However, the quality of ultrasound (US) images may be degraded due to the presence of noise, and other retinal conditions may cause membranous echoes. All these can influence the accuracy of diagnosis. Hence, to overcome the above, we are proposing an automated system to detect RD using texton, higher order spectral (HOS) cumulants and locality sensitive discriminant analysis (LSDA) techniques. Our developed method is able to classify the posterior vitreous detachment and RD using support vector machine classifier with highest accuracy of 99.13%. Our system is ready to be tested with more diverse ultrasound images and aid ophthalmologists to arrive at a more accurate diagnosis.
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http://dx.doi.org/10.1016/j.compbiomed.2020.103704DOI Listing
May 2020

Transcriptomic analyses of oncogenic hepatocytes reveal common and different molecular pathways of hepatocarcinogenesis in different developmental stages and genders in kras transgenic zebrafish.

Biochem Biophys Res Commun 2019 03 7;510(4):558-564. Epub 2019 Feb 7.

Department of Biological Sciences, National University of Singapore, Singapore. Electronic address:

Hepatocellular carcinoma (HCC), the most common type of primary liver cancer, is mainly due to genetic changes in hepatocytes. However, molecular expression in hepatocytes during hepatocarcinogenesis has not been characterized. In this study, using an inducible kras transgenic zebrafish models for HCC, transcriptomic profiles of oncogenic hepatocytes from larvae, male and female adult fish following a brief induction of oncogenic kras were investigated. We found that oncogenic hepatocytes from all the three sources possess most of the cancer hallmarks at molecular level, including Sustaining proliferative signaling, Evading growth suppressors, Resisting cell death, Avoiding immune destruction, Inflammation, Reprogramming of energy metabolism, Angiogenesis, and Activating invasion and metastasis, suggesting the malignant transformation at molecular level could occur at the early stage of hepatocarcinogensis and can be captured in hepatocytes. However, each group of oncogenic hepatocytes also had their own characteristics. Larval oncogenic hepatocytes have cancer stem cell features. Female oncogenic hepatocytes showed resemblance to a mild human HCC subtype while male oncogenic hepatocytes resembled a severe HCC subtype, consistent with the observed sex disparity of HCC in both zebrafish and human. Finally, the two adult groups were more similar to each other than to the larval group, indicating an overwhelming effect of development over the gender.
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http://dx.doi.org/10.1016/j.bbrc.2019.02.008DOI Listing
March 2019

Transcriptomic profiles of tumor-associated neutrophils reveal prominent roles in enhancing angiogenesis in liver tumorigenesis in zebrafish.

Sci Rep 2019 02 6;9(1):1509. Epub 2019 Feb 6.

Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

We have previously demonstrated the pro-tumoral role of neutrophils using a kras-induced zebrafish hepatocarcinogenesis model. To further illustrate the molecular basis of the pro-tumoral role, Tumor-associated neutrophils (TANs) were isolated by fluorescence-activated cell sorting (FACS) and transcriptomic analyses were carried out by RNA-Seq. Differentially expressed gene profiles of TANs from larvae, male and female livers indicate great variations during liver tumorigenesis, but the common responsive canonical pathways included an immune pathway (Acute Phase Response Signaling), a liver metabolism-related pathway (LXR/RXR Activation) and Thrombin Signaling. Consistent with the pro-tumoral role of TANs, gene module analysis identified a consistent down-regulation of Cytotoxicity module, which may allow continued proliferation of malignant cells. Gene Set Enrichment Analysis indicated up-regulation of several genes promoting angiogenesis. Consistent with this, we found decreased density of blood vessels accompanied with decreased oncogenic liver sizes in neutrophil-depleted larvae. Collectively, our study has indicated some molecular mechanisms of the pro-tumoral roles of TANs in hepatocarcinogenesis, including weakened immune clearance against tumor cells and enhanced function in angiogenesis.
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http://dx.doi.org/10.1038/s41598-018-36605-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365535PMC
February 2019

IFT88 mutations identified in individuals with non-syndromic recessive retinal degeneration result in abnormal ciliogenesis.

Hum Genet 2018 Jul 5;137(6-7):447-458. Epub 2018 Jul 5.

Shiley Eye Institute, University of California San Diego, 9415 Campus Point Drive, JRC 206, La Jolla, CA, 92093, USA.

Whole genome sequencing (WGS) was performed to identify the variants responsible for inherited retinal degeneration (IRD) in a Caucasian family. Segregation analysis of selected rare variants with pathogenic potential identified a set of compound heterozygous changes p.Arg266*:c.796C>T and p.Ala568Thr:c.1702G>A in the intraflagellar transport protein-88 (IFT88) gene segregating with IRD. Expression of IFT88 with the p.Arg266* and p.Ala568Thr mutations in mIMDC3 cells by transient transfection and in HeLa cells by introducing the mutations using CRISPR-cas9 system suggested that both mutations result in the formation of abnormal ciliary structures. The introduction of the IFT88 p.Arg266* variant in the homozygous state in HeLa cells by CRISPR-Cas9 genome-editing revealed that the mutant transcript undergoes nonsense-mediated decay leading to a significant depletion of IFT88 transcript. Additionally, abnormal ciliogenesis was observed in these cells. These observations suggest that the rare and unique combination of IFT88 alleles observed in this study provide insight into the physiological role of IFT88 in humans and the likely mechanism underlying retinal pathology in the pedigree with IRD.
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http://dx.doi.org/10.1007/s00439-018-1897-9DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6150774PMC
July 2018

Cytoplasmic polyadenylation-mediated translational control of maternal mRNAs directs maternal-to-zygotic transition.

Development 2018 01 8;145(1). Epub 2018 Jan 8.

Genome Institute of Singapore, Agency of Science Technology and Research, 138672 Singapore

In the earliest stages of animal development following fertilization, maternally deposited mRNAs direct biological processes to the point of zygotic genome activation (ZGA). These maternal mRNAs undergo cytoplasmic polyadenylation (CPA), suggesting translational control of their activation. To elucidate the biological role of CPA during embryogenesis, we performed genome-wide polysome profiling at several stages of zebrafish development. Our analysis revealed a correlation between CPA and polysome-association dynamics, demonstrating a coupling of translation to the CPA of maternal mRNAs. Pan-embryonic CPA inhibition disrupted the maternal-to-zygotic transition (MZT), causing a failure of developmental progression beyond the mid-blastula transition and changes in global gene expression that indicated a failure of ZGA and maternal mRNA clearance. Among the genes that were differentially expressed were those encoding chromatin modifiers and key transcription factors involved in ZGA, including , and , which have distinct CPA dynamics. Our results establish the necessity of CPA for ensuring progression of the MZT. The RNA-seq data generated in this study represent a valuable zebrafish resource for the discovery of novel elements of the early embryonic transcriptome.
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http://dx.doi.org/10.1242/dev.159566DOI Listing
January 2018

Glucocorticoids promote Von Hippel Lindau degradation and Hif-1α stabilization.

Proc Natl Acad Sci U S A 2017 09 29;114(37):9948-9953. Epub 2017 Aug 29.

Bateson Centre, Department of Biomedical Science, University of Sheffield, Sheffield S10 2TN, United Kingdom;

Glucocorticoid (GC) and hypoxic transcriptional responses play a central role in tissue homeostasis and regulate the cellular response to stress and inflammation, highlighting the potential for cross-talk between these two signaling pathways. We present results from an unbiased in vivo chemical screen in zebrafish that identifies GCs as activators of hypoxia-inducible factors (HIFs) in the liver. GCs activated consensus hypoxia response element (HRE) reporters in a glucocorticoid receptor (GR)-dependent manner. Importantly, GCs activated HIF transcriptional responses in a zebrafish mutant line harboring a point mutation in the GR DNA-binding domain, suggesting a nontranscriptional route for GR to activate HIF signaling. We noted that GCs increase the transcription of several key regulators of glucose metabolism that contain HREs, suggesting a role for GC/HIF cross-talk in regulating glucose homeostasis. Importantly, we show that GCs stabilize HIF protein in intact human liver tissue and isolated hepatocytes. We find that GCs limit the expression of Von Hippel Lindau protein (pVHL), a negative regulator of HIF, and that treatment with the c-src inhibitor PP2 rescued this effect, suggesting a role for GCs in promoting c-src-mediated proteosomal degradation of pVHL. Our data support a model for GCs to stabilize HIF through activation of c-src and subsequent destabilization of pVHL.
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http://dx.doi.org/10.1073/pnas.1705338114DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5604011PMC
September 2017

Ikk2 regulates cytokinesis during vertebrate development.

Sci Rep 2017 08 14;7(1):8094. Epub 2017 Aug 14.

Institute of Molecular and Cell Biology, Singapore, Singapore.

NFκB signaling has a pivotal role in regulation of development, innate immunity, and inflammation. Ikk2 is one of the two critical kinases that regulate the NFκB signaling pathway. While the role of Ikk2 in immunity, inflammation and oncogenesis has received attention, an understanding of the role of Ikk2 in vertebrate development has been compounded by the embryonic lethality seen in mice lacking Ikk2. We find that despite abnormal angiogenesis in IKK2 zygotic mutants of zebrafish, the maternal activity of Ikk2 supports embryogenesis and maturation of fertile animals and allows to study the role of IKK2 in development. Maternal-zygotic ikk2 mutants represent the first vertebrates globally devoid of maternal and zygotic Ikk2 activity. They are defective in cell proliferation as evidenced by abnormal cytokinesis, nuclear enlargement and syncytialisation of a significant portion of blastoderm. We further document that reduced phosphorylation of Aurora A by Ikk2 could underlie the basis of these defects in cell division.
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http://dx.doi.org/10.1038/s41598-017-06904-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5556003PMC
August 2017

Editor's Highlight: Transgenic Zebrafish Reporter Lines as Alternative In Vivo Organ Toxicity Models.

Toxicol Sci 2017 03;156(1):133-148

Institute of Molecular and Cell Biology (IMCB), A*STAR (Agency for Science, Technology and Research), 61 Biopolis Drive, 138673 Singapore.

Organ toxicity, particularly liver toxicity, remains one of the major reasons for the termination of drug candidates in the development pipeline as well as withdrawal or restrictions of marketed drugs. A screening-amenable alternative in vivo model such as zebrafish would, therefore, find immediate application in the early prediction of unacceptable organ toxicity. To identify highly upregulated genes as biomarkers of toxic responses in the zebrafish model, a set of well-characterized reference drugs that cause drug-induced liver injury (DILI) in the clinic were applied to zebrafish larvae and adults. Transcriptome microarray analysis was performed on whole larvae or dissected adult livers. Integration of data sets from different drug treatments at different stages identified common upregulated detoxification pathways. Within these were candidate biomarkers which recurred in multiple treatments. We prioritized 4 highly upregulated genes encoding enzymes acting in distinct phases of the drug metabolism pathway. Through promoter isolation and fosmid recombineering, eGFP reporter transgenic zebrafish lines were generated and evaluated for their response to DILI drugs. Three of the 4 generated reporter lines showed a dose and time-dependent induction in endodermal organs to reference drugs and an expanded drug set. In conclusion, through integrated transcriptomics and transgenic approaches, we have developed parallel independent zebrafish in vivo screening platforms able to predict organ toxicities of preclinical drugs.
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http://dx.doi.org/10.1093/toxsci/kfw250DOI Listing
March 2017

Sex differences in DNA methylation and expression in zebrafish brain: a test of an extended 'male sex drive' hypothesis.

Gene 2016 Sep 31;590(2):307-16. Epub 2016 May 31.

Department of Zoology, University of Otago, 340 Great King Street, Dunedin 9054, New Zealand; Evolution & Ecology Research Centre and School of Biological, Earth and Environmental Sciences, University of New South Wales, Kensington, 2052 Sydney, NSW, Australia; Diabetes and Metabolism Division, Garvan Institute of Medical Research, Darlinghurst, NSW 2010, Australia. Electronic address:

The sex drive hypothesis predicts that stronger selection on male traits has resulted in masculinization of the genome. Here we test whether such masculinizing effects can be detected at the level of the transcriptome and methylome in the adult zebrafish brain. Although methylation is globally similar, we identified 914 specific differentially methylated CpGs (DMCs) between males and females (435 were hypermethylated and 479 were hypomethylated in males compared to females). These DMCs were prevalent in gene body, intergenic regions and CpG island shores. We also discovered 15 distinct CpG clusters with striking sex-specific DNA methylation differences. In contrast, at transcriptome level, more female-biased genes than male-biased genes were expressed, giving little support for the male sex drive hypothesis. Our study provides genome-wide methylome and transcriptome assessment and sheds light on sex-specific epigenetic patterns and in zebrafish for the first time.
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http://dx.doi.org/10.1016/j.gene.2016.05.042DOI Listing
September 2016

Deep sequencing of small RNA facilitates tissue and sex associated microRNA discovery in zebrafish.

BMC Genomics 2015 Nov 16;16:950. Epub 2015 Nov 16.

Lee Kong Chian School of Medicine, Nanyang Technological University, 11 Mandalay Road, Singapore, 308232, Singapore.

Background: The role of microRNAs in gene regulation has been well established. The extent of miRNA regulation also increases with increasing genome complexity. Though the number of genes appear to be equal between human and zebrafish, substantially less microRNAs have been discovered in zebrafish compared to human (miRBase Release 19). It appears that most of the miRNAs in zebrafish are yet to be discovered.

Results: We sequenced small RNAs from brain, gut, liver, ovary, testis, eye, heart and embryo of zebrafish. In brain, gut and liver sequencing was done sex specifically. Majority of the sequenced reads (16-62 %) mapped to known miRNAs, with the exception of ovary (5.7 %) and testis (7.8 %). Using the miRNA discovery tool (miRDeep2), we discovered novel miRNAs from the unannotated reads that ranged from 7.6 to 23.0 %, with exceptions of ovary (51.4 %) and testis (55.2 %). The prediction tool identified a total of 459 novel pre-miRNAs. We compared expression of miRNAs between different tissues and between males and females to identify tissue associated and sex associated miRNAs respectively. These miRNAs could serve as putative biomarkers for these tissues. The brain and liver had highest number of tissue associated (22) and sex associated (34) miRNAs, respectively.

Conclusions: This study comprehensively identifies tissue and sex associated miRNAs in zebrafish. Further, we have discovered 459 novel pre-miRNAs (~30 % seed homology to human miRNA) as a genomic resource which can facilitate further investigations to understand miRNA-mRNA gene regulatory networks in zebrafish which will have implications in understanding the function of human homologs.
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http://dx.doi.org/10.1186/s12864-015-2135-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4647824PMC
November 2015

Genome-wide mapping of Hif-1α binding sites in zebrafish.

BMC Genomics 2015 Nov 11;16:923. Epub 2015 Nov 11.

Bateson Centre, Department of Biomedical Science, The University of Sheffield, Western Bank, Sheffield, UK.

Background: Hypoxia Inducible Factor (HIF) regulates a cascade of transcriptional events in response to decreased oxygenation, acting from the cellular to the physiological level. This response is evolutionarily conserved, allowing the use of zebrafish (Danio rerio) as a model for studying the hypoxic response. Activation of the hypoxic response can be achieved in zebrafish by homozygous null mutation of the von Hippel-Lindau (vhl) tumour suppressor gene. Previous work from our lab has focused on the phenotypic characterisation of this mutant, establishing the links between vhl mutation, the hypoxic response and cancer. To further develop fish as a model for studying hypoxic signalling, we examine the transcriptional profile of the vhl mutant with respect to Hif-1α. As our approach uses embryos consisting of many cell types, it has the potential to uncover additional HIF regulated genes that have escaped detection in analogous mammalian cell culture studies.

Results: We performed high-density oligonucleotide microarray analysis of the gene expression changes in von Hippel-Lindau mutant zebrafish, which identified up-regulation of well-known hypoxia response genes and down-regulation of genes primarily involved in lipid processing. To identify the dependency of these transcriptional changes on HIF, we undertook Chromatin Immunoprecipitation linked next generation sequencing (ChIP-seq) for the transcription factor Hypoxia Inducible Factor 1α (HIF-1α). We identified HIF-1α binding sites across the genome, with binding sites showing enrichment for an RCGTG motif, showing conservation with the mammalian hypoxia response element.

Conclusions: Transcriptome analysis of vhl mutant embryos detected activation of key hypoxia response genes seen in human cell models of hypoxia, but also suppression of many genes primarily involved in lipid processing. ChIP-seq analysis of Hif-1α binding sites unveiled an unprecedented number of loci, with a high proportion containing a canonical hypoxia response element. Whether these sites are functional remains unknown, nevertheless their frequent location near transcriptional start sites suggests functionality, and will allow for investigation into the potential hypoxic regulation of genes in their vicinity. We expect that our data will be an excellent starting point for analysis of both fish and mammalian gene regulation by HIF.
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http://dx.doi.org/10.1186/s12864-015-2169-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4642629PMC
November 2015

Zebrafish Rnf111 is encoded by multiple transcripts and is required for epiboly progression and prechordal plate development.

Differentiation 2015 Jan-Feb;89(1-2):22-30. Epub 2015 Jan 22.

Institute of Medical Biology, Agency for Science, Technology and Research (A⁎STAR), 8A Biomedical Grove, #06-06 Immunos, Singapore 138648, Singapore. Electronic address:

Arkadia (also known as RING finger 111) encodes a nuclear E3 ubiquitin ligase that targets intracellular effectors and modulators of TGFβ/Nodal-related signaling for polyubiquitination and proteasome-dependent degradation. In the mouse, loss of Arkadia results in early embryonic lethality, with defects attributed to compromised Nodal signaling. Here, we report the isolation of zebrafish arkadia/rnf111, which is represented by 5 transcript variants. arkadia/rnf111 is broadly expressed during the blastula and gastrula stages, with eventual enrichment in the anterior mesendoderm, including the prechordal plate. Morpholino knockdown experiments reveal an unexpected role for Arkadia/Rnf111 in both early blastula organization and epiboly progression. Using a splice junction morpholino, we present additional evidence that arkadia/rnf111 transcript variants containing a 3' alternative exon are specifically required for epiboly progression in the late gastrula. This result suggests that arkadia/rnf111 transcript variants encode functionally relevant protein isoforms that provide additional intracellular flexibility and regulation to the Nodal signaling pathway.
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http://dx.doi.org/10.1016/j.diff.2014.12.004DOI Listing
January 2016

Differential transcriptomic analyses revealed genes and signaling pathways involved in iono-osmoregulation and cellular remodeling in the gills of euryhaline Mozambique tilapia, Oreochromis mossambicus.

BMC Genomics 2014 Oct 23;15:921. Epub 2014 Oct 23.

NUS Environmental Research Institute, National University of Singapore, 5A Engineering Drive, 117411 Singapore, Singapore.

Background: The Mozambique tilapia Oreochromis mossambicus has the ability to adapt to a broad range of environmental salinities and has long been used for investigating iono-osmoregulation. However, to date most studies have focused mainly on several key molecules or parameters hence yielding a limited perspective of the versatile iono-osmoregulation in the euryhaline fish. This study aimed to capture transcriptome-wide differences between the freshwater- and seawater-acclimated gills of the Mozambique tilapia.

Results: We have identified over 5000 annotated gene transcripts with high homology (E-value <1.0E-50) to human genes that were differentially expressed in freshwater- and seawater-acclimated gills of the Mozambique tilapia. These putative human homologs were found to be significantly associated with over 50 canonical signaling pathways that are operating in at least 23 biological processes in relation to branchial iono-osmoregulation and cellular remodeling. The analysis revealed multiple signaling pathways in freshwater-acclimated gills acting in concert to maintain cellular homeostasis under hypo-osmotic environment while seawater-acclimated gills abounded with molecular signals to cope with the higher cellular turn-over rate, energetics and iono-regulatory demands under hyper-osmostic stress. Additionally, over 100 transcripts encoding putative inorganic ion transporters/channels were identified, of which several are well established in gill iono-regulation while the remainder are lesser known. We have also validated the expression profiles of 47 representative genes in freshwater- and seawater-acclimated gills, as well as in hypersaline-acclimated (two-fold salinity of seawater) gills. The findings confirmed that many of these responsive genes retained their expression profiles in hypersaline-acclimated gills as in seawater-acclimated gills, although several genes had changed significantly in their expression level/direction in hypersaline-acclimated gills.

Conclusions: This is the first study that has provided an unprecedented transcriptomic-wide perspective of gill iono-osmoregulation since such studies were initiated more than 80 years ago. It has expanded our molecular perspective from a relatively few well-studied molecules to a plethora of gene transcripts and a myriad of canonical signaling pathways driving various biological processes that are operating in gills under hypo-osmotic and hyper-osmotic stresses. These findings would provide insights and resources to fuel future studies on gill iono-osmoregulation and cellular remodeling in response to salinity challenge and acclimation.
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http://dx.doi.org/10.1186/1471-2164-15-921DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4213501PMC
October 2014

Functional mapping of the zebrafish early embryo proteome and transcriptome.

J Proteome Res 2014 Dec 26;13(12):5536-50. Epub 2014 Sep 26.

Institute of Molecular and Cell Biology, Agency for Science, Technology and Research , 61 Biopolis Drive, 138673, Singapore.

Zebrafish is a popular system for studying vertebrate development and disease that shows high genetic conservation with humans. Molecular level studies at different stages of development are essential for understanding the processes deployed during ontogeny. Here, we performed comparative analysis of the whole proteome and transcriptome of the early stage (24 h post-fertilization) zebrafish embryo. We identified 8363 proteins with their approximate cellular abundances (the largest number of zebrafish embryo proteins quantified thus far), through a combination of thorough deyolking and extensive fractionation procedures, before resolving the peptides by mass spectrometry. We performed deep sequencing of the transcripts and found that the expressed proteome and transcriptome displayed a moderate correlation for the majority of cellular processes. Integrative functional mapping of the quantified genes demonstrated that embryonic developmental systems differentially exploit transcriptional and post-transcriptional regulatory mechanisms to modulate protein abundance. Using network mapping of the low-abundance proteins, we identified various signal transduction pathways important in embryonic development and also revealed genes that may be regulated at the post-transcriptional level. Our data set represents a deep coverage of the functional proteome and transcriptome of the developing zebrafish, and our findings unveil molecular regulatory mechanisms that underlie embryonic development.
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http://dx.doi.org/10.1021/pr5005136DOI Listing
December 2014

Impaired development of neural-crest cell-derived organs and intellectual disability caused by MED13L haploinsufficiency.

Hum Mutat 2014 Nov 23;35(11):1311-20. Epub 2014 Sep 23.

Human Genetics, Genome Institute of Singapore, Singapore, 138672; Department of Paediatrics, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117597.

MED13L is a component subunit of the Mediator complex, an important regulator of transcription that is highly conserved across eukaryotes. Here, we report MED13L disruption in a translocation t(12;19) breakpoint of a patient with Pierre-Robin syndrome, moderate intellectual disability, craniofacial anomalies, and muscular defects. The phenotype is similar to previously described patients with MED13L haploinsufficiency. Knockdown of MED13L orthologue in zebrafish, med13b, showed early defective migration of cranial neural crest cells (NCCs) that contributed to cartilage structure deformities in the later stage, recapitulating craniofacial anomalies seen in human patients. Notably, we observed abnormal distribution of developing neurons in different brain regions of med13b morphant embryos, which could be rescued upon introduction of full-length human MED13L mRNA. To compare with mammalian system, we suppressed MED13L expression by short-hairpin RNA in ES-derived human neural progenitors, and differentiated them into neurons. Transcriptome analysis revealed differential expression of components of Wnt and FGF signaling pathways in MED13L-deficient neurons. Our finding provides a novel insight into the mechanism of overlapping phenotypic outcome targeting NCCs derivatives organs in patients with MED13L haploinsufficiency, and emphasizes a clinically recognizable syndromic phenotype in these patients.
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http://dx.doi.org/10.1002/humu.22636DOI Listing
November 2014

Normalization of RNA-sequencing data from samples with varying mRNA levels.

PLoS One 2014 25;9(2):e89158. Epub 2014 Feb 25.

BasAM, Norwegian School of Veterinary Science, Oslo, Norway.

Methods for normalization of RNA-sequencing gene expression data commonly assume equal total expression between compared samples. In contrast, scenarios of global gene expression shifts are many and increasing. Here we compare the performance of three normalization methods when polyA(+) RNA content fluctuates significantly during zebrafish early developmental stages. As a benchmark we have used reverse transcription-quantitative PCR. The results show that reads per kilobase per million (RPKM) and trimmed mean of M-values (TMM) normalization systematically leads to biased gene expression estimates. Biological scaling normalization (BSN), designed to handle differences in total expression, showed improved accuracy compared to the two other methods in estimating transcript level dynamics. The results have implications for past and future studies using RNA-sequencing on samples with different levels of total or polyA(+) RNA.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0089158PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3934880PMC
December 2014

Transcriptomic analysis of a transgenic zebrafish hepatocellular carcinoma model reveals a prominent role of immune responses in tumour progression and regression.

Int J Cancer 2014 Oct 4;135(7):1564-73. Epub 2014 Mar 4.

Department of Biological Sciences, National University of Singapore, Singapore; Human genetics 2, Genome Institute of Singapore, Singapore.

Using our previously established xmrk transgenic zebrafish, hepatocellular carcinoma (HCC) was generated by induced expression of xmrk, which encoded a hyperactive epidermal growth factor receptor (EGFR) homolog, and regressed by suppression of xmrk expression. To investigate molecular changes in liver tumour progression and regression, RNA-Seq was performed for induced HCC and early and late stages of liver tissues during tumour regression. We found that Xmrk-induced zebrafish HCC shared strong molecular characteristics with a human HCC subtype (S2), which shows activated Myc signalling, upregulated phosphor-S6 and epithelial cell adhesion molecule. In the HCC stage, there were enhanced proteasome, antigen processing and presentation, aminosugars metabolisms, p53 and cell cycle pathways. During tumour regression, the transcriptomic profile showed a reversed trend of molecular changes compared with human HCC progression. Interestingly, distinct immune responses in tumour progression and regression were observed, including increased major histocompatibility complex class I (MHCI) at the HCC stage, enriched immune cell trafficking signals and inflammation in early regression and enhanced MHCII in late regression. Both neutrophils and macrophages were enriched during tumour progression and regression; however, the distribution of neutrophils and macrophages in HCC was relatively uniform, whereas both types of immune cells were regionally clustered during tumour regression, especially with dominant blood vessel association of macrophage in late regression, suggesting differential functions of these immune cells in tumour progression and regression. As tumour regression in our model resembles the targeted inhibition of EGFR in cancer therapy, our observations may provide molecular insights into the targeted inhibition and highlight the importance of immune response in tumour regression.
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http://dx.doi.org/10.1002/ijc.28794DOI Listing
October 2014

Genome wide expression profiling during spinal cord regeneration identifies comprehensive cellular responses in zebrafish.

PLoS One 2014 20;9(1):e84212. Epub 2014 Jan 20.

Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, Kolkata, India.

Background: Among the vertebrates, teleost and urodele amphibians are capable of regenerating their central nervous system. We have used zebrafish as a model to study spinal cord injury and regeneration. Relatively little is known about the molecular mechanisms underlying spinal cord regeneration and information based on high density oligonucleotide microarray was not available. We have used a high density microarray to profile the temporal transcriptome dynamics during the entire phenomenon.

Results: A total of 3842 genes expressed differentially with significant fold changes during spinal cord regeneration. Cluster analysis revealed event specific dynamic expression of genes related to inflammation, cell death, cell migration, cell proliferation, neurogenesis, neural patterning and axonal regrowth. Spatio-temporal analysis of stat3 expression suggested its possible function in controlling inflammation and cell proliferation. Genes involved in neurogenesis and their dorso-ventral patterning (sox2 and dbx2) are differentially expressed. Injury induced cell proliferation is controlled by many cell cycle regulators and some are commonly expressed in regenerating fin, heart and retina. Expression pattern of certain pathway genes are identified for the first time during regeneration of spinal cord. Several genes involved in PNS regeneration in mammals like stat3, socs3, atf3, mmp9 and sox11 are upregulated in zebrafish SCI thus creating PNS like environment after injury.

Conclusion: Our study provides a comprehensive genetic blue print of diverse cellular response(s) during regeneration of zebrafish spinal cord. The data highlights the importance of different event specific gene expression that could be better understood and manipulated further to induce successful regeneration in mammals.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0084212PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3896338PMC
December 2014

SMN deficiency alters Nrxn2 expression and splicing in zebrafish and mouse models of spinal muscular atrophy.

Hum Mol Genet 2014 Apr 11;23(7):1754-70. Epub 2013 Nov 11.

Department of Biological Sciences, National University of Singapore, Singapore 117543, Singapore.

Spinal muscular atrophy (SMA) is a progressive neurodegenerative disease affecting lower motor neurons. SMA is caused by mutations in the Survival Motor Neuron 1 (SMN1) gene, which result in reduced levels of functional SMN protein. Biochemical studies have linked the ubiquitously expressed SMN protein to the assembly of pre-mRNA processing U snRNPs, raising the possibility that aberrant splicing is a major defect in SMA. Accordingly, several transcripts affected upon SMN deficiency have been reported. A second function for SMN in axonal mRNA transport has also been proposed that may likewise contribute to the SMA phenotype. The underlying etiology of SMA, however, is still not fully understood. Here, we have used a combination of genomics and live Ca(2+) imaging to investigate the consequences of SMN deficiency in a zebrafish model of SMA. In a transcriptome analyses of SMN-deficient zebrafish, we identified neurexin2a (nrxn2a) as strongly down-regulated and displaying changes in alternative splicing patterns. Importantly, the knock-down of two distinct nrxn2a isoforms phenocopies SMN-deficient fish and results in a significant reduction of motor axon excitability. Interestingly, we observed altered expression and splicing of Nrxn2 also in motor neurons from the Smn(-/-);SMN2(+/+) mouse model of SMA, suggesting conservation of nrxn2 regulation by SMN in mammals. We propose that SMN deficiency affects splicing and abundance of nrxn2a. This may explain the pre-synaptic defects at neuromuscular endplates in SMA pathophysiology.
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http://dx.doi.org/10.1093/hmg/ddt567DOI Listing
April 2014

Genome wide analysis reveals Zic3 interaction with distal regulatory elements of stage specific developmental genes in zebrafish.

PLoS Genet 2013 Oct 31;9(10):e1003852. Epub 2013 Oct 31.

Human Genetics, Genome Institute of Singapore, Singapore, Singapore.

Zic3 regulates early embryonic patterning in vertebrates. Loss of Zic3 function is known to disrupt gastrulation, left-right patterning, and neurogenesis. However, molecular events downstream of this transcription factor are poorly characterized. Here we use the zebrafish as a model to study the developmental role of Zic3 in vivo, by applying a combination of two powerful genomics approaches--ChIP-seq and microarray. Besides confirming direct regulation of previously implicated Zic3 targets of the Nodal and canonical Wnt pathways, analysis of gastrula stage embryos uncovered a number of novel candidate target genes, among which were members of the non-canonical Wnt pathway and the neural pre-pattern genes. A similar analysis in zic3-expressing cells obtained by FACS at segmentation stage revealed a dramatic shift in Zic3 binding site locations and identified an entirely distinct set of target genes associated with later developmental functions such as neural development. We demonstrate cis-regulation of several of these target genes by Zic3 using in vivo enhancer assay. Analysis of Zic3 binding sites revealed a distribution biased towards distal intergenic regions, indicative of a long distance regulatory mechanism; some of these binding sites are highly conserved during evolution and act as functional enhancers. This demonstrated that Zic3 regulation of developmental genes is achieved predominantly through long distance regulatory mechanism and revealed that developmental transitions could be accompanied by dramatic changes in regulatory landscape.
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http://dx.doi.org/10.1371/journal.pgen.1003852DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3814314PMC
October 2013

Molecular insights into 4-nitrophenol-induced hepatotoxicity in zebrafish: transcriptomic, histological and targeted gene expression analyses.

Biochim Biophys Acta 2013 Oct 17;1830(10):4778-89. Epub 2013 Jun 17.

Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

Background: 4-Nitrophenol (4-NP) is a prioritized environmental pollutant and its toxicity has been investigated using zebrafish, advocated as an alternative toxicological model. However, molecular information of 4-NP induced hepatotoxicity is still limited. This study aimed to obtain molecular insights into 4-NP-induced hepatotoxicity using zebrafish as a model.

Methods: Adult male zebrafish were exposed to 4-NP for 8, 24, 48 and 96h. Livers were sampled for microarray experiment, qRT-PCR and various histological analyses.

Results: Transcriptomic analysis revealed that genes associated with oxidative phosphorylation and electron transport chain were significantly up-regulated throughout early and late stages of 4-NP exposure due to oxidative phosphorylation uncoupling by 4-NP. This in turn induced oxidative stress damage and up-regulated pathways associated with tumor suppressors Rb and p53, cell cycle, DNA damage, proteasome degradation and apoptosis. Pathways associated with cell adhesion and morphology were deregulated. Carbohydrate and lipid metabolisms were down-regulated while methionine and aromatic amino acid metabolisms as well as NFKB pathway associated with chronic liver conditions were up-regulated. Up-regulation of NFKB, NFAT and interleukin pathways suggested hepatitis. Histological analyses with specific staining methods and qRT-PCR analysis of selected genes corroborated with the transcriptomic analysis suggesting 4-NP induced liver injury.

Conclusion: Our findings allowed us to propose a plausible model and provide a broader understanding of the molecular events leading to 4-NP induced acute hepatotoxicity for future studies involving other nitrophenol derivatives.

General Significance: This is the first transcriptomic report on 4-NP induced hepatotoxicity.
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http://dx.doi.org/10.1016/j.bbagen.2013.06.008DOI Listing
October 2013

Selective small molecule probes for the hypoxia inducible factor (HIF) prolyl hydroxylases.

ACS Chem Biol 2013 Jul 12;8(7):1488-96. Epub 2013 Jun 12.

Department of Chemistry, Chemistry Research Laboratory, University of Oxford , Mansfield Road, Oxford, OX1 3TA, United Kingdom.

The hypoxia inducible factor (HIF) system is central to the signaling of low oxygen (hypoxia) in animals. The levels of HIF-α isoforms are regulated in an oxygen-dependent manner by the activity of the HIF prolyl-hydroxylases (PHD or EGLN enzymes), which are Fe(II) and 2-oxoglutarate (2OG) dependent oxygenases. Here, we describe biochemical, crystallographic, cellular profiling, and animal studies on PHD inhibitors including selectivity studies using a representative set of human 2OG oxygenases. We identify suitable probe compounds for use in studies on the functional effects of PHD inhibition in cells and in animals.
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http://dx.doi.org/10.1021/cb400088qDOI Listing
July 2013

Differential transcript isoform usage pre- and post-zygotic genome activation in zebrafish.

BMC Genomics 2013 May 15;14:331. Epub 2013 May 15.

BasAM, Norwegian School of Veterinary Science, 0033 Dep, Oslo, Norway.

Background: Zebrafish embryos are transcriptionally silent until activation of the zygotic genome during the 10th cell cycle. Onset of transcription is followed by cellular and morphological changes involving cell speciation and gastrulation. Previous genome-wide surveys of transcriptional changes only assessed gene expression levels; however, recent studies have shown the necessity to map isoform-specific transcriptional changes. Here, we perform isoform discovery and quantification on transcriptome sequences from before and after zebrafish zygotic genome activation (ZGA).

Results: We identify novel isoforms and isoform switches during ZGA for genes related to cell adhesion, pluripotency and DNA methylation. Isoform switching events include alternative splicing and changes in transcriptional start sites and in 3' untranslated regions. New isoforms are identified even for well-characterized genes such as pou5f1, sall4 and dnmt1. Genes involved in cell-cell interactions such as f11r and magi1 display isoform switches with alterations of coding sequences. We also detect over 1000 transcripts that acquire a longer 3' terminal exon when transcribed by the zygote compared to their maternal transcript counterparts. ChIP-sequencing data mapped onto skipped exon events reveal a correlation between histone H3K36 trimethylation peaks and skipped exons, suggesting epigenetic marks being part of alternative splicing regulation.

Conclusions: The novel isoforms and isoform switches reported here include regulators of transcriptional, cellular and morphological changes taking place around ZGA. Our data display an array of isoform-related functional changes and represent a valuable resource complementary to existing early embryo transcriptomes.
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http://dx.doi.org/10.1186/1471-2164-14-331DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3747860PMC
May 2013

Dorsal activity of maternal squint is mediated by a non-coding function of the RNA.

Development 2012 Aug 21;139(16):2903-15. Epub 2012 Jun 21.

Temasek Life Sciences Laboratory, 1 Research Link, National University of Singapore, Singapore 117604.

Despite extensive study, the earliest steps of vertebrate axis formation are only beginning to be elucidated. We previously showed that asymmetric localization of maternal transcripts of the conserved zebrafish TGFβ factor Squint (Sqt) in 4-cell stage embryos predicts dorsal, preceding nuclear accumulation of β-catenin. Cell ablations and antisense oligonucleotides that deplete Sqt lead to dorsal deficiencies, suggesting that localized maternal sqt functions in dorsal specification. However, based upon analysis of sqt and Nodal signaling mutants, the function and mechanism of maternal sqt was debated. Here, we show that sqt RNA may function independently of Sqt protein in dorsal specification. sqt insertion mutants express localized maternal sqt RNA. Overexpression of mutant/non-coding sqt RNA and, particularly, the sqt 3'UTR, leads to ectopic nuclear β-catenin accumulation and expands dorsal gene expression. Dorsal activity of sqt RNA requires Wnt/β-catenin but not Oep-dependent Nodal signaling. Unexpectedly, sqt ATG morpholinos block both sqt RNA localization and translation and abolish nuclear β-catenin, providing a mechanism for the loss of dorsal identity in sqt morphants and placing maternal sqt RNA upstream of β-catenin. The loss of early dorsal gene expression can be rescued by the sqt 3'UTR. Our findings identify new non-coding functions for the Nodal genes and support a model wherein sqt RNA acts as a scaffold to bind and deliver/sequester maternal factors to future embryonic dorsal.
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http://dx.doi.org/10.1242/dev.077081DOI Listing
August 2012

Epigenetic complexity during the zebrafish mid-blastula transition.

Biochem Biophys Res Commun 2012 Jan 22;417(4):1139-44. Epub 2011 Dec 22.

Stem Cell Epigenetics Laboratory, Institute of Basic Medical Sciences, Faculty of Medicine, University of Oslo, 0317 Oslo, Norway.

The zebrafish developmental transcription program is determined by temporal post-translational histone modifications established in a step-wise and combinatorial manner on specific promoters around the time of zygotic genome activation (ZGA). Here, we characterize this increasing epigenetic complexity before, during and after ZGA. H3K4me3/H3K27me3 co-enrichment prevails over H3K4me3/H3K9me3 at the time of ZGA. Whereas most H3K4me3-marked promoters are devoid of transcriptionally repressive H3K9me3 or H3K27me3, the latter marks rarely occur in absence of H3K4me3. On co-enriched genomic regions, H3K4me3 and H3K27me3 can overlap regardless of H3K9me3 enrichment, but H3K4me3 and H3K9me3 are mutually exclusive. H3K4me3 and H3K9me3 may however overlap only when H3K27me3 also marks the overlapping domain, suggesting that H3K27me3 may modulate chromatin states. On metagenes, H3K27me3 enrichment correlates with local alteration in H3K4me3 density, and co-enrichment in H3K9me3 is linked to alterations in both H3K27me3 and H3K4me3 profiles. This suggests physical proximity of these marks and supports a view of existence of bi- or tri-valent chromatin domains. Thus enrichment in trimethylated H3K9 or H3K27 is associated with local remodeling of chromatin manifested by changes in H3K4me3 density. We propose that metagenes can provide information on the multivalency of chromatin sates.
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http://dx.doi.org/10.1016/j.bbrc.2011.12.077DOI Listing
January 2012

Toxicogenomic and phenotypic analyses of bisphenol-A early-life exposure toxicity in zebrafish.

PLoS One 2011 14;6(12):e28273. Epub 2011 Dec 14.

Department of Biological Sciences, National University of Singapore, Singapore, Singapore.

Bisphenol-A is an important environmental contaminant due to the increased early-life exposure that may pose significant health-risks to various organisms including humans. This study aimed to use zebrafish as a toxicogenomic model to capture transcriptomic and phenotypic changes for inference of signaling pathways, biological processes, physiological systems and identify potential biomarker genes that are affected by early-life exposure to bisphenol-A. Phenotypic analysis using wild-type zebrafish larvae revealed BPA early-life exposure toxicity caused cardiac edema, cranio-facial abnormality, failure of swimbladder inflation and poor tactile response. Fluorescent imaging analysis using three transgenic lines revealed suppressed neuron branching from the spinal cord, abnormal development of neuromast cells, and suppressed vascularization in the abdominal region. Using knowledge-based data mining algorithms, transcriptome analysis suggests that several signaling pathways involving ephrin receptor, clathrin-mediated endocytosis, synaptic long-term potentiation, axonal guidance, vascular endothelial growth factor, integrin and tight junction were deregulated. Physiological systems with related disorders associated with the nervous, cardiovascular, skeletal-muscular, blood and reproductive systems were implicated, hence corroborated with the phenotypic analysis. Further analysis identified a common set of BPA-targeted genes and revealed a plausible mechanism involving disruption of endocrine-regulated genes and processes in known susceptible tissue-organs. The expression of 28 genes were validated in a separate experiment using quantitative real-time PCR and 6 genes, ncl1, apoeb, mdm1, mycl1b, sp4, U1SNRNPBP homolog, were found to be sensitive and robust biomarkers for BPA early-life exposure toxicity. The susceptibility of sp4 to BPA perturbation suggests its role in altering brain development, function and subsequently behavior observed in laboratory animals exposed to BPA during early life, which is a health-risk concern of early life exposure in humans. The present study further established zebrafish as a model for toxicogenomic inference of early-life chemical exposure toxicity.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0028273PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3237442PMC
August 2012