De Novo Purine Synthesis Mediates Circadian Control of Cell Cycle in Zebrafish
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ABSTRACT:
ORGANISM(S): Danio rerio
SUBMITTER: Guang Li
PROVIDER: E-GEOD-51279 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Cell cycle and metabolism are two major outputs controlled by circadian rhythm in many organisms. Here we show that the three processes were linked through inosine 5'-phosphate dehydrogenase (impdh), a rate-limiting enzyme in de novo purine synthesis. We using adult zebrafish as a model system,we applied a genome-wide transcriptome approach that allowed us to investigate circadian gene expression. The whole-genome transcriptome profiles of adult brain in time-series were assayed on Agilent zebrafish microarrays. We used a similar statistical method to identify zebrafish circadian genes (ZCOG) as our previous study in larval zebrafish. Three isoforms of impdh show strong circadian oscillations in different tissues of zebrafish. impdh1a contributes to the ocular development and pigment synthesis, impdh2 promotes and impdh1b delays the development. By limiting the GTP required by DNA synthesis, impdh2 contributes to the daily rhythm of S phase in cell cycle. Multiple enzymes in the de novo purine synthesis pathway show the same circadian oscillations with peaks similar to impdh2. The circadian expression of this pathway is conserved in mouse liver. In summary, we show that the circadian regulation of de novo purine synthesis that supplies crucial building blocks for DNA replication is critical for gating cell cycle in circadian rhythm. Adult zebrafish were sacrificed and dissected at 4h intervals starting at CT0 in both LD and DD conditions for 12 time points. Total RNA of individual zebrafish brain was extracted using Trizol (Invitrogen, Carlsbad, CA) according to the manufacturerM-bM-^@M-^Ys instruction. Microarrays were manufactured by Agilent Technologies (Agilent Technologies, Palo Alto, CA), containing 43,603 probes for zebrafish whole-genome transcriptional profiling.
2013-10-01 | E-GEOD-51277 | biostudies-arrayexpress
Project description:Inosine 5'-phosphate dehydrogenase (impdh) has been well known as a key enzyme in GTP biosynthesis pathway. We found that three isoforms of impdh in zebrafish, namely impdh1a, impdh1b and impdh2, all show robust circadian expression.To examine the molecular functions of three impdh isoforms in zebrafish on the genome scale, we measured the global expression changes of impdh1a, impdh1b and impdh2 morpholino injected larvae (morphants) respectively using RNA-seq. Wild type (WT), control and three impdh morphants were collected at 32 hpf. In our RNA-seq result, we identified 468, 331 and 1166 significant genes affected by impdh1a, impdh1b and impdh2 morpholino (MO) knock-down respectively. Among them, there are limited overlaps between genes affected by different MOs and only 36 genes in common among all three MOs. This indicates that the three impdh isoforms have distinct molecular functions. To knock down the target genes, three impdh MOs and control MO were pressure-injected into 1- to 2-cell stage embryos. WT, control and three impdh morphants were raised at 28°C under 14h: 10h light/dark cycle from birth and sampled simultaneously at 32 hpf. Each group has at least 40 embryos.
2013-10-01 | E-GEOD-51278 | biostudies-arrayexpress
Project description:Host response to systemic bacterial challenge (sepsis) with E. coli strains CFT073 and F11 at 6 and 12 hours post inoculum 48 hpf embryos were manually dechorianated, briefly anesthetized with 0.77 mM ethyl 3-aminobenzoate methanesulfonate salt (tricaine) (Sigma-Aldrich), and embedded in 0.8% low melt agarose (MO BIO Laboratories) without tricaine. After the agarose solidified, embryos were immersed in E3 media lacking methylene blue. Prior to injection, 1 ml of bacterial culture was pelleted, washed with 1 ml sterile PBS, and re-suspended in PBS to obtain ~1X109 CFU/ml. PBS. One nl of this bacterial suspension containing ~1000 CFU was microinjected into the bloodstream via the circulation valley using an Olympus SZ61 or SZX10 stereomicroscope together with a YOU-1 micromanipulator (Narishige), a Narishige IM-200 microinjector, and a JUN-AIR model 3-compressor. For each experiment, the average CFU per injection was determined by adding 10 1-nl drops to 1 ml of 0.7% NaCl, which was then serial diluted and plated on Luria-Bertani (LB) agar plates. Mock-infected controls were inoculated with 1 nl sterile PBS. Following injection, embryos were removed from agar and placed individually into wells of a 48-well plate (Nunc) containing E3 medium and incubated at 28.5°C. Experiments were performed in biological quadruplicate.
2016-03-29 | E-GEOD-79665 | biostudies-arrayexpress
Project description:We report the high-throughput profiling of H3K9ac and SRT1 in MH63 and SRT1 RNAi. By obtaining about 8 million pair-end reads from H3K9ac and 6 million pair-end reads from SRT1, we find that about 45% of rice genes are marked by H3K9ac in 11 day-old seedlings. There are 1824 genes may be bound directly by OsSRT1. OsSRT1 is a site-specific deacetylase involved in deacetylation of H3K9 over specific genes. Overlapping between increased H3K9ac peaks in the RNAi plants and that of OsSRT1-binding was found over 37 genes, suggesting that OsSRT1 directly targeted to these genes for H3K9 deacetylation. The observations that H3K9ac was enriched at the 5M-bM-^@M-^Y end of genes larger than 1.5 kb and OsSRT1 RNAi enhanced the enrichment at that position. However, the OsSRT1-binding was found to be enriched over gene bodies. While the meaning of this binding profile is not yet clear, it is speculated that OsSRT1 may contribute to maintain the low level of H3K9ac in the gene body. In addition, this work revealed that OsSRT1 is enriched over retrotransposons. Examination of H3K9ac in MH63(WT) and SRT1-RNAi, OsSRT1 in MH63.
2013-02-10 | E-GEOD-40054 | biostudies-arrayexpress
Project description:Low temperatures may cause severe growth inhibition and mortality in fish. In order to understand the mechanism of cold tolerance, a transgenic zebrafish Tg (smyd1:m3ck) model was established to study the effect of energy homeostasis during cold stress. The muscle-specific promoter Smyd1 was used to express the carp muscle form III of creatine kinase (M3-CK), which maintained enzymatic activity at a relatively low temperature, in zebrafish skeletal muscle. In situ hybridization showed that M3-CK was expressed strongly in the skeletal muscle. When exposed to 13M-BM-0C, Tg (smyd1:m3ck) fish maintained their swimming behavior, while the wild-type could not. Energy measurements showed that the concentration of ATP increased in Tg (smyd1:m3ck) versus wild-type fish at 28M-BM-0C. After 2 h at 13M-BM-0C, ATP concentrations were 2.16-fold higher in Tg (smyd1:m3ck) than in wild-type (P < 0.05). At 13M-BM-0C, the ATP concentration in Tg (smyd1:m3ck) fish and wild-type fish was 63.3% and 20.0%, respectively, of that in wild-type fish at 28M-BM-0C. Microarray analysis revealed differential expression of 1249 transcripts in Tg (smyd1:m3ck) versus wild-type fish under cold stress. Biological processes that were significantly overrepresented in this group included circadian rhythm, energy metabolism, lipid transport, and metabolism. These results are clues to understanding the mechanisms underlying temperature acclimation in fish. Gene expression in triplicate samples of m3ck-13M-BM-0C, m3ck-28M-BM-0C, wt-13M-BM-0C, and wt-28M-BM-0C was assessed. Twelve microarray experiments were performed, each with three fish.
2014-12-23 | E-GEOD-58988 | biostudies-arrayexpress
Project description:Vanishing white matter (VWM) disease is a genetic leukodystrophy leading to severe neurological disease and early death. VWM is caused by bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (EIF2B). Although previous studies are being attempted to investigate the molecular mechanism of VWN by constructing variant models for each subunit of EIF2B that causes VWM disease. The underlying molecular mechanism of how mutations (MT) in EIF2B3 result in VWM is unknown. Based on our recently report, we constructed an eif2b3 knockout (eif2b3-/-) zebrafish (Danio rerio) model and performed quantitative proteomic analysis between the wide type (WT) and eif2b3-/- models to identify 25 differentially expressed proteins (DEPs). A total of 4 proteins were significantly up-regulated, and 21 proteins were significantly down-regulated in eif2b3-/- larvae compared to WT, respectively. Representatively, Lon protease and neutral amino acid transporter SLC1A4 were significantly increased in truncated eif2b3 zebrafish, and crystallin proteins were also significantly decreased. In conclusion, this study proposed the candidate proteins as a key regulator related to the progression of VWN disease through quantitative proteomic analysis in the first variant model of VWN disease.
2021-09-10 | PXD023933 | Pride
Project description:During early vertebrate development, a large number of noncoding RNAs are maternally inherited or expressed upon activation of zygotic transcription. The exact identity, expression levels, and function during early vertebrate development for most of these noncoding RNAs remains largely unknown. miRNAs (microRNAs) and piRNAs (piwi-interacting RNAs) are two classes of small non-coding RNAs that play important roles in gene regulation during early embryonic development. Here, we utilized Illumina next generation sequencing technology to determine temporal expression patterns for both miRNAs and piRNAs during four distinct stages of early vertebrate development using zebrafish as a model system. For miRNAs, the expression patterns for 192 known miRNAs and 12 novel miRNAs within 123 different miRNA families were determined. Significant sequence variation was observed at the 5' and 3' ends of miRNAs with a large number of extra nucleotides added in a non-template directed manner. We also identified a large and diverse set of piRNAs expressed during early development, far beyond that expected if piRNA expression is restricted to germ cells. Our analyses represent the deepest investigation to date of small RNA expression during early vertebrate development and suggest important novel functions for small RNAs during embryogenesis. Identify the expression of small RNAs in zebrafish embryos of four different developmental stages using high through-put sequencing
2012-03-01 | E-GEOD-27722 | biostudies-arrayexpress
Project description:Adult F0 female zebrafish were fed with control diet or diets containing 5-AZA, MeHg or TCDD. After breeding with unexposed males to produce the F1 generation, livers were sampled from the F0 females. F1 generation embryos were unexposed to test chemicals, were sampled, then bred to produce F2 fish, also unexposed to test chemicals. Methylated DNA immunoprecipitation was carried out on liver samples from F0 and F1 and F2 embryos and MeDIP samples were labeled with Cy5 and hybridised to a zebrafish CGI tiling array versus Cy3-labled zebrafish genomic DNA. The objective was to determine DNA methylation changes following chemical exposure and whether these persisted transgenerationally.
2014-03-01 | E-MTAB-2057 | biostudies-arrayexpress
Project description:To further development of the gene expression, we have employed whole genome microarray expression profiling as a discovery platform to identify genes that are regulated in miR-132 knockdown zebrafish. Zebrafish larvae were injected with Control MO or miR-132 MO at one-cell stage. Then the upregulated genes are combined to the miRNA prediction alagorithm. 21 genes were picked up and verifed by luciferase assay. The total RNA was extracted from 3 dpf zebrafish injected with 8 ng control or miR-132 MO MO. The RNA was applied for the microarray gene proliling
2016-08-09 | E-GEOD-85291 | biostudies-arrayexpress
Project description:Background: Hypoxia can affect aerobic organisms profoundly. Biological responses to extreme hypoxia have been well studied. However, it is not well characterized how living organisms respond to mild hypoxia, and how they distinguish different levels of hypoxia. Results: We examined the transcriptional responses of locusts using microarrays to reveal their strategies to cope with mild hypoxia. Mitochondrial activities were systemically suppressed, mainly involving energy production and mitochondrial biogenesis. The functions of endoplasmic reticulum were activated to clear the dysfunctional proteins and rescue newly synthesized proteins. Glucose in cytosol was shunted from glycolysis to pentose phosphate pathway probably to tackle the oxidative stress by enhancing the production of reductive forces. Conclusions: LocustsM-bM-^@M-^Y responses to mild hypoxia differ from that to severe hypoxia . In severe hypoxia, glucose resources are shunted to glycolysis to produce ATP and tackle energy crisis; however, in mild hypoxia, they are diverted to produce reductive forces and deal with oxidative stress. Locusts are capable of distinguishing different levels of hypoxia and initiating proper defenses. Simulated 4km altitude hypoxia treatment vs. normoxia control;direct comparison on 6 separate microarrays; each microarray compares one biological replicate of treatment and control; each biological replicate contains 10 individuals
2012-12-12 | E-GEOD-33898 | biostudies-arrayexpress