RNA-seq of whole zebrafish embryos at 6 and 10hr post fertilisation
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ABSTRACT:
INSTRUMENT(S): Illumina NovaSeq 6000
ORGANISM(S): Danio rerio
SUBMITTER: Hiroaki Miki
PROVIDER: E-MTAB-10167 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Quantitative proteomic data for 10 stages (including 4-cell, 256-cell, 1000-cell, Oblong, Dome, Bud, 16-hpf, 28-hpf, 3-dpf, 5-dpf) of early zebrafish development, each stage corresponds to two sets of biological repeats, and each set of biological repeats corresponds to two sets of technical repeats.
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Project description:We are performing microarray experiments for expression profiling of zebrafish embryogenesis, both as a baseline for future analysis of mutant and other conditions and to validate our microarray technology. For our purpose we used the Affymetrix zebrafish array which contains approximately 15,000 genes. This represents about 50 % of the estimated number of zebrafish genes. Total RNA was collected from embryos at 16 different stages (zygote, shield stage, 75 % epiboly, 90 % epiboly, bud stage, 5-somite stage, 14-somite stage, prim-5 stage, 32 hpf and long-pec stage, 4d post fertilization (dpf), 5 dpf, 14 dpf, 30 dpf, 90 dpf, adult). Microarray analysis was performed with these stages in single colour experiments. After normalization, differential expressed genes were selected and further analyzed with GeneSpring software. In order to validate the microarray data and to assign biological functions we chose a few genes to do semi-quantitative real-time PCR. Many of the differentially expressed genes are unknown and could be candidates for regulatory genes identified in mutagenesis experiments. We identified several genes known to be involved in zebrafish organogenesis as well as novel genes with unique temporal expression patterns.
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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.
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Project description:Transcription factors play diverse roles during embryonic development, combinatorially controlling multiple cellular states in a spatially and temporally defined manner. Resolving the dynamic transcriptional profiles that underlie these patterning processes is essential for understanding embryogenesis at the molecular level. Here we show how temporal, tissue-specific changes in embryonic transcription factor function can be discerned by integrating caged morpholinos (cMOs) with photoactivatable fluorophores, fluorescence-activated cell sorting (FACS), and microarray technologies. As a proof of principle, we have dynamically profiled No tail-a (Ntla)-dependent genes at different stages of axial mesoderm development in zebrafish, discovering and characterizing discrete sets of transcripts that are coincident with either notochord cell fate commitment or differentiation. Our studies demonstrate how optically controlled chemical tools can be use to probe developmental processes with spatiotemporal precision and reveal the sequential activation of distinct transcriptomes within a cell lineage by a single transcriptional factor. Zebrafish zygotes were injected with a mixture of ntla caged morpholino (cMO) and caged fluorescein dextran (cFD), and a 100 µm-diameter region of the shield was UV-irradiated at 6 hours post fertilization (hpf). The UV irradiation generated an active morpholino targeting the ntla 5'UTR and simultaneously labeled the cells with green fluorescence. By 36 hpf, the irradiated, green-fluorescent cells contributed to the medial floor plate rather than the notochord, consistent with earlier proposals that Ntla acts as a transcriptional switch between these two cell fates. Combining these caged reagents with microarray analysis identified transcriptional changes coincident with loss of ntla at 9 hpf and subsequent notochord fate respecification. Zebrafish zygotes were injected with a mixture of ntla cMO/cFD, and a 100 µm-diameter region of the shield was UV-irradiated at 6 hpf. Control embryos were injected with cFD and similarly irradiated at 6 hpf. Experimental and control sets of embryos were enzymatically dissociated at 9 hpf, green-fluorescent cells were isolated by FACS, and collected in trizol. Thirty embryos were used for each experiment or controls, each yielding approximately 8,000 green fluorescent cells. Five biological replicates were performed along with five paired controls. Total RNA was isolated from each sample, reverse transcribed, and amplified using WTA2 TransPlex Complete Whole Transcriptome Amplification kit (Sigma) using a miniaturized procedure and manufacturer-recommended incubation steps. The synthesized cDNA was sent to Nimblegen for dye labeling and hybridization. Samples were hybridized to the Nimblegen 2007 (Zv 7) Danio rerio Gene Expression Array chip using one dye per chip. Raw probe data (.pair file) was subjected to RMA, normalization, background correction, and generation of gene expression summary (.calls file) by Nimblegen. Processed data was analyzed in ArrayStar software (DNAStar). Significantly affected genes (fold change > 2 and p-value < 0.10) were identified using a moderated t-test.
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Project description:In zebrafish, ovulated oocytes contain both cortisol deposited from the maternal circulation and maternal mRNA for the glucocorticoid receptor (gr mRNA), which is spread as granular structures throughout the central ooplasm. At the 1-cell stage (0.2 hpf), this transcript is relocated by streamers in the blastodisc area and equally partitioned among blastomeres. At 15 hpf, it is replaced by the zygotic transcript. Morpholino knockdown was applied to block translation (grATG1MO or MO2-nr3c1 and grATG2MO or MO3-nr3c1) of both maternal and zygotic gr transcripts, while a missplicing morpholino (grmismMO or MO4-nr3c1) was used to block post-transcriptionally the zygotic transcript alone. MO2-nr3c1 and MO3-nr3c1 (but not MO4-nr3c1) treatment produced craniofacial and caudal malformations in 1-dpf embryos and 5-dpf larvae, which were also affected by pericardial oedema, persistent yolk sac, reduced subintestinal veins, altered neurogenesis and uninflated swim bladder. Such effects were rescued with trout gr2 mRNA. Pangenomic microarray analysis revealed that 114 and 37 highly expressed transcripts were up- and down-regulated, respectively, by maternal GR protein deficiency in 5-hpf embryos. Similar alterations were found at 10 hpf. These effects were confirmed by real-time PCR of 2 up- (casp8, grp1 and igf2a) and 1 down-regulated transcripts (mcm6) evaluated at 4, 8 and 12 hpf. As the contents of transcripts were modified already at 4 hpf, it seems that the lack of GR affects both ways the molecular machinery for the degradation of maternal mRNAs. These results indicate that the maternal gr transcript participates in the maternal programming of zebrafish development. MO2-nr3c1 morphants were compared with MO2-nr3c1-5m morphants at 5 hpf and 10 hpf. MO2-nr3c1 morphants were compared with wild type (WT) at 5 hpf and 10 hpf. MO2-nr3c1 is a morpholino selected to knockdown translation of gr mRNA. MO2-nr3c1-5m is a specific control morpholino.
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