Nascent transcriptome of Xenopus laevis embryos at mid-blastula transition (MBT) stages
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ABSTRACT:
PROVIDER: PRJNA545469 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9160380_1.fastq.gz | Fastqsanger.gz | |||
| SRR9160380_2.fastq.gz | Fastqsanger.gz | |||
| SRR9160390_1.fastq.gz | Fastqsanger.gz | |||
| SRR9160390_2.fastq.gz | Fastqsanger.gz | |||
| SRR9160381_1.fastq.gz | Fastqsanger.gz |
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Project description:To validate that EU-RNA imaging provides a direct readout of wide-spread zygotic transcription, we sought to identify the nascent transcriptome using RNA-seq. We micoinjected 5-ethynyl uridine (EU) into 1-cell Xenopus embryos, isolated total RNA from embryos at mid-blastula transition (MBT), biotinylated EU-RNA and purified it to generate cDNA libraries for sequencing. Total RNA from normal embyros were used as control. We found over 25,000 nascent genes in this EU-RNA dataset at mid-ZGA. The nascent transcriptome dataset captures nearly 90% of the transcripts present in the whole-embryo dataset and with similar or better read-depth, indicating that our EU-RNA imaging is truly representative of wide-spread zygotic transcription. Furthermore, of the known zygotic genes that are most highly induced at MBT, we detected all of them and at much higher levels than in the whole-embryo dataset. These results show that our labeling captures the nascent zygotic transcriptome. We found ~ 4-fold higher sensitivity for nascent transcripts in the EU-RNA dataset compared to the whole embryo dataset, and as excepted we did not detect thousands of maternal-only transcripts in the nascent dataset. Together, these results suggest that EU-labeling provides a visual readout of bona fide nascent zygotic transcripts.
2019-05-31 | GSE131962 | GEO
Project description:Sox31 is a member of the zebrafish SoxB1 subfamily, and its expression can be detected both pre- and post-MBT. To distinguish the function of its maternal and zygotic transcripts, a splice blocking morpholino (Sb MO) was designed to interfere with the processing of new, zygotically synthesised mRNAs without interfering with mRNAs of maternal origin. Developmental arrest was observed in Sb MO which could not bypass MBT. Mid-Blastula Transition (MBT) functions as a time window for zygotic genome activation and maternal mRNA degradation. To uncover whether the “zygotic up” and “maternal down” event during MBT is retarded in Sb morphants, we performed microarray experiment at the end of MBT (about 4.3 hours post fertilication/4.3 hpf) to compare mRNAs from Sb morphants and control embryos.
2011-10-13 | GSE32914 | GEO
Project description:Sox31 is a member of the zebrafish SoxB1 subfamily, and its expression can be detected both pre- and post-MBT. To distinguish the function of its maternal and zygotic transcripts, a splice blocking morpholino (Sb MO) was designed to interfere with the processing of new, zygotically synthesised mRNAs without interfering with mRNAs of maternal origin. Developmental arrest was observed in Sb MO which could not bypass MBT. Mid-Blastula Transition (MBT) functions as a time window for zygotic genome activation and maternal mRNA degradation. To uncover whether the “zygotic up” and “maternal down” event during MBT is retarded in Sb morphants, we performed microarray experiment at the end of MBT (about 4.3 hours post fertilication/4.3 hpf) to compare mRNAs from Sb morphants and control embryos. In one experiment, three flocks of zebrafish eggs were injected with the Sox19b morpholino immediately after fertilization, while another three control populations were injected with placebo. At 4.3 hpf, these six flocks of embryos were sent for gene expression profiling with six Affymetrix Zebrafish Genome Arrays. In another experiment, we compared two wildtype embryo samples at 4h (post-MBT) against two wildtype samples at 2.5 h (pre-MBT).
2011-10-12 | E-GEOD-32914 | biostudies-arrayexpress
Project description:The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilization wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the 7th cleavage and extending beyond the MBT at the 12th cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal genes whose mRNAs are deadenylated shortly after fertilization, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organization of gene regulatory networks in the early
2014-03-27 | GSE56242 | GEO
Project description:The Xenopus mid-blastula transition (MBT) marks the onset of large-scale zygotic transcription, as well as an increase in cell cycle length and a loss of synchronous cell divisions. Little is known about what triggers the activation of transcription or how newly expressed genes interact with each other. Here we use high-resolution expression profiling to identify three waves of gene activity: a post-fertilization wave involving polyadenylation of maternal transcripts; a broad wave of zygotic transcription detectable as early as the 7th cleavage and extending beyond the MBT at the 12th cleavage; and a shorter post-MBT wave of transcription that becomes apparent as development proceeds. Our studies have also allowed us to define a set of maternal genes whose mRNAs are deadenylated shortly after fertilization, and are likely to be degraded thereafter. Experimental analysis indicates that the polyadenylation of maternal transcripts is necessary for the establishment of proper levels of zygotic transcription at the MBT, and that genes activated in the second wave of expression, including Brachyury and Mixer, contribute to the regulation of genes expressed in the third. Together, our high-resolution time series and experimental studies have yielded a deeper understanding of the temporal organization of gene regulatory networks in the early Time series polyA+ and RiboZero RNA sequencing of Xenopus Embryos covering 0-9.5 hours post fertilization
2014-03-27 | E-GEOD-56242 | biostudies-arrayexpress
Project description:Externally deposited eggs begin development with an immense cytoplasm and a single overwhelmed nucleus. Rapid mitotic cycles restore normality as the ratio of nuclei to cytoplasm (N/C) increases. A threshold N/C has been widely proposed to activate zygotic genome transcription and onset of morphogenesis at the mid-blastula transition (MBT). To test whether a threshold N/C is required for these events, we blocked N/C-increase by downregulating cyclin/Cdk1 to arrest early cell cycles in Drosophila. Embryos that were arrested two cell cycles prior to the normal MBT activated widespread transcription of the zygotic genome including genes previously described as N/C-dependent. Zygotic transcription of these genes largely retained features of their regulation in space and time. Furthermore, zygotically regulated post-MBT events such as cellularization and gastrulation movements occurred in these cell-cycle-arrested embryos. These results are not compatible with models suggesting that these MBT events are directly coupled to N/C. Cyclin/Cdk1 activity normally declines in tight association with increasing N/C and is regulated by N/C. By experimentally promoting the decrease in cyclin/Cdk1, we uncoupled MBT from N/C increase, arguing that N/C guided down-regulation of cyclin/Cdk1 is sufficient for genome activation and MBT.
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Project description:In most embryos, the mid-blastula transition is a complex process featuring maternal RNA degradation, cell cycle pause, zygotic transcriptional activation and morphological changes. The nucleocytoplasmic (N/C) ratio has been proposed to control the multiple events at MBT. To understand the global transcriptional response to the changes of the N/C ratio, we profiled wild type and haploid embryos using cDNA microarrays at three developmental stages. Keywords: time course and different genotype
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Project description:Gene expression was analysed in normally developing Xenopus laevis embryos from early blastula to early neurula stages
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Project description:To characterize the effect of cell cyle elongation on nascent transcription during zygotic genome activation (ZGA) of Xenopus laevis embryos, we microinjected 5-ethynyl uridine (EU) into 1-cell stage embryos and treated the embryos with 0.2 mg/ml of cycloheximide (CHX) to arrest them in interphase from 5 hpf to 7.5 hpf. Control and CHX-arrested embryos were collected for isolating total RNAs, followed by biotinylation using disulfide biotin azide via click reaction and purification of nascent transcripts using streptavidin beads. Libraries were constructed and sequenced on illumina NextSeq 500.
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Project description:To characterize the nascent transcriptome during zygotic genome activation (ZGA) of Xenopus laevis embryos, we microinjected 5-ethynyl uridine (EU) into 1-cell stage embryos and isolated total RNAs from whole embryos at 5, 6, 7, 8 and 9 hours post-fertilization (hpf) at room temperature, respectively, covering the stages of pre-ZGA to widespread ZGA (stage 7-9). To purify nascent transcripts, total RNAs were biotinylated using disulfide biotin azide via click reaction and biotinylated nascent transcripts were purified using streptavidin beads. Libraries were constructed from using the total RNA ('All'), nascent transcripts ('Bead') and the flowthrough after purification of nascent transcripts ('FL'), respectively. To categorize maternal-zygotic (MZ) genes and zygotic-only (Z) genes, total RNAs from eggs were isolated for constructing libraries. All libraries were sequenced on illumina NextSeq 500.
2022-08-22 | GSE201835 | GEO