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Nascent Transcriptome of AP and VP from Xenopus laevis Embryos at 5-9 hpf (stage 7-9)

ABSTRACT: To characterize the spatial patterns of zygtoic transcription 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). The animal pole (AP, ~ 1/3 at the top region of embryo) and the vegetal pole (VP,~ 1/3 at the bottom region of embryo) regions were dissected and used for total RNA isolation. 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 nascent transcripts. All libraries were sequenced on illumina NextSeq 500.

ORGANISM(S): Xenopus laevis

PROVIDER: GSE201833 | GEO | 2022/08/22

REPOSITORIES: GEO

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Total and Nascent Transcriptome of Xenopus laevis Embryos at 5-9 hpf (stage 7-9)

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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.

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Dynamic RNA-protein interactions underlie the zebrafish maternal-to-zygotic transition

Project description:During the maternal-to-zygotic transition (MZT), transcriptionally silent embryos rely on post-transcriptional regulation of maternal mRNAs until zygotic genome activation (ZGA). RNA-binding proteins (RBPs) are important regulators of post-transcriptional RNA processing events, yet their identities and functions during developmental transitions in vertebrates remain largely unexplored. Using mRNA interactome capture, we identified 227 RBPs in zebrafish embryos before and during ZGA, hereby named the zebrafish MZT mRNAbound proteome. This protein constellation consists of many conserved RBPs, with additional embryo- and stage-specific mRNA interactors that likely reflect the dynamics of RNA-protein interactions during MZT. The enrichment of numerous splicing factors like hnRNP proteins before ZGA was surprising, because maternal mRNAs were found to be fully spliced. To address potentially unique roles of RBPs in embryogenesis, we focused on hnRNP A1. iCLIP and subsequent mRNA reporter assays revealed a function for hnRNP A1 in the regulation of poly(A) tail length and translation of maternal mRNAs through sequence-specific association with 3’UTRs before ZGA. Comparison of iCLIP data from two developmental stages revealed that hnRNP A1 dissociates from maternal mRNAs at ZGA and instead regulates the nuclear processing of pri-miR-430 transcripts, which we validated experimentally. The shift from cytoplasmic to nuclear RNA targets was accompanied by a dramatic translocation of hnRNP A1 and other pre-mRNA splicing factors to the nucleus in a transcription-dependent manner. Thus, our study identifies global changes in RNA-protein interactions during vertebrate MZT and shows that hnRNP A1 RNA-binding activities are spatially and temporally coordinated to regulate RNA metabolism during early development.

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Drosophila pole cell transcriptome in early embryogenesis (stage 4).

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Project description:How maternal factors in oocytes initiate zygotic genome activation (ZGA) remains elusive. Recent studies indicate that DPPA2 and DPPA4 are required for establishing a 2-cell embryo-like (2C-like) state in mouse embryonic stem cells (ESCs) in a DUX-dependent manner. These results suggest that DPPA2 and DPPA4 are essential maternal factors that regulate Dux and ZGA in embryos. By analyzing maternal knockout and maternal-zygotic knockout embryos, we unexpectedly found that Dux activation, ZGA, and preimplantation development are normal in embryos without DPPA2 or DPPA4. Thus, unlike in ESCs/2C-like cells, DPPA2 and DPPA4 are dispensable for ZGA and preimplantation development.

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HDAC1/2 is indispensable for mouse and bovine ZGA via regulating distribution of H3K27ac and H3K4me3 [ChIP-seq]

Project description:Zygotic genome activation (ZGA) is an impressed biological event following fertilization during early embryo development. Precise reprogramming of epigenetic modifications (e.g. histone H3 lysine 27 acetylation, H3K27ac) have been found contribute to safeguard ZGA in zebrafish and Drosophila, but the dynamic of H3K27ac and the underlying regulation mechanism in mammal embryos are still enigmatic yet. As the main erasers for histone acetylation, histone deacetylase 1 and 2 (HDAC1 and HDAC2) are reported essential for mouse oogenesis and embryo lineage development, while it is unclear if they are functional for ZGA. Here, we observed significant global decrease of H3K27ac during ZGA in both mouse and bovine embryos, and accumulation of HDAC1/2 is responsible for the decline of H3K27ac. Repression of HDAC1/2 by dominant negative mutation led to arrested development at late 2-cell stage in mouse embryos, accompanied by downregulation of genes supposed to be expressed during ZGA. ChIP-seq revealed abnormal distribution of H3K27ac, and DUX participates in H3K27ac aberrant hyper-acetylation in embryos overexpressed mutant HDAC1/2. Moreover, suppression of HDAC1/2 also restrains H3K4me3 removal at gene body regions via impeding expression of KDM5s during mouse ZGA, and compensatory exogenous Kdm5b mRNA can fix the exceeding H3K4me3 and failed ZGA. Intriguingly, function of HDAC1/2 seems to be conservative between mouse and bovine embryos. Together, this study reveals that HDAC1/2 is indispensable for mouse and bovine ZGA via regulating distribution of H3K27ac and H3K4me3.

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