Transcription profiling of Xenopus laevis early gastrulation embryos injected with alpha-amanitin against RNA polymerase II activity
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ABSTRACT: Xenopus laevis embryos were injected with alpha-amanitin to inhibit RNA polymerase II activity. Embryos were allowed to develop up to stage 10.5 (early gastrula, control and alpha-amanitin injected embryos) and subsequently collected for RNA isolation. The transcriptome profiles of alpha-amanitin injected and control embryos were compared.
Project description:Xenopus laevis embryos were injected with antisense oligonucleotides against TBP, TLF or TBP2. Embryos were allowed to develop up to stage 7 (non-injected control embryos) or stage 10.5 (early gastrula, control and antisense injected embryos) and subsequently collected for RNA and protein isolation. The transcriptome profile of antisense injected and control embryos was compared.
Project description:The Xenopus POU class V transcription factor XOct-25 has been shown to inhibit BMP-dependent epidermal differentiation and promote neural induction in the ectoderm during early embryogenesis. In order to identify genes that act downstream of XOct-25, transcriptional profile of Xenopus ectodermal cells overexpressing XOct-25 was compared with control cells by using a DNA microarray method. Two independent experiments. Each experiment contains ectodermal cells overexpressing XOct-25 and corresponding control cells. Xenopus embryos were injected at the 8-cell stage with mRNA encoding GR-XOct-25, a hormone-inducible version of XOct-25. Explants from stage 9 embryos were treated with or without dexamethazone (DEX) until the sibling embryos reached stage 10.5, when they were used for RNA extraction. The explants cultured without DEX was used as a control sample. biological replicates: Sample name XOct-exp 1, Sample name XOct-exp 2
Project description:Eye development is a multistep process that requires specific inductive signals and precise morphogenetic movements, starting early during development in the eye-field, a well-definite region of the anterior neural plate. It has been demonstrated that a gene network of eye field transcription factors (EFTFs) contributes to specify the neural and retinal fate of the eye field. Among these EFTFs, Xrx1 is involved in proliferation and neurogenesis in the eye field and is necessary for the correct development of the retina. By means of Affymetrix microarrays, a high throughput screening was performed, looking for genes that can mediate the function of Xrx1, by comparison of the expression profiles of whole embryos in which the Xrx1 function was either overexpressed or abolished by use of morpholino antisense oligonucleotides. Xenopus laevis embryos were harvested soon after fertilization. 50ng of capped Xrx1 mRNA or 0.5pmol of MoXrx1 antisense oligonucleotides were coinjected with 400ng of GFP synthetic mRNA as a tracer, in both dorsal blastomeres of 4-cells stage embryos. Embryos were let to develop until stage 13, in which the eye field induction steps are active, and selected for correct injection site by fluorescence topographic assessment. RNA was extracted from each single embryo and control PCRs were performed to assess the correct molecular signature of Xrx1 loss- and gain- of function.
Project description:DNA methylation is a tightly regulated epigenetic mark associated with transcriptional repression. Next-generation sequencing of purified methylated DNA obtained from early Xenopus tropicalis embryos demonstrates that this genome is heavily methylated during blastula and gastrula stages. Although DNA methylation is largely absent from transcriptional start sites marked with histone H3 lysine 4 trimethylation (H3K4me3), we find both promoters and gene bodies of active genes robustly methylated. By contrast, DNA methylation is absent in large H3K27me3 domains, indicating these two repression pathways have different roles. Comparison with chromatin state maps of human ES cells reveals strong conservation of epigenetic makeup and gene regulation between the two systems. Strikingly, genes that are highly expressed in human pluripotent cells and in Xenopus embryos but not in differentiated cells exhibit relatively high methylation in both promoters and gene bodies in embryos. Therefore we tested the repressive potential of DNA methylation using transient and transgenic approaches and show that methylated promoters are robustly transcribed in blastula and gastrula-stage embryos, but not in oocytes or late embryos where methylated templates are repressed efficiently. These findings have implications for reprogramming and the epigenetic regulation of pluripotency and differentiation, suggesting a relatively open, pliable chromatin state in early embryos followed by re-established methylation-dependent transcriptional repression during organogenesis and differentiation. MethylCap (methylated DNA affinity capture with the MBD domain of MeCP2), 500mM and 700mM elution fractions of stage 9 (blastula) and stage 12.5 (gastrula) Xenopus tropicalis DNA
Project description:Transcription initiation involves the recruitment of basal transcription factors to the core promoter. A variety of core promoter elements exists, however for most of these motifs the distribution across species is unknown. Here we report on the comparison of human and amphibian promoter sequences. We have used oligo-capping in combination with deep sequencing to determine transcription start sites in Xenopus tropicalis. To systematically predict regulatory elements we have developed a de novo motif finding pipeline using an ensemble of computational tools. A comprehensive comparison of human and amphibian promoter sequences revealed both similarities and differences in core promoter architecture. Some of the differences stem from a highly divergent nucleotide composition of Xenopus and human promoters. Whereas the distribution of some core promoter motifs is conserved independent of species-specific nucleotide bias, the frequency of another class of motifs correlates with the single nucleotide frequencies. This class includes the well-known TATA box and SP1 motifs, which are more abundant in Xenopus and human promoters, respectively. While these motifs are enriched above the local nucleotide background in both organisms, their frequency varies in step with this background. These differences are likely adaptive as these motifs can recruit TFIID to either CpG island or sharply initiating promoters. Our results highlight both conserved and diverged aspects of vertebrate transcription, most notably showing co-opted motif usage to recruit the transcriptional machinery to promoters with diverging nucleotide composition. This shows how sweeping changes in nucleotide composition are compatible with highly conserved mechanisms of transcription initiation. ChIP-seq profiles of TBP in Xenopus tropicalis stage 12 embryos and TSS-seq profiles of Xenopus oocytes and stage 12 embryos
Project description:We aimed to identify genes that are inhibited by Kctd15 overexpression during neural crest (NC) induction. Injection of RNAs encoding Wnt3a and chordin into the embryo followed by animal cap explant culture leads to the induction of NC marker genes as well as additional genes expressed in the neural plate border. Our previous study indicated that co-injection of kctd15 inhibits the induction of these markers. We injected four sets of RNAs, lacZ as control (L), wnt3a+chd (WC), wnt3a+chd+kctd15 (WCK), and kctd15 (K). After evaluation of marker gene expression with RT-PCR, we prepared probes with same RNA extract for Affymetrix DNA microarray. The hybridization, washing and scanning were perfomed following the manual from Affymetrix. Partek Genomics Suite was employed for data analysis. Xenopus embryos were injected with mRNAs at two-cell stage. When developing to stage 9, the animal caps were dissected for RNA extraction and hybridization on Affymetrix microarrays. According to the mRNAs injected, the animal caps (AC) were divided into four groups, including (1) AC injected with lacZ; (2) AC injected with kctd15; (3) AC injected with chordin and wnt3a; (4) AC injected with chordin, wnt3a and kctd15. The injected animal caps were cultured till stage 15 and then collected for RT-PCR and microarray assay. Each group had three repeats.
Project description:Recent developments in genomic sequencing technology have enabled comprehensive transcriptome analyses of single cells. In contrast, single cell proteomics analyses have been restricted to targeted analyses, for example using flow cytometry with GFP fusions or mass cytometry. Here, we performed global absolute protein quantification of single Xenopus laevis eggs using mass spectrometry-based proteomics. We quantified over 5800 proteins, thus representing the largest single cell proteome that has been characterized to date. Absolute protein amounts in single eggs are highly comparable, thus indicating a tight regulation of global protein abundance. Comparison between the single-cell proteome and transcriptome reveal poor expression correlation. Finally, we identified 439 proteins that significantly change in abundance during early embryogenesis. Many of these proteins do not show regulation at the transcript level. Altogether, our data reveal that the transcriptome is a poor indicator of the proteome and that protein levels are tightly controlled in Xenopus leavis eggs. RNA-seq in Xenopus laevis of 5 replicates of both single eggs and single embryos.
Project description:To analyze the dynamics and diversity of coding and non-coding transcripts during development, both polyadenylated mRNA and ribosomal RNA-depleted total RNA were harvested across six developmental stages and subjected to high throughput sequencing. The maternally loaded transcriptome is highly diverse and consists of both polyadenylated and deadenylated transcripts. Many maternal genes show peak expression in the oocyte and include genes which are known to be the key regulators of events like oocyte maturation and fertilization. Of all the transcripts that increase in abundance between an early blastula and larval stages, about 30% of the embryonic genes are induced by fourfold or more by the late blastula stage and another 35% by late gastrulation. Using a gene model validation and discovery pipeline, we identified novel transcripts, the majority of which show hallmarks of being long non-coding RNAs both in terms of their coding potential and gene structure. Profiles of polyadenylated mRNA (6 stages) and ribosomal RNA-depleted total RNA (3 stages) through early Xenopus tropicalis development
Project description:During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using α-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the α-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (α-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (α-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (α-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the α-amanitin experiments were generated. For RNA-seq samples of the α-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA