Project description:[PROJECT] After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition (MZT). This universal process coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem (ES) cells. To study the changes in chromatin structure that accompany zygotic genome activation and pluripotency, we mapped the genomic locations of histone H3 modifications before and after MZT in zebrafish embryos. Repressive H3 lysine 27 trimethylation (H3K27me3) and activating H3 lysine 4 trimethylation (H3K4me3) are only detected after MZT. H3K4me3 marks more than 80% of genes, including many developmental regulatory genes that are also occupied by H3K27me3. Sequential chromatin immunoprecipitation demonstrates that both methylation marks occupy the same promoter regions, revealing that the bivalent chromatin domains found in cultured ES cells also exist in embryos. In addition, we find a large group of genes that are monovalently marked by H3K4me3 but not H3K27me3. These H3K4me3 monovalent genes are neither expressed nor stably bound by RNA polymerase II. Closer inspection of in vitro data sets reveals similar monovalent H3K4me3 domains in ES cells. The analysis of an inducible transgene indicates that H3K4me3 domains can form in the absence of sequence-specific transcriptional activators or stable association with RNA pol II. These results suggest that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during MZT. [SAMPLES] ChIPchip analysis of histone modifications (H3K4me3, H3K27me3, H3K36me3) and RNA polymerase II in pre MZT (256-cell) and post MZT (4hpf; dome/30% epiboly) wt zebrafish embryos. H3K4me3, H3K27me3, H3K36me3 and PolII ChIP-chip at 256 cell stage (one replicate) and 4hpf (dome/30% epiboly) (two replicates)
Project description:Upon fertilization, the embryonic genome remains transcriptionally inactive until the mid-blastula transition. Zygotic genome activation (ZGA) of vertebrate embryos has been extensively studied using nucleic acid-based strategies, but proteomics data are still scarce, impeding the full mechanistic understanding of how ZGA is executed during the maternal-to-zygotic transition (MZT). Here, we performed quantitative proteomics to decipher the proteome landscape of zebrafish embryos during the MZT, quantifying nearly 5,000 proteins across four embryonic stages. The stage-specific clustering based on protein expression pattern revealed that helicases (i.e., eif4a2 and ruvbl1) facilitate pluripotency factors (i.e., nanog, pou5f3, ctcf, and hmga1) triggering ZGA in zebrafish, accompanied by the maternal product decay with P-bodies and ubiquitin dependent proteolytic pathway. Dozens of transcription factors show wave-like expression patterns during MZT, implying their diverse functions in triggering the ZGA and modulating differentiation for organ development. The combination of morpholino knockdown and quantitative proteomics demonstrated that maternal Nanog is required for proper embryogenesis by regulating 1) interactions with other pluripotency factors, 2) F-actin band formation, 3) cell cycle checkpoints and 4) maternal product degradation. This study represents the most systematic proteomics survey of developmentally regulated proteins and their expression profiles accompanying MZT in zebrafish, which is a valuable proteome resource for understanding ZGA.
Project description:[PROJECT] After fertilization the embryonic genome is inactive until transcription is initiated during the maternal-zygotic transition (MZT). This universal process coincides with the formation of pluripotent cells, which in mammals can be used to generate embryonic stem (ES) cells. To study the changes in chromatin structure that accompany zygotic genome activation and pluripotency, we mapped the genomic locations of histone H3 modifications before and after MZT in zebrafish embryos. Repressive H3 lysine 27 trimethylation (H3K27me3) and activating H3 lysine 4 trimethylation (H3K4me3) are only detected after MZT. H3K4me3 marks more than 80% of genes, including many developmental regulatory genes that are also occupied by H3K27me3. Sequential chromatin immunoprecipitation demonstrates that both methylation marks occupy the same promoter regions, revealing that the bivalent chromatin domains found in cultured ES cells also exist in embryos. In addition, we find a large group of genes that are monovalently marked by H3K4me3 but not H3K27me3. These H3K4me3 monovalent genes are neither expressed nor stably bound by RNA polymerase II. Closer inspection of in vitro data sets reveals similar monovalent H3K4me3 domains in ES cells. The analysis of an inducible transgene indicates that H3K4me3 domains can form in the absence of sequence-specific transcriptional activators or stable association with RNA pol II. These results suggest that bivalent and monovalent domains might poise embryonic genes for activation and that the chromatin profile associated with pluripotency is established during MZT. [SAMPLES] ChIPchip analysis of histone modifications (H3K4me3, H3K27me3, H3K36me3) and RNA polymerase II in pre MZT (256-cell) and post MZT (4hpf; dome/30% epiboly) wt zebrafish embryos.
Project description:Ikk2 regulates cytokinesis during vertebrate development (Trancriptome profiling from the wild-type and Ikk2 maternal-zygotic mutant zebrafish)
Project description:Classical embryological studies revealed that during mid-embryogenesis vertebrates show similar morphologies. This âphylotypic stageâ has recently received support from transcriptome analyses, which have also detected similar stages in nematodes and arthropods. A conserved stage in these three phyla has led us to ask if all animals pass through a universal definitive stage as a consequence of ancestral constraints on animal development. Previous work has suggested that HOX genes may comprise such a âzootypicâ stage, however this hypothetical stage has hitherto resisted systematic analysis. We have examined the embryonic development of ten different animals each of a fundamentally different phylum, including a segmented worm, a flatworm, a roundworm, a water bear, a fruitfly, a sea urchin, a zebrafish, a sea anemone, a sponge, and a comb jelly. For each species, we collected the embryonic transcriptomes at ~100 different developmental stages and analyzed their gene expression profiles. We found dynamic gene expression across all of the species that is structured in a stage like manner. Strikingly, we found that animal embryology contains two dominant modules of zygotic expression in terms of their protein domain composition: one involving proliferation, and a second involving differentiation. The switch between these two modules involves induction of the zootype; which in addition to homeobox containing genes, also involves Wnt and Notch signaling as well as forkhead domain transcription factors. Our results provide a systematic characterization of animal universality and identify the points of embryological constraints and flexibility. 106 single embryo samples
Project description:The maternal-to-zygotic transition is crucial in embryonic development, marked by the degradation of maternally provided mRNAs and initiation of zygotic gene expression. However, the changes occurring at the protein level during this transition remain unclear. Here, we conducted protein profiling throughout zebrafish embryogenesis using quantitative mass spectrometry, integrating transcriptomics and translatomics datasets. Our data shows that unlike RNA changes, protein changes are less dynamic. Further, increases in protein levels correlate with mRNA translation, whereas declines in protein levels do not, suggesting active protein degradation processes. Interestingly, proteins from pure zygotic genes are present at fertilization, challenging existing mRNA-based gene classifications. As a proof of concept, we utilized CRISPR-Cas13d to target znf281b mRNA, a gene whose protein significantly accumulates within the first two hours post-fertilization, demonstrating its crucial role in development. Consequently, our protein profiling, coupled with CRISPR-Cas13d, offers a new approach to unravel maternal mRNAs function during embryonic development.