ABSTRACT: Ikk2 regulates cytokinesis during vertebrate development (Trancriptome profiling from the wild-type and Ikk2 maternal-zygotic mutant zebrafish)
Project description:Purpose: The Ikk2 maternal-zygotic mutants are the only vertebrates animals completely depleted globally of the Ikk2 function which is expected to block an activity of the canonical NFkB signaling pathway. Transcriptome profiling of embryos before the midblastula transition (MBT) and after MBT may provide a clean strategy to identify the NFkB target genes. Methods: Zebrafish lines were maintained under standard laboratory procedures. Results: Using an optimized data analysis workflow, we identified 54,276 transcripts in the embryos at 2 hours postfertilization (hpf) and 4 hpf. RNA-seq data confirmed lack of expression of a number of genes in the mutant both prior to and after the MBT, including genes linked to angiogenesis, skin development, cytokinesis, innate immunity and cytoskeletonT, and 4 of these were validated with qRT–PCR. M. add here if required. Conclusions: Our study represents the first detailed analysis of transcriptomes of vertebrates globally depleted of activity of Ikk2, with two biologic replicates, generated by RNA-seq technology.The data reported here should provide a framework for understanding of maternal and zygotic genes which expression is controlled by Ikk2 activity. Our results expands a list of transcripts which expression may be controlled by the canonical NFkB signaling. We conclude that RNA-seq based transcriptome characterization improves analysis of NFkB regulated genes.
Project description:MicroRNAs comprise 1-3% of all vertebrate genes, but their in vivo functions and mechanisms of action remain largely unknown. Zebrafish miR-430 is expressed at the onset of zygotic transcription and regulates morphogenesis during early development. Using a microarray approach and in vivo target validation, we find that miR-430 directly regulates several hundred target mRNAs. Targets are highly enriched for maternal mRNAs that accumulate in the absence of miR-430. We also show that miR-430 accelerates the deadenylation of target mRNAs. These results suggest that miR-430 facilitates the deadenylation and clearance of maternal mRNAs during early embryogenesis. Keywords: Dicer, MZdicer, miR-430, miRNA target, maternal, zygotic
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)