Reduced representation bisulfite sequencing (RRBS) for primordial germ cells and somatic cells during zebrafish early embryogenesis
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Danio rerio
SUBMITTER: Fabio D'Orazio
PROVIDER: E-MTAB-9858 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:Zebrafish Primordial Germ Cells (PGCs) were tracked in the Tg(Buc-GFP) line where the germ plasm protein Buc is fused to GFP. GFP-positive cells were isolated via FACS and RNA-seq was performed on polyadenylated transcripts for PGCs and somatic cells at 256-cell, high, dome, 10 somites and prim-5 stages. Two hundred cells were used for each biological replicate.
2020-12-24 | E-MTAB-8741 | biostudies-arrayexpress
Project description:The small non-coding RNAs (sncRNAs) are considered as postranscriptional key regulators of male germ cell development. In addition to microRNAs (miRNAs) and PIWI-interacting RNAs (piRNAs), other sncRNAs generated from small nucleolar RNAs (snoRNAs), tRNAs or rRNAs processing may also play important regulatory roles in spermatogenesis. By next generation sequencing (NGS), we characterized the different sncRNA populations detected at three milestone stages in male germ differentiation: primordial germ cells (PGCs) at 13.5 dpc, pubertal spermatogonia cells, and mature spermatozoa. In order to assess the potential transmission of the sncRNAs through the mature spermatozoa during fertilization, the sncRNA population detected in male germ cells was also compared with sncRNAs detected in unfertilized mouse oocytes and zygotes. Combining the data obtained by NGS and microarrays from whole PGC and spermatogonia transcriptome, we defined the potential regulatory roles of specific miRNAs and their validated targets. Similar to miRNAs, both the small RNAs derived from snoRNAs and the piRNAs, could be involved in the postranscriptional regulation of mRNA transcripts during the male germ development. Finally, our results strongly suggest that the small RNAs-derived from tRNAs and rRNAs are interacting with PIWI proteins, and specifically with MILI. These new classes of piRNAs are not generated by the ping-pong pathway and could be the source of primary piRNAs. mRNA analysis of Primordial Germ Cells (PGCs), Spermatogonia cells (SPG), adult testis (AdT) and Gonad-less (GL) embryos. Indirect comparisons were made across multiple arrays with raw data pulled from different channels for data analysis and comparison to the control data.
2015-03-26 | E-GEOD-59252 | biostudies-arrayexpress
Project description:Zebrafish Primordial Germ Cells (PGCs) were tracked in the Tg(Buc-GFP) line where the germ plasm protein Buc is fused to GFP. GFP-positive cells were isolated via FACS and RNA-seq was performed on polyadenylated transcripts for PGCs and somatic cells at 256-cell, high, dome, 10 somites and prim-5 stages. Two hundred cells were used for each biological replicate. RNA-seq was also performed on embryos in where Tdrd7 translation was inhibited via morpholino treatment.
2020-12-24 | E-MTAB-8707 | biostudies-arrayexpress
Project description:The present investigation was to identify the signaling and metabolic pathways of expressed genes by microarray comparison between Primordial Germ Cells (PGCs) and their somatic counterpart, chicken embryonic fibroblasts (CEFs). We identified a total of 2,605 expressed transcripts. Among these, 1,197 were predominantly expressed in PGCs, and 1,408 were predominantly expressed in CEFs. Cell culture and microarray data generation were performed in triplicate. Blood PGCs from E2.5 embryos (N=20 for each replication) and CEFs from E6.5 embryos (N=5 for each replication) were cultured in appropriate culture medium. Total RNA was extracted from cultured PGCs (1,500,000 cells for each replication) and cultured CEFs (6,000,000 cells for each replication) with a Qiagen RNeasy kit. About 5 M-BM-5g of total RNA from each replication was used for labeling. Probe synthesis from total RNA samples, hybridization, detection, and scanning were performed according to standard protocols from Affymetrix.
2013-01-10 | E-GEOD-38168 | biostudies-arrayexpress
Project description:Purpose: To identify molecular pathways underlying epigenetic reprogramming in early germ cell precursors, we examined global gene expression of wild type primordial germ cells using mRNA sequencing. Methods: Given the limited number of PGCs collected from E9.5 to E13.5 (ranging from 300 to 5000), we used a low-input RNA sequencing method, Smart-Seq. RNA libraries were pooled and sequenced by Illumina Hiseq. Results: We generated >22 million uniquely mapped reads per sample and identified >10 thousand transcripts per genotype (RPKM>0.1). Hierarchical clustering and correlation analysis on gene expression indicates samples were clearly separated according to their genotypes with Spearman correlation coefficient of 0.98/0.99 within biological replicates. Compared with E9.5 PGCs, 479 genes were significantly up-regulated and 248 genes were down-regulated in E11.5 PGCs. When compared with E11.5 PGCs, male E13.5 PGCs had 362 up-regulated, and 239 down-regulated genes, whereas female E13.5 PGCs had 1163 up-regulated and 333 down-regulated genes. Overall, the number of up-regulated genes was greater than that of the down-regulated genes in every comparison, suggesting that gene expression is generally activated during PGC reprogramming. mRNA profiles of primordial germ cells derived from developmental embryos stages (E9.5, E11.5 and E13.5) were generated by deep sequencing, in duplicates (E9.5 and E11.5) or triplicates (E13.5f and E13.5m), using Illumina Hiseq.
2012-10-31 | E-GEOD-41908 | biostudies-arrayexpress
Project description:Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, develop pre-meiotic germ cell characteristics, including X-reactivation, imprint erasure, and cyst formation. Upon transplantation under ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which, through in vitro maturation and fertilization, contribute to fertile offspring. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro. We performed expression analysis of female PGC-like cells [PGCLCs; day 3 (d3), day 6 (d6), and day 3 followed by day 6 in aggregation with female embryonic day (E) 12.5 gonadal somatic cells (d3ag6)] in comparison to in vivo embryonic PGCs (E12.5 female PGCs and E9.5 PGCs) and male d6 PGCLCs. PGCLCs were marked with fluorescence of Blimp1-mVenus, stella-ECFP transgenic reporters (BVSC). PGCs were marked with fluorescence of the stella-EGFP transgenic reporter.
2012-11-05 | E-GEOD-40716 | biostudies-arrayexpress
Project description:The precise positioning of organ progenitor cells constitutes an essential, yet poorly understood step in organ development and function. Using primordial germ cells and the process of gonadogenesis as an in vivo model, we present the developmental mechanisms facilitating the maintenance of a motile progenitor cell population at the site where the organ develops. We find that the action of repulsive cues coupled with the generation of physical barriers confine the cells to the correct bilateral positions within the embryo. Using computer particle-based simulations we could also demonstrate the role of reflecting barriers, from which cells turn away upon contact, and the importance of proper level of cell-cell interaction for maintaining the tight cell clusters and their correct positioning at the target region. The cellular mechanisms operating during those events were determined using high-resolution live imaging microscopy. This analysis revealed cell polarity change upon interaction with the physical barrier and the establishment of compact clusters that involves increased cell-cell interaction as a result of collapse of cell protrusions and enhanced adhesion. The combination of these developmental and cellular RNA profiling in zebrafish embryos of 7 hpf and 36 hpf in sorted germ cells and randomly sorted somatic cells
2016-04-05 | E-GEOD-77077 | biostudies-arrayexpress
Project description:We developed a ChIP protocol for the analysis of histone marks using less than 10,000 cells per IP, and used it to investigate the chromatin state of E11.5 mouse primordial germ cells (PGCs). A genome-wide ChIP-Seq analysis of E11.5 PGCs revealed a distribution of H3K4me3/H3K27me3 bivalent domains highly enriched for developmental regulatory genes. H3K4me3 and H3K27me3 ChIP-Seq from mouse E11.5 primordial germ cells.
2013-05-30 | E-GEOD-46396 | biostudies-arrayexpress
Project description:We performed gene expression profiling on in vitro derived PGCs, undifferentiated ESCs, and somatic cells from the EB to examine germ cell expression in ESC-derived cells All cells were collected using fluorescence activated cell sorting to isolate SSEA1+/cKit+ ESCs, SSEA1+/cKitbright PGCs, Oct4-gfp+/cKitbright PGCs, and SSEA1-/cKit- somatic cells and Oct4-gfp-/cKit- somatic cells.
2011-12-15 | E-GEOD-33121 | biostudies-arrayexpress
Project description:Specification of germ cell fate is fundamental in development. With a highly representative single-cell microarray and rigorous quantitative-PCR analysis, we defined the genome-wide transcription dynamics that create primordial germ cells (PGCs) from the epiblast, a process that exclusively segregates them from their somatic neighbors. We also analyzed the effect of the loss of Blimp1, a key transcriptional regulator, on these dynamics. Our analysis revealed that PGC specification involves complex, yet highly ordered regulation of a large number of genes, proceeding under the strong influence of mesoderm induction with active repression of specific programs such as epithelial-mesenchymal transition, Hox gene activation, cell-cycle progression and DNA methyltransferase machinery. Remarkably, Blimp1 is essential for repressing nearly all the genes normally down-regulated in PGCs relative to their somatic neighbors, whereas it is dispensable for the activation of approximately half of the genes up-regulated in PGCs. Experiment Overall Design: Embryo isolation, dissection, and single cell cDNA amplification were performed as described (Kurimot et al., 2006, Nucleic Acids Res 34: e42; Kurimoto et al., 2007, Nature protocols, 2007, 2:739-52). Randomly picked cells were screened with gene-specific primers for Blimp1 and fragilis to select Blimp1-positive embryonic cells (which marks lineage-restricted PGC precursors or PGCs). For Blimp1 KO embryos, non-functional transcript from the Blimp1null allele was detected with the same primer set.
2008-10-25 | E-GEOD-11128 | biostudies-arrayexpress