Expression profiling of FACS-purified fetal germ cells
ABSTRACT: We used microarrays to profile global gene expression changes of Pou5f1-GFP-positive germ cells between E11.5 to E15.5. Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. Three timepoints were included in this study: E11.5 (male/female), E13.5 (male) and E15.5 (male). For each timepoint, three biological replicates were analyzed. The Pou5f1-GFP-negative (non-germ cell) fraction of E13.5 (male) gonads was also included as a control.
Project description:The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale ChIP-Seq method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac and H2BK20ac), facilitating the identification of active and repressed cis-regulatory elements in germ cells in vivo. Comparison of active chromatin state between somatic, embryonic stem cells (ESC) and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. The nuclear receptor Nr5a2 motif is enriched at a subset of cis-regulatory regions and we confirm its role in germ cell differentiation. Interestingly, germ cells have comparatively more H3K27me3-marked sites that are absent in ESC and other somatic cell types. These repressed regions are enriched for retrotransposons and MHC genes and this indicates that these loci are specifically silenced in germ cells. Together, our study provides the first genome-wide histone modification maps of in vivo germ cells and revealed the molecular chromatin signatures unique to germ cells. Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. ChIP-seq of Histone modification was done for two timepoints in this study: E11.5 (male/female), E13.5 (male). For E13.5 timepoint, two biological replicates were analyzed. In order to validate small scale ChIP-seq method limited number of ES cells were used to check consistency of ChIP-seq data.
Project description:Single-cell RNA-Seq RNA from medial ganglionic eminence at E11.5, E13.5, E15.5 or E17.5. The "SAMPLE_ID" sample characteristic is a sample identifier internal to Genentech. The ID of this project in Genentech's ExpressionPlot database is PRJ0007389 Overall design: Single-cell RNA-Seq from medial ganglionic eminence at E11.5, E13.5, E15.5 or E17.5.
Project description:RNAs were isolated from FACS sorted ScxGFP positive cells of hindlimbs at E11.5, E13.5 and E15.5, and characterized by RNAseq Overall design: 18 hindlimbs, 12 hindlimbs and 11 hindlimbs were pooled together for E11.5, E13.5 and E15.5, respectively.GFP positive cells were FACS sorted, then for RNA extraction, cDNA library preparation and proceeded for RNAseq
Project description:We have characterized by small-RNAseq the miRNA expression pattern of mouse male and female Primordial Germ Cells (PGCs) and somatic stromal cells from gonads at E11.5, E12.5 and E13.5. MiRNA accumulation was higher in somatic cells than in PGCs and more stable across the different developmental stages analyzed. Differential expression analyses showed differences in the regulation of key miRNA clusters such as miR-199-214, miR-182-183-96 and miR-34c-5p whose targets have defined roles in both germ and somatic cells in gonadal sexual determination. Extensive analyses of miRNA sequences revealed an increase in non-canonical isoforms in PGCs at E12.5 compared to E11.5 and E13.5 and a dramatic change in isomiR expression and non-template 3' nucleotide additions in female PGCs at E13.5 respect to the other samples. Overall design: miRNA profiles of 12 different mice samples, corresponding to PGCs and gonadal somatic cells from E11.5, E12.5 and E13.5 male and female gonads, generated by deep sequencing using MiSeq Sequencing System (Illumina).
Project description:Satellite cells are the primary source of stem cells for skeletal muscle growth and regeneration. Since adult stem cell maintenance involves a fine balance between intrinsic and extrinsic mechanisms, we performed genome-wide chronological expression profiling to identify the transcriptomic changes involved in acquisition of muscle stem cell characteristics. Muscle samples were isolated from the trunk during development, postnatally and in adult and aged Pax3GFP/+ mice. After digestion, GFP cells were purified via FACS and process for RNA extraction and hybridization on Affymetrix microarrays (Affymetrix Mouse Genome 430 2.0 Arrays). The different ages selected for sample isolation were E11.5-E12.5-E13.5-E14.5-E15.5-E17.5-P1-P12-1MO-2MO-18MO (E, Embryonic days; P, Postnatal days; MO, age in months), covering embryonic and fetal progenitors and proliferant, quiescent satellite cells. The eleven stages were done in triplicate for E11.5-E12.5-E14.5-E15.5-1MO-2MO-18MO, twice for E13.5, 4 times for P1-P12 and 5 times for E17.5, so 36 samples included in the microarray.
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.
Project description:Limb development is a powerful model to investigate transcriptional complexity during differentiation and development, as tissue types appear at various stages, with unique transcriptional signatures. So far, most studies have used conventional expression arrays, which can identify only a predetermined set of cDNAs. However there is growing evidence that most of the non-coding part of the genome is transcribed as well. We took advantage of genomic tiling arrays, which cover most of the mouse genome, to look at limb transcriptomes in an unbiased manner, by identifying all genic- and intergenic transcripts. We selected three developmental stages (E11.5 ; E13.5 ; E15.5), covering a large period of limb organogenesis, during which the limb evolves from a mostly homogenous mesenchymal cell population to more specialized tissue types such as cartilage, bone, muscle and skin. Overall design: Two biological replicates for each of three time points (E11.5, E13.5, E15.5).
Project description:Primordial germ cells (PGCs) are the foundation of totipotency and vital for reproduction and heredity. PGCs in mice arise from the epiblast around Embryonic Day (E) 7.0, migrate through the hindgut endoderm, and colonize and proliferate in the embryonic gonads until around E13.5 prior to their differentiation either into pro-spermatogonia or oogonia. PRDM1, a transcriptional repressor, plays an essential role in PGC specification that includes robustly repressing a somatic mesodermal program. Using an inducible conditional knockout system, we show here that PRDM1 is critically required throughout PGC development. When Prdm1 was deleted in migrating PGCs at E9.5/E10.5 or in male gonadal PGCs at E11.5, PGCs were eliminated by apoptosis from around E10/5/E11.5 or E13.5, respectively. When Prdm1 was deleted in female gonadal PGCs at E11.5, PGCs progressed into the first meiotic prophase in an apparently normal fashion, but the oogonia exhibited an aberrant pachytene phenotype, undergoing abrupt apoptosis from around E16.5. The escape of a fraction of PGCs (~10%) from the Prdm1 deletion was sufficient to recover fairly normal germ-cell pools both in male and female adults. The key targets of PRDM1 in migrating/gonadal PGCs, including genes for development, apoptosis, and pro-spermatogonial differentiation, showed only a modest overlap with those upon PGC specification and were enriched with histone H3 lysine 27 tri-methylation (H3K27me3). Our findings provide a critical insight into the mechanism for maintaining the transcriptional integrity of PGCs. Overall design: Examination of Prdm1 conditional knock-out primordial germ cells
Project description:This experiment depicts RNA-Seq datasets from wild type XY male and XX female, as well as sex chromosomally abnormal XO female (Turner syndrome) and XX male (Klinefelter variant syndrome) mouse germ cells before, during and after germline reprogramming. This range from E6.5 epiblasts, fluorescence activated cell sorted (FACS) highly purified populations of germ cells (EGFP-positive) and gonadal somatic cells (EGFP-negative) from both sexes at E9.5, E11.5, E12.5, E14.5, E15.5, E16.5 and E18.5, as well as purified spermatogonia and leptotene / zygotene spermatocytes from P2 and P11 males, respectively. Non-gonadal somatic cell control datasets were generated from male and female E14.5 liver and tail. Germ cells from individual embryos were processed to make cDNA libraries and served as biological replicates. We generated in total 184 libraries for our analysis from 60 separate conditions.
Project description:Adult neural stem cells (NSCs) derive from embryonic precursors, but little is known about how or when this occurs. We have addressed this issue using single cell RNAseq at multiple developmental timepoints to analyze the embryonic murine cortex, one source of adult forebrain NSCs. We computationally identify all major cortical cell types, including the embryonic radial precursors (RPs) that generate adult NSCs. We define the initial emergence of RPs from neuroepithelial stem cells at E11.5. We show that by E13.5 these RPs express a transcriptional identity that is maintained and reinforced throughout their transition to a non-proliferative state between E15.5 and E17.5. These slowly-proliferating late embryonic RPs share a core transcriptional phenotype with quiescent adult forebrain NSCs. Together, these findings support a model where cortical RPs maintain a core transcriptional identity from embryogenesis through to adulthood, and where the transition to a quiescent adult NSC occurs during late neurogenesis. Overall design: We applied the high-throughput single-cell mRNA sequencing technique, Drop-seq, to the embryonic mouse cortex. 2000-5000 single cells from wildtype CD1 embryos of gestational ages E11.5, E13.5, E15.5 and E17.5 were characterized.