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Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq

ABSTRACT: 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.

ORGANISM(S): Mus musculus

PROVIDER: GSE41908 | GEO | 2012/10/31

SECONDARY ACCESSION(S): PRJNA178509

REPOSITORIES: GEO

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Transcriptome profiling of wild type primordial germ cells (PGC) by RNAseq

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

Persistent Requirement and Alteration of the Key Targets of PRDM1 during Primordial Germ Cell Development in Mice

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.

2015-12-02 | GSE74094 | GEO

MicroRNAs-wide analysis of primordial germ and somatic cells reveals sexual and developmental differential functional regulation in mouse embryonic gonads

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.

2018-02-28 | GSE98713 | GEO

Comprehensive profiling of migratory primordial germ cells reveals niche-specific differences in non-canonical Wnt and Nodal-Lefty signaling in anterior vs posterior migrants

Project description:Mammalian primordial germ cells (PGCs) migrate asynchronously through the embryonic hindgut and dorsal mesentery to reach the gonads. To characterize transcriptional heterogeneity of migrating PGCs and their niches, we performed single-cell RNA sequencing of 13,262 mouse PGCs and 7,868 surrounding somatic cells during migration (E9.5, E10.5, E11.5) and in anterior versus posterior locations to enrich for leading and lagging migrants. Analysis of PGCs by position revealed dynamic gene expression changes between faster or earlier migrants in the anterior and slower or later migrants in the posterior at E9.5. At E10.5, we found that anterior PGCs upregulate Nodal transcriptional targets including Lefty1/2 and validated this LEFTY1/2 upregulation via whole-mount immunofluorescence staining. This positional and temporal atlas of mouse PGCs supports the idea that niche interactions along the migratory route elicit changes in proliferation, actin dynamics, pluripotency and epigenetic reprogramming.

2024-10-08 | GSE274603 | GEO

Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline in Vivo

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

Single-cell RNA-seq of the developing C57BL/6 mouse ovary from E11.5 to E14.5

Project description:We profiled a time-course single-cell RNA-seq on C57BL/6 mouse ovary from E11.5 to E14.5 to study the cell population heterogeneity and regulation of meiosis initiation. We collected single cell from XX gonads at E11.5, E12.5, E13.5 and E14.5, and were further captured with 10x Genomics based single-cell system. By using single-cell RNA seq, we recapitulated the progression of meiosis using pseudotime ordering of all germ cells and delineated key molecular network among all germ cell clusters. Besides, we provide candidate meiosis stage-specific master regulons along the progression of meiosis and provide detailed transcriptome profile information for controversial Xist+ PGCs. Our data here offers the opportunity for exploring mechanisms underlying germ cell meiosis progression at single cell resolution.

2020-06-30 | GSE128553 | GEO

Bivalent Chromatin Marks Developmental Regulatory Genes in the Mouse Embryonic Germline in Vivo

2013-05-30 | GSE46396 | GEO

Proteome landscape of mouse primordial germ cells

Project description:Primordial germ cells (PGCs) are precursors of both male and female gametes as fundamental materials for organism development. The transcriptome, methylome and chromatin accessibility profiles of PGCs in both mice and humans have been recently reported. However, little is known about the characteristics of PGCs at the protein levels, which directly exert cellular functions. Here, we construct landscapes of both proteome of mouse PGCs at E11.5, E13.5 and E16.5 days, the three critical developmental windows for PGCs’ sex differentiation, female meiosis initiation and male mitotic arrest. In each developmental stage of PGCs nearly 2000-3000 proteins are identified, among which specific functional pathways such as oxidative phosphorylation, DNA damage repair, and meiotic cell cycle are involved for different events during PGCs development. Thus, our work offers a valuable resource for the systematic investigations of PGC development at protein level.

2020-05-18 | PXD019222 |

RNA-seq profiling of mouse PGC (Mus musculus)

Project description:PGCs undergo two distinct stages of demethylation before reaching a hypomethylated ground state at E13.5. Stage 1 occurs between E7.25- E9.5 in which PGCs experience a global loss of cytosine methylation. However, discreet loci escape this global loss of methylation and between E10.5-E13.5, stage 2 of demethylation takes place. In this stage these loci are targeted by Tet1 and Tet2 leading to the loss of the remaining methylation and resulting in the epigenetic ground state. Our data shows that Dnmt1 is responsible for maintaining the methylation of loci that escape stage 1 demethylation, and that it functions in a UHRF1 independent manner. Our data further demonstrates that when these loci lose methylation prior to stage 2 it results in early activation of the meiotic program, which leads to precocious differentiation of the germ line resulting in a decreased pool of PGCs in the embryo and subsequent infertility in adult mice.

2016-09-13 | GSE74938 | GEO

Molecular sexual identity of chicken in vitro derived pre-gonadal primordial germ cells

Project description:In poultry, in vitro derived primordial germ cells (PGCs) represent an important tool for management of genetic resources. However, several studies have highlighted sexual differences exhibited by PGCs through in vitro steps, which may compromise their reproductive capacities. To understand this phenomenon, we compared the proteome of pregonadal chicken male (ZZ) and female (ZW) PGCs expanded in vitro by quantitative proteomic analysis using a GeLC-MS/MS strategy. The proteins found to be differentially abundant in chicken male and female PGCs indicated their early sexual identity. Many of the proteins up-accumulated in male PGCs were encoded by genes strongly enriched in the sexual chromosome Z. This suggests that the known lack of dosage compensation of the transcription of Z-linked genes between sexes persists at protein level in PGCs, and that this may be a key factor of their autonomous sex differentiation. Male and female PGCs up-accumulated protein sets were associated with differential biological processes, and contained proteins biologically relevant for male and female germ cell development respectively. This study presents first evidence on early predetermined sex specific cell fate of chicken PGCs that will help to understand their sexual physiological specificities and enable more precise sex-specific adaptation of in vitro culture conditions.

2021-11-03 | PXD022452 | Pride

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