Transcriptome analysis of medaka germ cells and gonadal somatic cells during the gonadal sex differentiation.
ABSTRACT: Microarray analysis was performed using germ cells and gonadal somatic cells isolated by Fluorescence Activated Cell Sorter (FACS) from XX and XY embryos at st.33 and st.35. Medaka germ cells (olvas-EGFP) and somatic cells (sox9b-DsRed) were isolated by FACS. The microarray experiments were done using three independent biological replicates.
Project description:In many metazoans, germ cells are separated from somatic lineages early in development and maintain their identity throughout life. Here we show that a Polycomb group (PcG) component, Enhancer of Zeste [E(z)] H3K27me3-specific methyltransferase, maintains germline identity in Drosophila adult testes. We find excessive early-stage somatic gonadal cells in E(z) mutant testes, which originate from both over-proliferative cyst stem cells and germ cells turning on an early-stage somatic cell marker. Using complementary lineage-tracing experiments in E(z) mutant testes, a portion of excessive early-stage somatic gonadal cells are found to derive from early-stage germ cells, including germline stem cells. Interestingly, knocking down E(z) specifically in somatic cells caused this germline-to-soma change, suggesting a non-cell autonomous role of E(z) to antagonize somatic identity in germ cells. Using fly testis specifically expressing E(z) shmiR RNAi in germ cells by nos promoter driven GAL4>UAS system, ChIPseq with H3K27me3 antibody was performed, where H3K27me3 is only detected in somatic cells.
Project description:With the goal of understanding the epigenetic regulation required for germ cell-specific gene expression, we devised a method of DNA methylation analysis adapted for a small number of developing germ cells. This microarray-based method provides the genome-wide assay of DNA methylation using a sub-nanogram quantity of genomic DNA. Using this technique, we obtained DNA methylation profiles for mouse germ cells in various developmental stages including primordial germ cells (PGC) and for stem cells derived from embryos or germ cells. Cluster analysis of the data revealed that each cell type possesses its own characteristic DNA methylation profile, enabling classification of the cell types. This classification is generally consistent with that based on gene expression profiles except for primordial germ cells, whose genome is globally hypomethylated. Among the differentially methylated sites thus identified, we focused on a group of genomic sequences hypomethylated specifically in germline cells. These hypomethylated sequences tend to be clustered, forming large (10 kb to ~9 Mb) genomic domains. Most of these hypomethylated regions designated here as Large Hypomethylated Domain (LoD) correspond to segmentally duplicated regions that contain gene families showing germ cell-specific expression. These include mouse orthologues of human cancer testis antigen genes. Most LoDs appear to be enriched with H3 lysine 9 dimethylation (H3K9me2), usually regarded as a repressive histone modification. It thus appears t hat such a unique epigenomic state (i.e., DNA hypomethylation with H3K9me2 enrichment) may be a prerequisite for the expression of genes contained in these genomic domains. Gene expression data sets from early developmenatal stage of mouse, cultured cell, and somatic tissue cells.
Project description:To identify the transcripts preferentially expressed in type A spermatogonia in rainbow trout immature testis, we compared the transcriptome between type A spermatogonia and testicular somatic cells by microarry analysis. We used fluorescence-activated cell sorting (FACS) to isolate type A spermatogonia or testicular somatic cells from the fishes carrying the transgene, pvasa-GFP, which expresses GFP in spermatogonia. RNA from type A spermatogonia and from testicular somatic cells were hybridized to a microarray after Cy3 labeling.
Project description:Male fertility and testis function changes with age and so it was sought to determine if these changes are accompanied by changes in gene expression in the spermatogonial cells at the foundation of spermatogenesis A full genome microarray was used to determine if distinct pathways of genes were altered in expression in CD9-positive germ cells with age Testes from young (4 months) and aged (21 months) Sprague Dawley rats were subjected to dissociation to isolate a singel cell suspension of germ cells. These cells were used to isolate CD9 positive spermatogonial cells. RNA was isolated from these cells for hybridization on agilent miroarrays.
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:Expression profiles of XX and XY somatic gonadal compartment during sex determination. This experiment was updated in March 2011 to correct the incorrect age value for sample Female E10.5_6 (was 11 days should be 10.5).
Project description:Male germ cells from young and aged Rats were Isolated and cultured and then treated with pro-oxidant SIN-1 and antioxidant EUK134 Study of the response of isolated male germ cells from young and aged Brown Norway Rats to oxidative stress. Treatment of Isolated and cultured germ cells with pro-oxidant and antioxidant.
Project description:To identify the gene expressing profiles of TIE2 expressing Monocytes(TEMs), we have employed the Agilent lncRNA Gene Expression 4×180K(Design ID:042818) microarray. Human peripheral blood mononuclear cells (PBMCs) in venous blood from healthy donors were isolated by Lymphoprep (Axis-Shield, Norway). Human monocytes in PBMCs, identified as cells that expressed CD14, were enriched by positive immunomagnetic selection using anti-CD14 MicroBeads (Miltenyi, Germany). TEMs (TIE2+CD14+) and TIE2-Monocytes (Tie2-CD14+) were then isolated by FACS-sorting (Aria II, BD Biosciences) using FITC-conjugated anti-CD14 (BD Biosciences, USA) and APC-conjugated anti-TIE2 (R&D System, USA) antibodies.Three TEMs samples together with their paried TIE2-Monocytes were detected.Expressions of sixteen genes (CDKN1A, FDXR, SESN1, BBC3 and PHPT1) from this signature was quantified in the same RNA samples by real-time PCR, confirming low variability between donors as well as the predicted radiation response pattern. The gene expressions of three independent paried TEMs and TIE2- Monocytes samples from different donors were measured.
Project description:The male germ cell differentiation is a subtle and complex regulation processes, currently its regulatory mechanism is not fully understood. In our experiment, we performed the first comprehensive genome wide analyses of the crucial genes in three kinds of crucial cells (ESCs, PGCs and SSCs) associated with the male germ cells differentiation. We identified thousands of differentially expressed genes (DEGs) in this process andwe choosed 173 candidate genes involved in the differentiation and metabolic processes, like GAL9, AMH, PLK1, PSMD7 and so on. Further exploration found that the candidate genes express patterns were the same between in vitro induction experiments and transcriptome results. Our results clues to the mechanistic basis of male germ cell differentiation and provides an important reference for future studies. Gene expression in chicken germ stem cells was measured at different stages of development. Three independent experiments were performed at each stage (ESCs, PGCs and SSCs).
Project description:To identify germ cell- and somatic cell-specific gene expression profiles, we performed expression microarray analysis of the mouse gonads of the Nanos3+/-, Nanos3-/- female and male embryos from E12.5 to E15.5. Biological duplicates were examined at each stage, genotype, and sex for each experiment.