Transcription profiling by array of mouse testis development to compare the expression of mRNAs and noncoding RNAs
ABSTRACT: This experiment compared the expression of mRNAs and noncoding RNAs during the development of mouse testis. The primary aim of the experiment was to identify ncRNAs that were differentially expressed during this process.
Project description:This experiment compared the expression of mRNAs and noncoding RNAs during the development of mouse ovary. The primary aim of the experiment was to identify ncRNAs that were differentially expressed during this process.
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.
Project description:Transcriptional profiling of individual mouse embryonic (e11.5) XY gonads comparing inbred strains that are sensitive (C57BL/6J) and resistant (129S1/SvImJ) to XY sex reversal, and their reciprocal F1 hybrids. Experiment Overall Design: Two-color Agilent microarray profiles of 20 individual pairs of e11.5 XY gonads, including 5- C57BL/6J, 5- 129S1/SvImJ, 5- (B6x129S1)F1, and 5- (129S1xB6)F1 samples. Within each strain, samples were collected from multiple litters to account for potential litter biases. All samples were processed following the same protocol.
Project description:To examine the transcriptome of early testicular somatic cells during gonadogenesis at 12.5dpc RNA sequencing (RNA-Seq) was performed on murine primary testicular cell lineages isolated from the Sf1-eGFP line by FACS. The three main somatic cell lineages of the testis were isolated: the Sertoli cells which direct male development; the fetal Leydig cells (FLCs) that produce steroid hormones and virilise the XY individual and a heterogenous population of interstitial cells, some of which give rise to the adult Leydig cells (ALCs). This dataset provides a platform for exploring the biology of FLCs and understanding the role of these cells in testicular development and masculinization of the embryo, and a basis for targeted studies designed to identify causes of idiopathic XY DSD. RNA-Seq of 3 enriched cell populations from 12.5dpc mouse gonad (Sertoli cells, Leydig cells and Interstitial cells isolated by FACS-sorting) on an Illumina HiSeq 1500, in triplicate.
Project description:For a pathogen such as Streptococcus suis serotype 2, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analysis of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we used analysis of the global expression profile in response to acidic pH in vitro and identified a set of regulated genes involved in diverse cellular processes. 196 (11%) genes were differentially regulated by the acid stress: 92 (47%) were down-regulated at low acid (pH 5.8) relative to the neutral condition (pH 7.2), whereas 104 (53%) were up-regulated at pH 5.8 versus pH 7.2. To confirm the microarray data, 16 genes were measured by quantitative RT-PCR. There was a strong positive correlation (r = 0.96) between the results obtained by microarray and quantitative RT-PCR. The data showed that S. suis S2 is equally capable of inducing an acid tolerance response with maximal protection provided after adaptation at pH 5.8 for survival. A cDNA microarray imprinted with 2156 genes representing about 98% of the Streptococcus suis serotype 2 genome was used for transcriptome analysis. For two-sample (reference vs. test) microarray hybridization, four independent bacterial cultures from each condition (pH 5.8, pH 7.2) were prepared as biological replicates for RNA isolation. Accordingly, four dual-fluorescence-labeled cDNA probes were prepared to hybridize with four slides. Pairwise comparisons were made using dye-swaps to avoid labeling bias. A ratio of mRNA levels (test/reference) was calculated for each gene. Significant changes of gene expression were identified with the SAM software. After the SAM analysis, only genes with at least 2-fold changes in expression were collected for further analysis.
Project description:Testicular cord formation in male gonadogenesis involves assembly of several cell types, the precise molecular mechanism is still not well known.With the high-throughput quantitative proteomics technology, a comparative proteomic profile of mouse embryonic male gonads were analyzed at three time points (11.5, 12.5 and 13.5 days post coitum), corresponding to critical stages of testicular cord formation in gonadal development.
Project description:RNA expression microarray analysis of prospermatogonia in 15 day post-conceptus (dpc) fetuses, a stage when they are undergoing rapid de novo DNA methylation. For comparison, we also analysed 15 dpc pachytene oogonia, 15 dpc female and male gonadal somatic cells, and adult pachytene spermatocytes.
Project description:This series represents murine dorsal neural tube bisected along the midline with one half from each embryo used for control and the other half treated with 10-6M RA dissolved in ethanol for 6, 12, 24 or 48 h. For 6 h exposures, the explants were cultured overnight on fibronectin coated 35mm dishes (Biocoat, Becton Dickinson Labware, Bedford, MA) in DMEM with 10% horse serum in order to allow for sufficient outgrowth of neural crest cells. The RA was added the following morning; RMA Express 0.2 used to initially normalize data; GeneSpring (Silicon Genetics, Inc.) used for subsequent analysis; Samples were analyzed at 6, 12, 24, and 48 hours.
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.