Project description:Prostate organogenesis involves epithelial growth in response to inductive signalling from specialised subsets of mesenchyme. To identify regulators and morphogens active in mesenchyme, we performed transcriptomic analysis using Tag-seq, RNA-seq, and single cell RNA-seq and defined new mesenchymal subsets and markers. We documented transcript expression using Tag-seq and RNA-seq in female rat Ventral Mesenchymal Pad (VMP) as well as adjacent urethra comprised of smooth muscle and peri-urethral mesenchyme. Transcripts enriched in VMP were identified in Tag-seq data from microdissected tissue, and RNA-seq data derived from cell populations and single cells. We identified 400 transcripts as enriched or specific to the VMP using bio-informatic comparisons of Tag-seq and RNA-seq data. Comparison with single cell RNA-seq identified transcripts yielded 45 transcriptscommon to both approaches. Cell subset analysis showed that VMP and adjacent mesenchyme were composed of distinct cell types and that each tissue was comprised of two subgroups. Markers for these subgroups were highly subset specific. Thirteen transcripts were validated by qPCR to confirm cell specific expression in microdissected tissues, as well as expression in neonatal prostate. Immunohistochemical staining demonstrated that Ebf3 and Meis2 showed a restricted expression pattern in VMP condensed mesenchyme. Taken together, we demonstrate that the VMP shows limited cellular heterogeneity and that our high-resolution transcriptomic analysis identified new mesenchymal subset markers associated with prostate organogenesis.
Project description:Prostate organogenesis is regulated by interactions between mesenchymal and epithelial cells, and via androgen signalling though androgen receptors (AR) expressed in mesenchymal cells. However, there is little knowledge of AR target genes and we have used high resolution genomic methods to examine AR function in subsets of mesenchyme during prostate development. Using ChIP-seq, we defined genomic AR binding sites (ARBS) in microdissected mesenchymal tissues of neonatal male and female rats during prostate growth. By comparing female tissue subsets (ventral mesenchymal pad and urethral smooth muscle) with male tissues (ventral prostate and dorsolateral prostate), we were able to identify sexually dimorphic ARBS. Females had a distinct AR binding profile from males with enrichment of ARBS proximal to gene transcriptional start sites as well as binding to non-classical AR binding sequence motifs. Using both tissue RNA-sequencing and single-cell RNA-sequencing of the same mesenchymal subsets, we identified 128 differentially expressed transcripts between males and females. Comparison of these to ChIP-seq ARBS allowed us to define sexually dimorphic AR target genes. Selected candidates were validated as sexually dimorphic at both the mRNA and protein level in both rat and human developing prostate tissues by qPCR and western blot. Response to testosterone stimulation was examined using ex vivo rat tissue organ cultures and qPCR. In single cell RNAseq data we observed subpopulations of mesenchymal cells in males and females as well as subpopulations common to both. The distribution of AR and target genes did not follow the subpopulation distribution, suggesting that AR action is not restricted by subset identity.Our study is the first to apply multiple transcriptomic approaches to prostate mesenchyme, and we have identified unique AR targets. We have identified sexually dimorphic, androgen responsive mesenchymal target genes that may underlie sexually dimorphic development of the prostate. We suggest that the analysis of transcription factor binding and transcriptomes in cell subsets will be informative when applied to tumour cells and stroma – since these show similar cellular heterogeneity and subset specific activity.
Project description:Inflammation is a key component of pathological angiogenesis. Here we induce cornea neovascularisation using sutures placed into the cornea, and sutures are removed to induce a regression phase. We used whole transcriptome microarray to monitor gene expression profies of several genes
Project description:Background: The mesenchymal compartment plays a key role in organogenesis and cells within the mesenchyme/stroma are a source of potent molecules that control epithelia during development and tumourigenesis. We have used Serial Analysis of Gene Expression (SAGE) to profile a key subset of prostatic mesenchyme that regulates prostate development and is enriched for growth-regulatory molecules. Results: SAGE libraries were made of prostatic inductive mesenchyme (VMP) and the complete prostatic rudiment (including inductive mesenchyme, epithelium and smooth muscle; VSU). By comparing these two SAGE libraries we generated a list of 219 transcripts that were enriched or specific to inductive mesenchyme and which may act as mesenchymal regulators of organogenesis and tumourigenesis. Conclusions: The use of a precisely defined subset of cells, and a back-comparison approach, allowed us to identify rare mRNAs that might be overlooked using other approaches. Keywords: SAGE, gene profiling Two SAGE libraries were prepared and compared to each other. The VMP forms a subset of the VSU.