RNA-SEQ analysis of the developing lower urogenital region
ABSTRACT: We will use micro-dissection and FACS techniques to isolate cell types from the developing lower urogenital region and perform a transcriptional analysis using RNA-SEQ. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. Total RNA was obtained from FACS isolated embryonic day 17.5 bladders using Tg(Upk2-cre)6Xrw x Gt(ROSA)26Sortm4(ACTB-tdTomato,-EGFP)Luo transgenic mice. The long term goal is to generate a transcriptional atlas of developing lower urogenital region.
Project description:Developmental exposure of mouse fetuses to estrogens results in dose-dependent permanent effects on prostate morphology and function. Fetal prostatic mesenchyme cells express estrogen receptor alpha (ERα) and androgen receptors and convert stimuli from estrogens and androgens into signaling to regulate epithelial cell proliferation and differentiation. To obtain mechanistic insight into the role of different doses of estradiol (E2) in regulating mesenchymal cells, we examined E2-induced transcriptomal changes in primary cultures of fetal mouse prostate mesenchymal cells. Urogenital sinus mesenchyme cells were obtained from male mouse fetuses at gestation day 17 and exposed to 10 pM, 100 pM or 100 nM E2 in the presence of a physiological concentration of dihydrotestosterone (0.69 nM) for four days. Gene ontology studies suggested that low doses of E2 (10 pM and 100 pM) induce genes involved in cell adhesion, morphological tissue development, and sterol biosynthesis but suppress genes involved in growth factor signaling and cell adhesion. Genes showing inverted-U-shape dose responses (enhanced by E2 at 10 pM E2 but suppressed at 100 pM) were identified, and their enrichment in the glycolytic pathway was demonstrated. At the highest dose (100 nM), E2 induced genes enriched not only for cell adhesion but also steroid hormone signaling and metabolism, cytokines and their receptors, cell-to-cell communication, Wnt signaling, and TGF-β signaling. These results suggest that prostate mesenchymal cells may regulate epithelial cells through direct cell contacts when estrogen level is low whereas soluble growth factors might play significant roles when estrogen level is high. Primary culture urogenital sinus mesenchymal cells were isolated from prostate glands of gestation day 17 CD1 male mouse fetuses. Cells were then exposed to 10 pM or 100 pM 17beta-estradiol or vehicle (0.05% ethanol) for four days in the presence of 690 pM 5alpha-dihydrotestosterone. Total cellular RNA was then isolated for determination of transcriptomal profiles by Affymetrix mouse gene 1.0 ST array.
Project description:We used micro-dissection techniques and/or FACS to isolate cell types from the developing and adult kidney (E11.5 ureteric buds, E12.5, P1 and P4 cap mesenchyme, E15.5 collecting ducts, proximal tubules, ureter, Adult renal proximal tubules, podocytes, endothelial and mesangial cells). RNA-SEQ analysis was performed to determine the transcriptional profile of each cell type, identify component specific transcripts and isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. Total RNA is obtained from micro-dissected and/or FACS isolated embryonic and adult kidney components. The long term goal is to generate a transcriptional atlas of developing kidney.
Project description:Analysis of transcriptome of tissue recombinants (mouse seminal vesicle epithelial [SVE] cells or prostate epithelial [PE] cells, and rat urogenital sinus [UGS] mesenchymal cells) grown under the kidney capsule in athymic nude mice for 3 months. Total RNA obtained from tissue recombinants generated from combining engineered mouse epithelial cells (SVE or PE from 2-month-old C57Bl/6J mice) and rat UGS mesenchymal cells. Tissue recombinants were harvested and processed for RNA isolation and transcriptome analysis using the RNeasy kit (Qiagen).
Project description:We isolated fetal murine urogenital sinus epithelium and urogenital sinus mesenchyme and determined their global gene expression profiles to define their differentially expressed regulators. To distinguish gene expression patterns that are shared by other developing epithelial/mesenchymal compartments in the embryo from those that pertain to the prostate stem cell niche, we also determine the global gene expression of epidermis and dermis of the same embryos. We identified a distinctive core of transcripts that were differentially regulated in the prostate stem cell niche. Our analysis indicates that several of the key transcriptional components that are likely to be active in the embryonic prostate stem cell niche regulate processes such as self-renewal (e.g., E2f and Ap2), lipid metabolism (e.g., Serbp1) and cell migration (e.g., Areb6 and Rreb1). Several of the promoter binding motifs that are enriched in the profiles are shared between the prostate epithelial/mesenchymal compartments and their epidermis/dermis counterparts, indicating their likely relevance in epithelial/mesenchymal signaling in primitive cellular compartments. We also focused on defining ligand-receptor interactions that may be relevant in controlling signals in the stem cell niche and identified the Wnt/beta-catenin, ephrin, Notch, sonic hedgehog, FGF, TGF-beta and bone morphogenic signaling pathways as being of likely relevance in the prostate stem cell niches. Members of the integrins family including those that bind extracellular matrix proteins such as laminin and activate latent TGF-beta are also expressed in the prostate niche.development. Keywords: Differential gene expression Six biological replicate experiments were performed for UGE. Five biological replicate experiments were performed for UGM. Four biological replicate experiments were performed for Epidermis. Four biological replicate experiments were performed for Dermis.
Project description:Induced pluripotent stem (iPS) cells are a valuable resource for discovery of epigenetic changes critical to cell type-specific differentiation. Although iPS cells have been generated from other terminally differentiated cells, the reprogramming of normal adult human basal prostatic epithelial (E-PZ) cells to a pluripotent state has not been reported. Here, we attempted to reprogram E-PZ cells by forced expression of Oct4, Sox2, c-Myc, and Klf4 using lentiviral vectors and obtained embryonic stem cell (ESC)-like colonies at a frequency of 0.01%. These E-PZ-iPS-like cells with normal karyotype gained expression of pluripotent genes typical of iPS cells (Tra-1-81, SSEA-3, Nanog, Sox2, and Oct4) and lost gene expression characteristic of basal prostatic epithelial cells (CK5, CK14, and p63). E-PZ-iPS-like cells demonstrated pluripotency by differentiating into ectodermal, mesodermal, and endodermal cells in vitro, although lack of teratoma formation in vivo and incomplete demethylation of pluripotency genes suggested only partial reprogramming. Importantly, E-PZ-iPS-like cells re-expressed basal epithelial cell markers (CD44, p63, MAO-A) in response to prostate-specific medium in spheroid culture. Androgen induced expression of androgen receptor (AR), and co-culture with rat urogenital sinus further induced expression of prostate-specific antigen, a hallmark of secretory cells, suggesting that E-PZ-iPS-like cells have the capacity to differentiate into prostatic basal and secretory epithelial cells. Finally, when injected into mice, E-PZ-iPS-like cells expressed basal epithelial cell markers including CD44 and p63. When co-injected with rat urogenital mesenchyme, E-PZ-iPS-like cells expressed AR and expression of p63 and CD44 was repressed. DNA methylation profiling identified key pathways and regulators of prostatic differentiation including Wnt5a, BPM7, PTEN, and NKx3.1 using E-PZ-iPS-lke cells. Our results suggest that iPS-like cells can be derived from prostatic epithelial cells. These cells are pluripotent and capable of prostatic differentiation, and therefore provide a novel model for investigating epigenetic changes involved in prostate cell lineage specification. Bisulphite converted DNA from the 10 samples were hybridised to the Illumina Infinium HumanMethylation450K Beadchip v1.2
Project description:Cancer cells differentiate along specific lineages that largely determine their clinical and biologic behavior. Distinct cancer phenotypes from different cells and organs likely result from unique gene expression repertoires established during lineage commitment in the embryo and maintained after malignant transformation. We used comprehensive gene expression analysis to examine this concept in the prostate, an organ with a readily manipulable developmental program and a high propensity for cancer. We focused on gene expression changes in the murine prostate rudiment (Urogenital Sinus, UGS) at three time points during the first 48 hours of exposure to androgen, which initiates proliferation and invasion of prostate epithelial buds into surrounding mesenchyme. Keywords: time course, prostate gland development Differential gene expression was assessed by direct comparisons of labeled moieties, using dye-swaps as technical replicates. Hybridizations were performed on the Agilent (Santa Clara, CA) Whole Mouse Genome DNA microarray (mgug4122a). Three different time points were analyzed using RNA pooled from different embryos: • UGS of female embryos upon 6 hours of androgen treatment compared to UGS of vehicletreated mice (H6 comparison, 8 different slides); • UGS of female embryos upon 12 hours of androgen treatment compared to UGS of vehicletreated mice (H12 comparison, 6 different slides); • UGS of male embryos compared to UGS of female embryos (MvsF comparison, 10 different slides). Pools from male tissues were hybridized against pools from female litter-mates;
Project description:We used micro-dissection with FACS sorting techniques to isolate renal vesicle single cell types from post natal (P4) kidneys. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. Kidneys are harvested from Tg(Crym-EGFP)GF82Gsat mice. Single cells are extracted from P4 renal vesicles using micro-dissection with FACS sorting techniques. A subset of these cells is analyzed individually via Fluidigm single cell analysis. The long term goal is to generate a transcriptional atlas of the developing kidney.
Project description:We used micro-dissection and trypsinization techniques to isolate single cells from the E12.5 total kidney. A subset of these single cell populations is analysed individually via Fluidigm single cell analysis. This analysis will determine the transcriptional profile of each cell type, identify compartment specific transcripts, compartment specific transcript isoforms and cell-type specific long-noncoding RNAs. In addition the unbiased nature of RNA-SEQ will potentially identify novel transcripts that have not been annotated in the database. E12.5 kidneys are dissected; the kidneys are made into a single cell suspension via trypsinization. A subset of these cells is analysed individually via Fluidigm C1 single cell analysis. The long term goal is to generate a single cell resolution transcriptional atlas of the developing kidney.
Project description:Prostate cancer is one of the most common malignancies and the second leading cause of death from cancer in men. The molecular mechanisms driving prostate carcinogenesis are complex; with several lines of evidence suggesting that the re-expression of conserved developmental programs play a key role. Through conditional gene targeting and organ grafting, we describe conserved roles for the transcription factor Sox9 in the initiation of both prostate organogenesis and prostate carcinogenesis in murine models. Abrogation of Sox9 expression prior to the initiation of androgen signaling blocks the initiation of prostate development. Similarly, Sox9 deletion in two genetic models of prostate cancer (TRAMP and Hi-Myc) blocks cancer initiation. Expression profiling of Sox9-null prostate epithelial cells reveals that the role of Sox9 in the initiation of prostate development may relate to its regulation of multiple cytokeratins and/or calcium-related proteins. Due to its essential role in cancer initiation, manipulation of Sox9 targets in at-risk men may prove useful in the chemoprevention of prostate cancer. Sox9 differential gene expression in prostaspheres derived from the urogenital sinus epithelium was assessed by tow-colors direct comparisons of labeled moieties. Hybridizations were performed on the Agilent (Santa Clara, CA) Whole Mouse Genome DNA microarray (mgug4122a). Sox9 targets were assessed in murine prostate epithelial cells with targeted deletion.