ABSTRACT: Gene expression profiles of detrusor, stroma and urothelium isolated from SMGA (Actg2) transgenic neonatal bladders using LCM. (GUDMAP Series ID: 20)
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing bladder. The central thesis is straightforward. The combination of microdissected and laser capture microdissection (LCM) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing urogenital system. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. The data submitted here represents the gene expression profiles of compartmental bladder tissues collected through laser capture microscopy. Experiment Overall Design: Bladders were isolated from newborn SMGA/EGFP transgenic mice, embedded in OCT, frozen and sectioned (8 microns). Detrusor, stroma and urothelium were isloated using laser capture microscopy. The caps were frozen on dry ice and stored at -80 degrees C until RNA was extracted for gene expression analysis. Laser captured and total RNA isolated for gene expression analysis using the Affymetrix MOE430 microarray chip.
Project description:Kruppel-like transcription factor 5 (Klf5) is expressed during late embryogenesis in the forming murine bladder urothelium. Targeted disruption of the Klf5flox alleles by the ShhGfpCre transgene resulted in failure of the bladder urothelium to mature accompanied by hydronephrosis, hydroureter, and vesicoureteric reflux in all E18.5 fetuses. The bladder urothelium did not stratify nor did it express terminal differentiation markers characteristic of basal, intermediate, and umbrella cells including keratins 20, 14, and 5, and uroplakins. At E18.5, an ectopic alpha smooth muscle actin positive layer of cells was identified subjacent to the undifferentiated Klf5-deficient urothelium. The effects of Klf5 deficiency were unique to the urothelium since maturation of the epithelium comprising the bladder neck and urethra were unaffected by the lack of KLF5. mRNA microarray analysis of whole E14.5 control and Klf5 deficient bladders identified Ppar? and Grhl3 as putative downstream intermediary transcription factors that regulate urothelial maturation. Transient transfection assays demonstrated that KLF5 regulated expression of the mGrhl3 promoter. These observations show that alterations in maturation of the bladder urothelium alone are sufficient to induce bladder dysfunction leading to prenatal hydronephrosis. Total RNA from E14.5 whole bladders isolated from Klf5D/D and Klf5+/+ShhGfpCre+ embryos (n=3 samples/genotype, each sample a pool of two bladders) was isolated using the Qiagen MicroRNA Kit. The cDNA was then hybridized according to the manufacturer’s protocol to the Affymetrix Mouse Gene 1.0 ST Array
Project description:The long term objective is to create an encyclopedia of the expression levels of all genes in multiple components of the developing bladder. The central thesis is straightforward. The combination of laser capture microdissection (LCM) plus microarray analysis offers a powerful, efficient and effective method for the creation of a global gene expression atlas of the developing bladder. Microarrays with essentially complete genome coverage can be used to quantitate expression levels of every gene in laser capture microdissected components of the developing bladder. The ensuing rapid read-out provides an expression atlas that is more sensitive, more economical and more complete than would be possible by in situ hybridizations alone. As a first step we sought to determine if expression of the reporter gene (EGFP or EYFP) used to identify smooth muscle cells altered the gene expression profile of the bladder. Keywords: Comparison of postnatal day 1 bladders. Postnatal day 1 bladders were isolated from transgenic and wild-type mice to determine the effects of transgene expression on the gene expression profile in the bladder. Details of the GUDMAP project can be found at http://www.gudmap.org/index.html