Expression profiling of 3D 2D MDCKII epithelial morphogenesis
ABSTRACT: Formation of epithelial tissues requires the generation of apical-basal polarity and the co-ordination of this polarity between neighboring cells to form a central lumen. MDCK cell line has proven to be a powerful model to study mammalian polarized epithelia in vitro. MDCK cells plated in extracellular matrix (ECM) form cysts, a spherical structure of polarized cells enclosing a central lumen which resembles epithelial tubular structures. The morphogenetic process requires drastic changes in cell architecture, which are regulated by change in gene expression. We used microarrays to identify genes up-regulated in lumen formation. The identification of up-regulated genes could lead us to characterize novel pathways needed for this process. MDCKII cells were plated in two different conditions: Cells cultured in confluence in plastic dishes, forming polarized monolayers (2D); or cells cultured in plastic dishes covered with Matrigel (ECM) forming three dimensional cysts (3D). Comparison of both transcriptomic profiles would lead us to identify up-regulated genes in the 3D condition, which would be good candidates to be key regulators of novel processes involved in lumen morphogenesis.
Project description:Differentiation of monolayered epithelia is characterized by the formation of a basoapical polarity axis, except during the early stages of cancer development. Using mammary glandular structures (acini) produced in a three-dimensional cell culture system we have demonstrated that, in order for mammary epithelial cells to exit quiescence and enter the cell cycle, acini have to lose apical polarity. In order to identify the genes dependent on apical polarity that could control cell quiescence, and possibly other aspects of tissue homeostasis, we have used Affymetrix technology microarray analysis of the 22,277 features/genes of the Human Genome U133A 2.0 array Chip in apically polarized and non-polarized breast epithelial acinar cells in three-dimensional culture. Genes commonly down-regulated in two treatments that altered apical polarity compared to control were considered to be dependent on apical polarity status for their transcription. Apically polarized (Control) and two different populations of non-polarized human mammary epithelial cells (18-alpha-glycyrrhetinic acid =AGA, and 5-aza-2'-deoxycytidine =Aza) were used for microarray analysis containing four biological replicates (1-4) for each treatment. The two treatments that cause loss of apical polarity were compared to reference apically polarized control samples using two different statistical analysis methods (FDR and Holm’s).
Project description:To gain more information on the expression of microRNAs during EMT induced by oncogenic Ras in MDCK cells, we performed deep sequencing (Illumina HiSeq2000) of MDCK and MDCK-Ras cells and compared their genome-wide microRNA expression profiles. RNA-Seq data from the same study has also been deposited at ArrayExpress under accession number E-MTAB-3301 (https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-3301/).
Project description:In order to gain insight into epithelial morphogenesis and the influence of culture geometry on gene expression patterns, Madin Darby Canine Kidney (MDCK) epithelial cells where grown in 2-dimensional (2D) culture or 3-dimensional (3D) culture . MDCK cells cultured in 2D were plated atop a pre-solidified type I collagen gel. Cells cultured under these conditions grew as flat monolayer sheets. In 3D culture, cells are embedded within type I collagen gel. Cells grown under these conditions form large spherical cysts with hollow central lumens. We anticipate, therefore, that these results provide insight into the mechanisms that regulate epithelial cystogenesis. Cells grown in the 2D or 3D geometries were collected from digested type I collagen gels on day 8. The 2D MDCK cells were treated as the control condition and there gene expression patterns were compared to those of 3D grown cells, which served as the experimental condition.
Project description:Gene expression in Madin Darby canine kidney cells grown for 7 days to confluence on Transwell filters and exposed to HGF +/- inhibitors of the MAPK pathway (MAPK is one of the pathways activated when HGF binds to the CMET receptor tyrosine kinase). We used microarrays to detail the program of gene expression in MDCK cells and identified genes specifically regulated by HGF via the MAPK pathway. Keywords: signaling pathway analysis MDCK cells were selected for RNA extraction and hybridization on Affymetrix microarrays. We sought to obtain expression of genes regulated by the MAPK pathway following HGF induction.
Project description:Expression profile analysis in MDCK-E47 cells in comparisom with MDCK CMV control cells Keywords: Genetic Modification (overexpression of E-cadherin repressors) The Oncochip microarray platform v1.1 contains 9,726 clones corresponding to 6,386 different genes, and it includes 2,489 duplicate clones. Duplicate samples (different extrations of RNA) were labeled with dUTP-Cy5 and hybridized against dUTP-Cy3-MDCK control cells. In addittion, one RNA extraction was labelled with dUTP-Cy3 and was hybridized againts dUTP-Cy5- CMV control cells
Project description:Loss of expression of the cell-cell adhesion molecule E-cadherin is an essential event for epithelial-mesenchymal transition (EMT), a process that allows cell migration in the developing embryo and during tumour invasion. Transcriptional repression has emerged as the main mechanism responsible for E-cadherin downregulation in most carcinomas. Recently, we have identified the class I HLH transcriptional regulator E2-2 (ITF2), in a yeast one hybrid screen designed to identify transcriptional repressors interacting with the E-pal element of the murine E-cadherin promoter. The E2-2 gene codifies two isoforms that differ in their N-terminal regions but their specific functions remain unknown. In the present work we show that both E2-2A and E2-2B induce a complete EMT, in MDCK cells, with loss of E-cadherin expression, gain of mesenchymal markers and acquisition of motile and invasive properties. Although both isoforms repress E-cadherin promoter in MDCK cells, only the E2-2B isoform does it in other epithelial cell lines. Notably, we found that E2-2B is upregulated at the mRNA level in MDCK cells after treatment with TGF-1, a key regulator of EMT. Upregulation of E2-2A/B factors was confirmed in MDCK cells overexpressing other EMT inducers, Snai1, Snai2 or E47. Interestingly, gene profiling studies indicate that bHLH E2-2 factors induce similar, yet distinct, genetic programs from those induced by bHLH E47 in MDCK cells. These results, together with the expression pattern observed in early mouse embryos, support a new role for E2-2A/B in E-cadherin regulation and EMT, and suggest an interesting interplay between bHLH factors and other E-cadherin repressors. Keywords: Genetic Modification (overexpression of E-cadherin repressors) The Oncochip microarray platform v2.0 contains 13,824 clones printed by duplicate corresponding to 9,300 different genes. Each of the MDCK transfectants (E22A, E22B) were labeled with dUTP-Cy5 and hybridized against the dUTP-Cy3-labeled MDCK-CMV controls. One additional hybridizations using reciprocal fluorochrome labeling were performed (dye-swap) in each clone. Thus, a total of two hybridizations were performed for each condition.
Project description:Two biological replicates of Madin-Darby Canine Kidney Epithelial Cells grown as 3D cysts in Collagen Type I (7 days old) were exposed to six different concentrations of Hepatocyte Growth Factor (HGF) (0, 1.03, 2.07, 4.15, 8.33 and 16.67 ng/ml). Total RNA was isolated from the cysts after 12 hours of HGF induction. The data submitted here are the raw sequence files of the single read lengths of 50 bp for the 12 samples (2 replicates X 6 conditions) after RNA sequencing experiment using Illumina HiSeq 2000.15
Project description:Our group is interested in epithelial-to-mesenchymal transition (EMT), in particular, TGF-beta induced EMT. TGF-beta signalling has been shown to be an important factor in the induction of EMT and it has been demonstrated that adding TGF-beta to epithelial cells in culture is a convenient way to study the process of EMT. “In response to TGF-beta, Smad2 and 3 are activated, and form complexes with Smad4, which then regulate transcription of target genes through interactions with other DNA binding transcription factors. In the induction of EMT, the activated Smads mediate transcriptional regulation through three families of transcription factors, resulting in repression of epithelial marker gene expression and activation of mesenchymal gene expression” (Xu J, et al. 2009) <br></br> Also investigated in this study is the role of H2A.Z in EMT. H2A.Z is an evolutionary conserved and a metazoan essential histone variant of the H2A class. Mice deficient in H2A.Z die during early development but the reason for this is unknown (Faast et al. 2001). Previously, our laboratory showed that the loss of H2A.Z in Xenpous laevis impaired cell movement required for the formation of the mesoderm and neural crest (Ridgway et al. 2004). Given that mesoderm formation is critically dependent upon EMT, we therefore wondered whether H2A.Z might be a chromatin regulator of EMT. We transfected MDCK cells with a lentiviral vector to express a construct encoding an shRNA targeting canine H2A.Z as we wanted to test the hypothesis that H2A.Z is involved in the maintenance of cellular identity and that its loss might trigger de-differentiation. <br></br> In order to investigate changes in gene expression associated with TGF-beta induced epithelial-to-mesenchymal transition (EMT) we performed paired end RNA-Seq of poly-A selected mRNA in untreated and TGFb-treated MDCK cells. The MDCK cell line has been extensively used as a model system for EMT because they convert fully from the epithelial to the mesenchymal state in response to TGF-beta. Gene expression profiles were also generated from MDCK cells in which H2A.Z was knocked down using shRNA.<br></br>Please note that ChIP-seq data generated in conjunction to this RNA-seq data set were also deposited at ArrayExpress under accession number E-MTAB-5637 ( https://www.ebi.ac.uk/arrayexpress/experiments/E-MTAB-5637 ).
Project description:In order to gain insight into epithelial morphogenesis and the influence of culture geometry on gene expression patterns, Madin Darby Canine Kidney (MDCK) epithelial cells where grown in 2-dimensional (2D) culture or 3-dimensional (3D) culture . MDCK cells cultured in 2D were plated atop a pre-solidified type I collagen gel. Cells cultured under these conditions grew as flat monolayer sheets. In 3D culture, cells are embedded within type I collagen gel. Cells grown under these conditions form large spherical cysts with hollow central lumens. We anticipate, therefore, that these results provide insight into the mechanisms that regulate epithelial cystogenesis. Overall design: Cells grown in the 2D or 3D geometries were collected from digested type I collagen gels on day 8. The 2D MDCK cells were treated as the control condition and there gene expression patterns were compared to those of 3D grown cells, which served as the experimental condition.