146 UROEPITHELIAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS IS MEDIATED BY RETINOIC ACID AND DEPENDENT ON GATA-4 AND GATA-6 SIGNALING

You have accessJournal of UrologyStem Cell Research1 Apr 2010146 UROEPITHELIAL DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS IS MEDIATED BY RETINOIC ACID AND DEPENDENT ON GATA-4 AND GATA-6 SIGNALING Joshua Mauney, Aruna Ramachandran, Rosalyn Adam, and Carlos Estrada Joshua MauneyJoshua Mauney More articles by this author , Aruna RamachandranAruna Ramachandran More articles by this author , Rosalyn AdamRosalyn Adam More articles by this author , and Carlos EstradaCarlos Estrada More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2010.02.199AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The urinary bladder is lined by the uroepithelium, a transitional epithelium which acts as a specialized permeability barrier that protects the underlying tissue from urine. To date our understanding of the developmental processes responsible for uroepithelial (UE) differentiation has been hampered due to the lack of suitable in vitro cell culture models. In this study, we describe a novel in vitro cell culture system for derivation of UE cells from mouse embryonic stem cells (ESCs) in response to all trans retinoic acid (RA) and demonstrate a role for GATA4/6 signaling mechanisms in this process. METHODS Murine ESCs (C57/BL6) were seeded on 2-D native collagen type I films and cultured for up to 9 days in DMEM/15% fetal calf serum in the presence or absence of RA (0.01-10μM). The extent of ESC differentiation was assessed by quantitative RT-PCR (qPCR), immunocytochemical (ICC), and immunoblot analyses for markers of pluripotency: OCT-4; pan-endoderm: SOX17, FOXA2, GATA4, GATA6; extra-embryonic endoderm: α-fetoprotein (AFP), SOX7, PEM; definitive hindgut endoderm (DHE): p63, CXCR4, HOXA13; and UE lineage: UPs and cytokeratins (CK). Naïve ESCs prior to differentiation were evaluated in parallel as controls. To ascertain the role of GATA4/6 in UE differentiation, GATA4-/- and GATA6-/- null ESCs were subjected to RA stimulation and compared to wild type (WT) controls. In addition, electromobility shift assays (EMSA) were performed on 2kb UP1B and UP2 promoter fragments containing putative GATA binding sites in order to evaluate the recruitment of GATA4 and GATA6 to transcriptional complexes. RESULTS qPCR and ICC analyses demonstrated that RA (10μM) treatment resulted in significant upregulation of all UPs, CK18 and CK20 as well as loss of OCT-4 expression in WT ESCs. In addition, RA enrichment of UP+ populations coincided with peak expression of pan-endoderm and DHE markers as well as downregulation of extra-embryonic endoderm specification. qPCR analysis revealed that RA-treated GATA6-/- ESCs failed to upregulate UP expression while GATA4-/- ESCs displayed significantly attenuated UP levels in comparison to WT controls. Competition and supershift EMSA analysis of WT ESCs revealed specific transcriptional complex formation at each GATA binding site within the murine UP1B and 2 promoters which consisted of both GATA4/6 following RA stimulation. CONCLUSIONS We show for the first time that UE differentiation of ESCs is mediated by RA signaling mechanisms and demonstrate a role for GATA4/6 in this process. Boston, MA© 2010 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 183Issue 4SApril 2010Page: e60 Advertisement Copyright & Permissions© 2010 by American Urological Association Education and Research, Inc.MetricsAuthor Information Joshua Mauney More articles by this author Aruna Ramachandran More articles by this author Rosalyn Adam More articles by this author Carlos Estrada More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...