TROP2 Expressed in the Trunk of the Ureteric Duct Regulates Branching Morphogenesis during Kidney Development Yuko Tsukahara, Minoru Tanaka*, Atsushi Miyajima* Institute of Molecular and Cellular Biosciences, The University of Tokyo, Tokyo, Japan Abstract TROP2, a cell surface protein structurally related to EpCAM, is expressed in various carcinomas, though its function remains largely unknown. We examined the expression of TROP2 and EpCAM in fetal mouse tissues, and found distinct patterns in the ureteric bud of the fetal kidney, which forms a tree-like structure. The tip cells in the ureteric bud proliferate to form branches, whereas the trunk cells differentiate to form a polarized ductal structure. EpCAM was expressed throughout the ureteric bud, whereas TROP2 expression was strongest at the trunk but diminished towards the tips, indicating the distinct cell populations in the ureteric bud. The cells highly expressing TROP2 (TROP2high) were negative for Ki67, a proliferating cell marker, and TROP2 and collagen-I were co-localized to the basal membrane of the trunk cells. TROP2high cells isolated from the fetal kidney failed to attach and spread on collagen-coated plates. Using MDCK cells, a well-established model for studying the branching morphogenesis of the ureteric bud, TROP2 was shown to inhibit cell spreading and motility on collagen-coated plates, and also branching in collagen-gel cultures, which mimic the ureteric bud’s microenvironment. These results together suggest that TROP2 modulates the interaction between the cells and matrix and regulates the formation of the ureteric duct by suppressing branching from the trunk during kidney development. Citation: Tsukahara Y, Tanaka M, Miyajima A (2011) TROP2 Expressed in the Trunk of the Ureteric Duct Regulates Branching Morphogenesis during Kidney Development. PLoS ONE 6(12): e28607. doi:10.1371/journal.pone.0028607 Editor: Tsutomu Kume, Feinberg Cardiovascular Research Institute, Northwestern University, United States of America Received August 1, 2011; Accepted November 11, 2011; Published December 14, 2011 Copyright: 2011 Tsukahara et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by research grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, Ministry of Health, Labour and Welfare of Japan, the Core Research for Evolutional Science and Technology (CREST) program from the Japan Science and Technology Agency and Takeda Science Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: tanaka@iam.u-tokyo.ac.jp (MT); miyajima@iam.u-tokyo.ac.jp (AM) Introduction TROP2, also known as EGP-1, M1S1, GA733-1, and TACSTD2, is a 36-kDa membrane glycoprotein, initially identified as a tumor-associated calcium signal transducer (TACSTD) expressed in gastrointestinal, bladder, lung, and cervix carcinomas and later found to be highly expressed in many other tumors including pancreatic cancers and squamous cell carcinomas of the oral cavity [1]. TROP2 is strongly expressed in trophoblasts forming the outer layer of the blastocyst which develops into the placenta [2]. In the prostate, TROP2 is specifically expressed in prostate stem cells located in the basal region [3]. We have recently found that TROP2 is expressed in hepatic progenitor cells known as oval cells, which are produced in response to severe liver injury and form duct-like structures surrounding the portal vein [4]. TROP2 is structurally related to epithelial cell adhesion molecule (EpCAM), also known as TACSTD1. Both TROP2 and EpCAM are type I transmembrane proteins, consisting of a hydrophobic leader peptide, a thyroglobulin-like domain, a single transmembrane domain and a short cytoplasmic domain. The cytoplasmic tail of TROP2, but not EpCAM, contains a region homologous to phosphatidylinositol 4,5-bisphosphate (PIP2)-bind- ing motif [5]. EpCAM is widely expressed in epithelial cells and plays roles in cell-cell adhesion, cell proliferation and cell motility [6–10]. On the other hand, TROP2 was suggested to enhance the proliferation of tumor cells in an anchorage-independent manner [11] and the formation of tight junctions by interacting with Claudin-1 and -7 in the corneal epithelium [12]. However, TROP2 expression in normal tissues and its functions still remain largely unstudied. In the development of the mammalian kidney, the ureteric bud epithelium proliferates and branches to form a tree-like structure [13,14]. This process is known as branching morphogenesis and depends on interactions of the ureteric bud with the metanephric mesenchyme and also components of the extracellular matrix (ECM). Factors secreted from the metanephric mesenchyme enhance the proliferation, elongation and branching of the ureteric bud cells, resulting in differentiation to form the collecting duct system. On the other hand, ECM components around the ureteric bud modulate growth factor signaling and provide an anchorage for cell migration via the ECM-specific integrin receptors on the ureteric bud cells [15,16]. The cells forming the tip and trunk of the ureteric bud exhibit different characteristics [17]. The tip cells are proliferating immature cells that are located at the branching points and interact with the metanephric mesenchyme. By contrast, the trunk cells are differentiating into the collecting duct with a rigid structure consisting of polarized epithelial cells. While the molecular mechanisms underlying the establishment of the tip structure have been well studied, little is known about the mechanisms of trunk morphogenesis. In particular, the factors PLoS ONE | www.plosone.org 1 December 2011 | Volume 6 | Issue 12 | e28607