linking trunk cells to the extracellular environment remain totally unknown. To gain insights into the functions of TROP2, we examined the expression of TROP2 together with EpCAM during mouse development and found that the two differ significantly. During development, EpCAM was widely expressed in epithelial tissues, whereas TROP2 expression was restricted to EpCAM+ cells in a few organs. In particular, a unique pattern of TROP2 expression was found in fetal kidney, i.e., TROP2 expression increases from the tip toward the trunk of the ureteric bud, whereas EpCAM was expressed uniformly. In this study, we show that the expression pattern does not appear to correlate with the known functions of TROP2 in cell proliferation and tight junction formation. Using anti-TROP2 and anti-EpCAM antibodies (Abs), we isolated the tip cells and trunk cells of the ureteric buds, and revealed that their morphology on collagen-gel differs significantly, i.e. tip cells spread, whereas trunk cells remain a rounded shape. We further demonstrate that TROP2 alters cell shape and motility on collagen-gel using a primary culture of ureteric bud cells and that it also inhibits branching of MDCK cells. Our findings suggest that TROP2 plays a role in the development of a ductal structure of ureteric trunks by suppressing the formation of new branches. Results Expression of TROP2 during kidney development We performed an immunohistochemical analysis of mouse embryos by using anti-EpCAM and anti-TROP2 Abs. At embryonic day 14.5 (E14.5), EpCAM was expressed in a variety of epithelial tissues including lung, gut, kidney, pancreas and epidermis. By contrast, TROP2 expression was restricted to a few tissues including kidney, gut and epidermis, and the TROP2+ cells were a subpopulation of EpCAM+ cells (Fig. 1A–C). Because TROP2 was highly expressed in the kidney, we focused on the expression and role of TROP2 in kidney development. In mice, the ureteric bud emerges at E10.5 from the Wolffian duct, which is induced by GDNF from metanephric mesenchyme [18,19]. Subsequently, the ureteric buds are also induced to form the first branch at E11.5 and undergo further branching until E14.5,15.0 by the signals from metanephric mesenchyme. Immunostaining in the fetal kidney at E11.5 revealed that EpCAM was uniformly expressed in the ureteric bud and the Wolffian duct (Figure 2A, arrow and arrowhead, respectively) [20]. Interestingly, TROP2 was expressed at the trunk of the ureteric bud and the Wolffian duct, but its expression was weak at the tip of the ureteric bud that is surrounded by metanephric mesenchyme (Figure 2A, yellow arrowhead). At E14.5, although EpCAM was expressed uniformly throughout the ureteric buds, TROP2 was expressed at the trunk but barely detectable at the tip (Figure 2B, yellow arrowhead). This gradient of TROP2 expression at the ureteric bud suggests TROP2 to be involved in morphogenesis of the ureteric bud, especially in the trunk. Characterization of TROP2+ and EpCAM+ cells in fetal kidney To characterize the cells expressing TROP2 in the ureteric bud, we performed a flow cytometric analysis and revealed that EpCAM+ cells in E12.5 kidney were divided into at least 2 populations, TROP2+ and TROP22 cells (Figure 2C). At E14.5, TROP2 expression was up-regulated, resulting in a broad distribution of TROP2 expression from TROP2low to TROP2high. In addition, the EpCAM+TROP22 cells increased during development, which is consistent with the results of immunostain- ing. These results suggested that the expression profiles of EpCAM TROP2 Defines Branch Formation in Fetal Kidney and TROP2 define the distinct cell types in the developing urinary duct. To characterize E14.5 kidney cells, EpCAM+ cells were + fractionated into three populations by cell sorting, EpCAM - TROP22, EpCAM + TROP2low and EpCAM + TROP2high cells, based on the expression levels of TROP2 (Figure 2D). The tip and trunk cells of the ureteric bud can be distinguished by the expression of specific markers. Quantitative RT-PCR revealed that Ret, Wnt11 and GFRa, tip cell markers [21–24], were highly expressed in EpCAM+TROP2low cells, whereas Wnt9b, a trunk cell marker [25], was strongly expressed in EpCAM + TROP2high cells (Figure 2E). These results indicate that the EpCAM+TROP2low and EpCAM+TROP2high populations include the tip and trunk cells, respectively, and that ureteric bud cells at different locations can be isolated by cell sorting based on TROP2 expression. It has been reported that EpCAM is expressed in the ureteric bud and also in the tissue derived from the metanephric mesenchyme by the mesenchymal-epithelial transition (MET) [20]. We found that EpCAM+TROP22 cells expressed Sall1, a marker of metanephric mesenchymal cells [26](Figure 2E), and that the number of EpCAM + TROP22 cells increased from E12.5 to E14.5, corresponding to the timing of + MET [27](Figure 2C). These results suggest that the EpCAM - TROP22 population is mainly composed of epithelial cells derived from the metanephric mesenchyme. Expression of TROP2 in ureteric buds TROP2 is highly expressed in a variety of carcinomas and involved in cancer cell proliferation [11,28]. In kidney develop- ment, a previous study showed that cell proliferation is restricted mainly to the tip of the ureteric bud in fetal kidney, which corresponds to TROP2low cells [17]. In fact, immunostaining of Ki67, a marker of cell proliferation, showed the expression to be higher in the tip cells than in the trunk cells (Figure S1). This result suggests that, unlike in carcinomas, the TROP2 expression level is not positively correlated with cell proliferation in fetal kidney. Furthermore, in the corneal epithelia, TROP2 enhances the expression and localization of tight junctional proteins, including Claudin-1 and Claudin-7 [12]. However, immunostaining showed that in contrast to the gradient of TROP2 expression, Claudin-7 was uniformly expressed throughout the ureteric bud cells (Figure S2A). Moreover, Claudin-7 was found only in the lateral membrane of the ureteric duct, while TROP2 was located in both the lateral and basal membranes (Figure S2B). These results indicate that expression of TROP2 and Claudin-7 are not fully correlated in the fetal kidney, suggesting that TROP2 is not only involved in the formation of tight junctions in the ureteric bud. The ureteric bud is surrounded by collagen-I in fetal kidney The extracellular matrix (ECM) is important for the growth and branching morphogenesis of embryonic organs [29]. In fetal kidney, the ureteric bud and the collecting duct express low and high levels of collagen-binding integrins a1 and a2, respectively, suggesting collagen to play a role in the differentiation and maturation of the ureteric bud by binding to integrins [30,31]. Because trunk cells in the ureteric bud differentiate to form the collecting duct, we examined the expression of collagen and TROP2 in the developing kidney by immunostaining. In E14.5 kidney, type I collagen (collagen-I) was abundant surrounding the TROP2+ trunks as well as in the mesenchyme and stroma around the ureteric bud trunk (Figure 3A). By contrast, little collagen-I was detected surrounding EpCAM+ tip cells (Figure S3). Integrins are major cell-surface receptors that mediate interac- tions between cells and the ECM and play a crucial role in various PLoS ONE | www.plosone.org 2 December 2011 | Volume 6 | Issue 12 | e28607