TROP2 Defines Branch Formation in Fetal Kidney Figure 6. Branch formation in collagen-gel culture of MDCK cells. (A) Phase-contrast images of MDCK-control cells and MDCK-TROP2 cells cultured in 3-dimensional collagen gels. The cells were cultured without (upper panels) or with 20 ng/mL of HGF (lower panels). Both cells proliferated and formed round cysts without HGF (upper panels). Branches were formed in MDCK-control cells but suppressed in MDCK-TROP2 cells (lower panels). Scale bars = 100 mm. (B) The percentage of branching cysts in the culture with HGF. (n = 3). Error bars, s.d. * p,0.05. doi:10.1371/journal.pone.0028607.g006 Branching morphogenesis is regulated by not only the ECM but also growth factor signaling. In collagen-gel cultures of MDCK cells, a well-established model of branch formation, growth factors such as HGF and GDNF are necessary for branching [18,38]. Studies with fetal kidney organ cultures and knockout mice have revealed the essential roles for many growth factors in branching morphogenesis. In response to growth factors, tip cells proliferate, migrate and finally form new branches, whereas trunk cells exhibit cell polarization, elongation and anti-apoptotic activity without branching [39–41]. In this paper we showed that TROP2 inhibits not only the attachment of cells onto collagen-coated plates but also branching in collagen-gel cultures. These results strongly suggest that TROP2 suppresses the formation of new branches at the trunk of the ureteric bud. Focal adhesion kinase (Fak) is phosphorylated by the activation of integrins through binding to ECM and also growth factor stimulation [42]. p-Fak promotes cell extension and migration through actin polymerization and the connection of F-actin to integrin and growth factor receptors at the edge of cells, contributing to branching. In cell spreading assays, the phosphorylation level of Fak in MDCK-TROP2 cells was declined (Figure S6A), suggesting that TROP2 may inhibit the Fak signaling pathway at the ureteric trunk to avoid extra branching. While immunostaining showed that p-FAK levels are comparable in the tip and trunk cells (Figure S6B), it may be difficult to capture the transient event in static sections. TROP2 and EpCAM are expressed in normal epithelial tissues, while they are known as oncogene and promote cancer cell proliferation and migration. EpCAM up-regulates cell motility by binding to several membrane proteins, Claudins, CD44 and tetraspanins, and a-actinin, a linker of F-actins [8,43]. Furthermore, in cancer cells, intracellular domain of EpCAM cleaved by TACE and presenilin-2 accelerates cell proliferation through the activation of b-catenin signaling [9]. By contrast, the molecular mechanisms of TROP2 in normal and cancer tissue are not known. Immunopre- cipitation assays showed that TROP2 binds EpCAM (data not shown), suggesting the possibility that TROP2 interacts with EpCAM and regulates the cell proliferation and migration activity. In ureteric bud development, TROP2 expression shows a gradient pattern in contrast to uniform expression of EpCAM. Furthermore, the TROP2 expression declines toward tip where cells are actively proliferating and migrating, suggesting that TROP2 is involved in regulation of cell proliferation, possibly via interaction with other proteins like EpCAM. Thus, the difference in expression profiles of TROP2-interacting proteins between normal and cancer cells may modulate the function of TROP2. The precise role of the TROP2 in normal and cancer tissue needs further investigation. In the lung, which also has a tree-like structure, branching morphogenesis is induced by the interaction between mesenchyme and epithelial cells, similar to the ureteric bud [44]. However, TROP2 is not expressed in the lung (Figure 1A) and the branching pattern is clearly different from the ureteric buds in the early stages. Lung epithelial cells generate several lateral branches from trunks. By contrast, the ureteric bud generates two epithelial tips after the first branching and repeats this dichotomous branching process, and trunks form a rigid duct structure by establishing tight cell-cell junctions and suppressing the formation of new branches [45]. Specific expression of TROP2 in the kidney may result in the difference in branching patterns between the lung and kidney. In conclusion, this study revealed a unique pattern of TROP2 expression in the ureteric bud, i.e., the expression was strongest in the trunk and gradually declined toward the tip. TROP2 and collagen-I were co-localized to the basal membrane of the trunk cells and TROP2 suppressed cell spreading and migration on collagen-I. Based on these findings, we propose that TROP2 plays a role in the morphogenesis of the ureteric bud by inhibiting unnecessary branching from the trunk. Further studies on the function of TROP2 forming a specific pattern of tubular structures should shed light on the molecular basis of branching in various organs. Materials and Methods Ethics Statement The mice were maintained and mated in the institutional animal facility according to the guidelines of the University of Tokyo. The experimental procedures in this study were approved by the Animal Research Committee of the Institute of Molecular and Cellular Biosciences, the University of Tokyo (approval number is 23003). Immunohistochemistry Mouse embryonic kidneys were fixed for 16 h at 4uC in Zamboni’s solution [46], which was replaced with a 10, 15 and PLoS ONE | www.plosone.org 8 December 2011 | Volume 6 | Issue 12 | e28607