TROP2 Defines Branch Formation in Fetal Kidney Figure 1. Expression of TROP2 and EpCAM in mouse at E14.5. (A–C) Immunostaining of E14.5 mouse frozen sections with anti-EpCAM (green) and anti-TROP2 (red) Abs. EpCAM was expressed in epithelial cells of various organs. By contrast, TROP2 expression was restricted to EpCAM+ cells in the kidney, gut and epidermis. L ; Lung, Lv ; Liver, P ; Pancreas, G ; Gut, K ; Kidney, Epi ; Epidermis, D ; Dermis, Scale bars = 200 mm. doi:10.1371/journal.pone.0028607.g001 cellular functions such as adhesion, migration, proliferation and anti-apoptosis [32,33]. Integrins consist of a and b subunits, with the extracellular domain of the a subunit conferring their specificity. To compare the expression of collagen-binding integrins such as integrin a1b1 and integrin a2b1 between the trunk and tip cells in the ureteric bud, we performed a quantitative + RT-PCR analysis of integrins using the sorted EpCAM TRO- P2low and EpCAM+TROP2high cells from E14.5 kidney (Figure 3B). There was no significant difference in the levels of collagen-binding integrin subunits, a1, a2 and b1 between the trunk and tip cells, suggesting that the tip and trunk cells have a similar potential to bind collagen via integrins. Considering the co- localization of TROP2 and collagen-I at the basal membrane of the ureteric bud, TROP2 may play a role in the development of trunk cells such as branching by modulating the function of collagen-I. Tip and trunk cells exhibit different potential to attach to and spread on collagen-I To further characterize the collagen-binding properties of fetal ureteric bud cells, sorted tip and trunk cells in the ureteric buds were cultured on collagen-coated dishes. The numbers of cells + attached to collagen-coated dishes were smaller for EpCAM - TROP2high cells than EpCAM+TROP2low cells, indicating that the trunk cells bind to collagen-I less efficiently (Figure 4A). Furthermore, while forward and side scatter by flow cytometry showed no significant difference in cell size between tip and trunk cells before plating (data not shown), the EpCAM + TROP2low cells were spread on the collagen-coated plates and exhibited membrane ruffles, resulting in an increase in cell size compared with the EpCAM+TROP2high trunk cells (Figure 4 B, C). It is known that new branches emerge from the tip and trunk cells form the mature ducts during the formation of ureteric bud branches, suggesting that the characteristics of tip and trunk cells in the same branch are different. These findings indicate that our primary cultures of ureteric bud cells recapitulate the differentiation process in vivo, i.e., the tip cells spread and migrate into the metanephric mesenchyme, whereas the differentiating trunk cells are less active. Furthermore, although there was no difference in the expression of collagen-binding integrins between the tip and trunk cells in the ureteric bud, the potential of EpCAM + TROP2high trunk cells to attach to and spread on collagen-coated plates was low, suggesting that factors other than the integrins regulate the interaction between trunk cells and collagen-I. Because TROP2 was expressed at a higher level in the trunk than tip cells and co- localized with collagen-I in the basal membrane of ureteric ducts, TROP2 may affect the potential of trunk cells to adhere to collagen-coated plates. TROP2 expression suppresses cell spreading To reveal the role of TROP2 in the cell spreading on collagen-I, we sorted EpCAM + TROP2low cells and overexpressed TROP2 by expression vector in the primary culture. Compared with the control cells transfected with the empty vector, the size of cells overexpressing TROP2 was markedly reduced, strongly suggesting that TROP2 affects the potential of EpCAM + TROP2low tip cells to spread on collagen-I (Figure 4D, E). To further confirm the role of TROP2 in the formation of ureteric bud branches, we utilized Madin-Darby canine kidney (MDCK) cells [34], established as a model for studying the PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28607