TROP2 Defines Branch Formation in Fetal Kidney Figure 3. Expression of collagen-I and integrins in the fetal kidney. (A) Immunostaining of E14.5 kidney with anti-TROP2 (green) and anti- collagen-I (red) Abs and nuclei (blue). Higher magnification images of the boxed regions are shown underneath. Arrowheads indicate collagen-I surrounding the ureteric trunks. Scale bars = 100 mm. (B) Quantitative RT-PCR analysis of integrin subunits in freshly isolated EpCAM+TROP2low(+) and EpCAM+TROP2high(++) cells from E14.5 kidney. The relative gene expression of integrin/GAPDH is shown. Error bars are s.d. doi:10.1371/journal.pone.0028607.g003 formation of branches of ureteric bud. As no endogenous canine TROP2 was detectable in MDCK cells by RT-PCR (Figure S4A), we established MDCK cell lines stably expressing TROP2 using retroviral vectors (Figure S4B and S4C). To confirm the results obtained with the primary cultures of ureteric bud cells, the effect of TROP2 expression on the spreading of MDCK cells was examined. Cells detached from a confluent monolayer were plated on a collagen-coated glass cover slip and images were captured at 1 and 2.5 h after plating. Control MDCK cells started to spread at least 30 min earlier than MDCK-TROP2 cells (data not shown). At 2.5 h after plating, approximately 50% of the control MDCK cells had spread (Figure 5 A, B). By striking contrast, 70% of MDCK-TROP2 cells retained a round shape without cytoplasmic extensions. This result together with the results obtained using the primary cultures indicates that the expression of TROP2 significantly suppressed spreading of kidney cells. MDCK cells displayed membrane ruffles with cortical actin filaments and stress fibers when cultured on collagen-coated glass cover slips, whereas TROP2 expression suppressed the membrane ruffling and stress fibers (Figure 5C). Since membrane ruffling is implicated in cell motility, we examined the effect of TROP2 on cell motility with the wound healing assay. The gap in cell sheets formed by a scratch was measured at 0 and 6 h. Expression of TROP2 suppressed the migration of MDCK cells 6 h after the scratch was made (Figure 5 D, E), suggesting that TROP2 also plays a role in suppressing cell spreading and migration on collagen. TROP2 inhibits the branching of MDCK cells in collagen- gel culture As the spreading and migration of ureteric bud cells are necessary for the development of renal tubules, we examined the effect of TROP2 on tubulogenesis and branch morphogenesis using MDCK cells in three-dimensional collagen-gel cultures. There was no significant difference in cyst size between clusters of control MDCK and MDCK-TROP2 cells in the collagen-gel cultures (Figure 6A, the top panels). The addition of 20 ng/mL of hepatocyte growth factor (HGF), a cell scattering factor, induced morphological changes in control MDCK cells, forming branches (Figure 6A, the left bottom panel). By contrast, MDCK-TROP2 cells formed round cysts without branching (Figure 6A, the right PLoS ONE | www.plosone.org 5 December 2011 | Volume 6 | Issue 12 | e28607