TROP2 Defines Branch Formation in Fetal Kidney Figure 4. Characterization of ureteric tip and trunk cells in vitro. (A) Attachment of tip and trunk cells to collagen-coated plates. An equal number (56103 cells/well) of freshly isolated EpCAM+TROP2low and EpCAM+TROP2high cells from E14.5 kidney were plated on a collagen-coated 96- well plate and stained for actin filaments with Alexa488-conjugated phalloidin (green) after 24 h of culture. The fluorescence of GFP and the actin filaments was visualized under a microscope. Nuclei (blue). Scale bars = 100 mm. (B) Cell morphology of ureteric bud tip and trunk cells. Higher- magnification images of (A) are shown. Arrowheads indicate the ruffles forming at the edge of membranes. Scale bars = 50 mm. (C) Spreading of EpCAM+TROP2low(+) and EpCAM+TROP2high(++) cells. Cell size was measured using imageJ (n = 3). Error bars are s.d., * p,0.005. (D) Cell morphology of EpCAM+TROP2low cells after enforced expression of TROP2. EpCAM+TROP2low cells cultured for 24 h were transiently transfected with pTIB741 or pTIB741-TROP2 plasmid. At 24 hours after transfection, cells were stained with Alexa555-phalloidin (red). Arrowheads indicate the transfected cells expressing GFP. (E) The ratio of spreading in transfected cells. The cell of which diameter is more than two-fold of nuclear is counted as ‘‘spreading cell’’. All the experiments were repeated three times and the similar results were obtained. Total number of tested transfectants is as follows: Control; 66, TROP2; 94. Representative results are shown. doi:10.1371/journal.pone.0028607.g004 bottom panel) and ectopic TROP2 significantly suppressed the branching formation (Figure 6B). These results suggest that TROP2 antagonizes the cell scattering activity of HGF during branch formation. These results together reveal that TROP2 suppresses cell spreading and migration on collagen-I, suggesting that it contributes to the complicated branching structures of the kidney by suppressing the formation of branches from the ureteric bud trunks. Discussion TROP2 and EpCAM are structurally related molecules, however, their expression profiles differ significantly. In the E14.5 fetus, EpCAM is widely expressed in various epithelial tissues, whereas TROP2 expression is restricted to some epithelial cells in the kidney, lung and epidermis (Figure 1). During the branching morphogenesis of ureteric buds, new branches emerge from the tip through the proliferation and migration of tip cells. Meanwhile, the trunk elongates to form a rigid structure like a stalk without branching. TROP2 expression shows a gradient, increasing from the tip to trunk, suggesting that TROP2 contributes to the trunk structure (Figure 2). ECM components play fundamental roles in the branching morphogenesis of the ureteric bud, exerting their functions through ECM-specific integrin receptors. Mice deficient in a3- integrin, a laminin-binding integrin subunit, fail to form branches of ureteric buds from the Wolffian duct, indicating laminin to be an important ECM component for branching morphogenesis [16]. On the other hand, the function of collagen-I in fetal kidney remains unknown, because mice deficient in either a1 or a2 PLoS ONE | www.plosone.org 6 December 2011 | Volume 6 | Issue 12 | e28607