Figure S6 Phosphorylated-FAK in the tip and trunk of the ureteric bud. (TIF) Table S1 Primary antibodies used in immunofluores- cence chemistry and flow cytometric analysis. (TIF) Acknowledgments We thank Dr. Naoki Tanimizu for the critical reading of the manuscript. We also thank Drs. Tohru Itoh and Shiro Suetsugu for their valuable References 1. Cubas R, Li M, Chen C, Yao Q (2009) Trop2: a possible therapeutic target for late stage epithelial carcinomas. Biochim Biophys Acta 1796: 309–314. 2. Yagel S, Shpan P, Dushnik M, Livni N, Shimonovitz S (1994) Trophoblasts circulating in maternal blood as candidates for prenatal genetic evaluation. Hum Reprod 9: 1184–1189. 3. Goldstein AS, Lawson DA, Cheng D, Sun W, Garraway IP, et al. (2008) Trop2 identifies a subpopulation of murine and human prostate basal cells with stem cell characteristics. Proc Natl Acad Sci U S A 105: 20882–20887. 4. Okabe M, Tsukahara Y, Tanaka M, Suzuki K, Saito S, et al. (2009) Potential hepatic stem cells reside in EpCAM+ cells of normal and injured mouse liver. Development 136: 1951–1960. 5. El Sewedy T, Fornaro M, Alberti S (1998) Cloning of the murine TROP2 gene: conservation of a PIP2-binding sequence in the cytoplasmic domain of TROP-2. Int J Cancer 75: 324–330. 6. Litvinov SV, Balzar M, Winter MJ, Bakker HA, Briaire-de Bruijn IH, et al. (1997) Epithelial cell adhesion molecule (Ep-CAM) modulates cell-cell interactions mediated by classic cadherins. J Cell Biol 139: 1337–1348. 7. Munz M, Kieu C, Mack B, Schmitt B, Zeidler R, et al. (2004) The carcinoma- associated antigen EpCAM upregulates c-myc and induces cell proliferation. Oncogene 23: 5748–5758. 8. Nubel T, Preobraschenski J, Tuncay H, Weiss T, Kuhn S, et al. (2009) Claudin- 7 regulates EpCAM-mediated functions in tumor progression. Mol Cancer Res 7: 285–299. 9. Maetzel D, Denzel S, Mack B, Canis M, Went P, et al. (2009) Nuclear signalling by tumour-associated antigen EpCAM. Nat Cell Biol 11: 162–171. 10. Maghzal N, Vogt E, Reintsch W, Fraser JS, Fagotto F (2010) The tumor- associated EpCAM regulates morphogenetic movements through intracellular signaling. J Cell Biol 191: 645–659. 11. Wang J, Day R, Dong Y, Weintraub SJ, Michel L (2008) Identification of Trop- 2 as an oncogene and an attractive therapeutic target in colon cancers. Mol Cancer Ther 7: 280–285. 12. Nakatsukasa M, Kawasaki S, Yamasaki K, Fukuoka H, Matsuda A, et al. (2010) Tumor-associated calcium signal transducer 2 is required for the proper subcellular localization of claudin 1 and 7: implications in the pathogenesis of gelatinous drop-like corneal dystrophy. Am J Pathol 177: 1344–1355. 13. Costantini F, Kopan R (2010) Patterning a complex organ: branching morphogen- esis and nephron segmentation in kidney development. Dev Cell 18: 698–712. 14. Dressler GR (2006) The cellular basis of kidney development. Annu Rev Cell Dev Biol 22: 509–529. 15. De Arcangelis A, Mark M, Kreidberg J, Sorokin L, Georges-Labouesse E (1999) Synergistic activities of alpha3 and alpha6 integrins are required during apical ectodermal ridge formation and organogenesis in the mouse. Development 126: 3957–3968. 16. Kreidberg JA, Donovan MJ, Goldstein SL, Rennke H, Shepherd K, et al. (1996) Alpha 3 beta 1 integrin has a crucial role in kidney and lung organogenesis. Development 122: 3537–3547. 17. Michael L, Davies JA (2004) Pattern and regulation of cell proliferation during murine ureteric bud development. J Anat 204: 241–255. 18. Chi X, Michos O, Shakya R, Riccio P, Enomoto H, et al. (2009) Ret-dependent cell rearrangements in the Wolffian duct epithelium initiate ureteric bud morphogenesis. Dev Cell 17: 199–209. 19. Costantini F, Shakya R (2006) GDNF/Ret signaling and the development of the kidney. Bioessays 28: 117–127. 20. Trzpis M, Popa ER, McLaughlin PM, van Goor H, Timmer A, et al. (2007) Spatial and temporal expression patterns of the epithelial cell adhesion molecule (EpCAM/ EGP-2) in developing and adult kidneys. Nephron Exp Nephrol 107: e119–131. 21. Tsuzuki T, Takahashi M, Asai N, Iwashita T, Matsuyama M, et al. (1995) Spatial and temporal expression of the ret proto-oncogene product in embryonic, infant and adult rat tissues. Oncogene 10: 191–198. 22. Pachnis V, Mankoo B, Costantini F (1993) Expression of the c-ret proto- oncogene during mouse embryogenesis. Development 119: 1005–1017. 23. Kispert A, Vainio S, Shen L, Rowitch DH, McMahon AP (1996) Proteoglycans are required for maintenance of Wnt-11 expression in the ureter tips. Development 122: 3627–3637. TROP2 Defines Branch Formation in Fetal Kidney advice and providing reagents, and Nahoko Miyata and Shigeru Saito for technical assistance. Author Contributions Conceived and designed the experiments: YT MT AM. Performed the experiments: YT. Analyzed the data: YT. Contributed reagents/materials/ analysis tools: YT MT AM. Wrote the paper: YT MT AM. 24. Enomoto H, Araki T, Jackman A, Heuckeroth RO, Snider WD, et al. (1998) GFR alpha1-deficient mice have deficits in the enteric nervous system and kidneys. Neuron 21: 317–324. 25. Karner CM, Chirumamilla R, Aoki S, Igarashi P, Wallingford JB, et al. (2009) Wnt9b signaling regulates planar cell polarity and kidney tubule morphogenesis. Nat Genet 41: 793–799. 26. Nishinakamura R, Matsumoto Y, Nakao K, Nakamura K, Sato A, et al. (2001) Murine homolog of SALL1 is essential for ureteric bud invasion in kidney development. Development 128: 3105–3115. 27. Quaggin SE, Kreidberg JA (2008) Development of the renal glomerulus: good neighbors and good fences. Development 135: 609–620. 28. Cubas R, Zhang S, Li M, Chen C, Yao Q (2010) Trop2 expression contributes to tumor pathogenesis by activating the ERK MAPK pathway. Mol Cancer 9: 253. 29. Adams JC, Watt FM (1993) Regulation of development and differentiation by the extracellular matrix. Development 117: 1183–1198. 30. Chen D, Roberts R, Pohl M, Nigam S, Kreidberg J, et al. (2004) Differential expression of collagen- and laminin-binding integrins mediates ureteric bud and inner medullary collecting duct cell tubulogenesis. Am J Physiol Renal Physiol 287: F602–611. 31. Korhonen M, Ylanne J, Laitinen L, Virtanen I (1990) The alpha 1-alpha 6 subunits of integrins are characteristically expressed in distinct segments of developing and adult human nephron. J Cell Biol 111: 1245–1254. 32. Hynes RO (2002) Integrins: bidirectional, allosteric signaling machines. Cell 110: 673–687. 33. Schwartz MA, Ginsberg MH (2002) Networks and crosstalk: integrin signalling spreads. Nat Cell Biol 4: E65–68. 34. Madin SH, Darby NB (1958) Established kidney cell lines of normal adult bovine and ovine origin. Proc Soc Exp Biol Med 98: 574–576. 35. Gardner H, Kreidberg J, Koteliansky V, Jaenisch R (1996) Deletion of integrin alpha 1 by homologous recombination permits normal murine development but gives rise to a specific deficit in cell adhesion. Dev Biol 175: 301–313. 36. Chen J, Diacovo TG, Grenache DG, Santoro SA, Zutter MM (2002) The alpha(2) integrin subunit-deficient mouse: a multifaceted phenotype including defects of branching morphogenesis and hemostasis. Am J Pathol 161: 337–344. 37. Saelman EU, Keely PJ, Santoro SA (1995) Loss of MDCK cell alpha 2 beta 1 integrin expression results in reduced cyst formation, failure of hepatocyte growth factor/scatter factor-induced branching morphogenesis, and increased apoptosis. J Cell Sci 108(Pt 11): 3531–3540. 38. Montesano R, Matsumoto K, Nakamura T, Orci L (1991) Identification of a fibroblast-derived epithelial morphogen as hepatocyte growth factor. Cell 67: 901–908. 39. Liu Y, Chattopadhyay N, Qin S, Szekeres C, Vasylyeva T, et al. (2009) Coordinate integrin and c-Met signaling regulate Wnt gene expression during epithelial morphogenesis. Development 136: 843–853. 40. Zhang Z, Pascuet E, Hueber PA, Chu L, Bichet DG, et al. (2010) Targeted inactivation of EGF receptor inhibits renal collecting duct development and function. J Am Soc Nephrol 21: 573–578. 41. Miyazaki Y, Oshima K, Fogo A, Hogan BL, Ichikawa I (2000) Bone morphogenetic protein 4 regulates the budding site and elongation of the mouse ureter. J Clin Invest 105: 863–873. 42. Mitra SK, Hanson DA, Schlaepfer DD (2005) Focal adhesion kinase: in command and control of cell motility. Nat Rev Mol Cell Biol 6: 56–68. 43. Balzar M, Bakker HA, Briaire-de-Bruijn IH, Fleuren GJ, Warnaar SO, et al. (1998) Cytoplasmic tail regulates the intercellular adhesion function of the epithelial cell adhesion molecule. Mol Cell Biol 18: 4833–4843. 44. Thesleff I, Vaahtokari A, Partanen AM (1995) Regulation of organogenesis. Common molecular mechanisms regulating the development of teeth and other organs. Int J Dev Biol 39: 35–50. 45. Lin Y, Zhang S, Tuukkanen J, Peltoketo H, Pihlajaniemi T, et al. (2003) Patterning parameters associated with the branching of the ureteric bud regulated by epithelial-mesenchymal interactions. Int J Dev Biol 47: 3–13. 46. Stefanini M, De Martino C, Zamboni L (1967) Fixation of ejaculated spermatozoa for electron microscopy. Nature 216: 173–174. 47. Morita S, Kojima T, Kitamura T (2000) Plat-E: an efficient and stable system for transient packaging of retroviruses. Gene Ther 7: 1063–1066. PLoS ONE | www.plosone.org 10 December 2011 | Volume 6 | Issue 12 | e28607