EphB Receptors Coordinate Migration and Proliferation in the Intestinal Stem Cell NicheIntroductionResultsReduced Proliferation in Colon Crypts in Mice Lacking EphB2 and EphB3Redistribution of Proliferative Cells in the Crypts of the Small Intestine in Mice Lacking EphB2 and EphB3 Masks Altered ProliferationAcute Inhibition of EphB Signaling in the IntestineAcute Inhibition of EphB Signaling Reduces Proliferation in the Small Intestine and ColonEnhanced EphB Signaling Increases Progenitor ProliferationEphB Kinase-Dependent Signaling Conveys the Mitogenic SignalEphB Signaling Regulates Cell-Cycle ReentryEphB Signaling Promotes Proliferation in AdenomasEphB Signaling Controls the Size of the Proliferative Domain but Not the Number of Stem or Progenitor CellsDiscussionCoupling of Migration and Proliferation in the Intestinal Stem Cell NicheEphrins and Eph Receptors in Intestinal Homeostasis and CancerEphB Receptors Extend the Range of Wnt-Mediated ProliferationExperimental ProceduresAnimalsGeneration of Mice with Constitutively Active EphB2Analysis of Proliferation and Cell DeathImmunohistochemistryDelineation of Crypt CompartmentsProtein InjectionsImmunoprecipitation and ImmunoblottingElectroporation and Explant CultureSupplemental DataAcknowledgmentsReferencesEphB Receptors CoordinateMigration and Proliferationin the Intestinal Stem Cell NicheJohan Holmberg,1,3Maria Genander,1,3Michael M. Halford,2Cecilia Annere´n,1Mariann Sondell,1Michael J. Chumley,2Robert E. Silvany,2Mark Henkemeyer,2and Jonas Frise´n1,*1Department of Cell and Molecular Biology, Medical Nobel Institute, Karolinska Institute, SE-171 77 Stockholm, Sweden2Center for Developmental Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA3These authors contributed equally to this work.*Contact: [email protected] 10.1016/j.cell.2006.04.030SUMMARYMore than 1010cells are generated every day inthe human intestine. Wnt proteins are key regu-lators of proliferation and are known endoge-nous mitogens for intestinal progenitor cells.The positioning of cells wit hin the stem cellniche in the intestinal epit helium is controlledby B subclass ephrins through their interactionwith EphB receptors. We report that EphB re-ceptors, in addition to directing cell migration,regulate proliferation in the intestine. EphB sig-naling prom otes cell-cycle reentry of progenitorcells and accounts for approximately 50% ofthe mitogenic activity in the adult mouse smallintestine and colon. These data establish EphBreceptors as key coordinators of migration andproliferation in the intestinal stem cell niche.INTRODUCTIONThe proliferation of stem cells and their generation of prog-eny need to be tightly regulated, as an overproductionmay result in tumor formation and an underproduction inatrophy of the tissue. The proliferation of stem cells is or-chestrated, in part, by neighboring cells, forming the stemcell niche (Fuchs et al., 2004; Mikkers and Frise´n, 2005;Watt and Hogan, 2000). The study of stem and progenitorcells and their niches in model organisms have providedimportant insights into their interplay through extracellularsignals (Spradling et al., 2001). The often more complexstructure of mammalian organs has made it more difficultto elucidate how cells in a niche communicate to maintainhomeostasis.The comparatively simple organization of the intestineoffers an attractive model in mammalian stem cell biology.In the intestine, the epithelial lineage is contained withina sheet of cells. Epithelial stem cells reside at or nearthe bottom of crypts that are formed by the convolutionof the epithelial sheet. Members of the BMP (Haramiset al., 2004; He et al., 2004) and hedgehog (Madisonet al., 2005; Ramalho-Santos et al., 2000; van denBrink et al., 2004) families direct the positioning of thecrypts through an interplay between epithelial and subepi-thelial cells.Wnt proteins present at the bottom of the crypts inducenuclear translocation of b-catenin, which is a pivotal fea-ture of intestinal epithelial stem/progenitor cells (van deWetering et al., 2002). In concert with the Notch pathway,b-catenin maintains intestinal stem cells and controls theirdifferentiation (Fre et al., 2005; Stanger et al., 2005; van deWetering et al., 2002; van Es et al., 2005). Experimental in-hibition of the b-catenin pathway abolishes proliferation inintestinal crypts, and mutations causing overactivation arefound in the vast majority of human intestinal cancers,underscoring the central role of this pathway in controllingcell proliferation and differentiation in the intestine (Reyaand Clevers, 2005).As the progeny of the epithelial stem/progenitor cellsdifferentiate, the majority of cells migrate out of the cryptand are shed into the lumen within one week. b-catenincontrols the positioning of cells within crypts by regulatingthe expression of members of the ephrin and Eph families(Batlle et al., 2002). Ephrin ligands and their Eph tyrosinekinase receptors regulate cell migration in many contexts(Holmberg and Frise´n, 2002; Palmer and Klein, 2003; Po-liakov et al., 2004). Both ephrins and Eph receptors aremembrane bound proteins, restricting their interactionsto sites of direct cell-cell contacts. The ephrin-Eph recep-tor interaction allows bidirectional communication, witha signal being conveyed in both the receptor-expressing(forward signaling) as well as in the ligand-expressing (re-verse signaling) cell (Cowan and Henkemeyer, 2002;Holmberg and Frise´n, 2002; Palmer and Klein, 2003; Pas-quale, 2005).Ephrins and Eph receptors negatively regulate the num-ber of neurons generated from stem/progenitor cells inthe brain (Depaepe et al., 2005; Holmberg et al., 2005).The transition from adenoma to colon carcinoma is asso-ciated with loss of EphB receptor expression, and this isCell 125, 1151–1163, June 16, 2006 ª2006 Elsevier Inc. 1151an important step in the progression to invasive cancer(Batlle et al., 2005; Guo et al., 2005; Jubb et al., 2005; Lugliet al., 2005). This prompted us to analyze whether ephrinsand Eph receptors, in addition to their role in directing cellmigration, may participate in the control of proliferativehomeostasis in the intestinal stem cell niche.We find by gain- and loss-of-function experiments thatB subclass ephrins and Eph receptors, independently oftheir influence on cell positioning, promote proliferationin the crypts of the small intestine and colon and accountfor about 50% of the mitogenic activity. EphB2