Mapping Wnt͞␤-catenin signaling during mouse development and in colorectal tumors Silvia Maretto*, Michelangelo Cordenonsi*, Sirio Dupont*, Paola Braghetta*, Vania Broccoli†, A. Bassim Hassan‡, Dino Volpin*, Giorgio M. Bressan*, and Stefano Piccolo*§ *Histology and Embryology Section, Department of Histology, Microbiology, and Medical Biotechnology, University of Padua, 35131 Padua, Italy; †Stem Cell Research Institute, H. S. Raffaele, 20132 Milan, Italy; and ‡Cell and Development Group, Department of Zoology, University of Oxford, South Parks Road, Oxford OX1 3PS, United Kingdom Edited by Eric N. Olson, University of Texas Southwestern Medical Center, Dallas, TX, and approved January 3, 2003 (received for review July 31, 2002) Wnt͞␤-catenin signaling plays key roles in several developmental tissues. Although contributing important information, genetic and pathological processes. Domains of Wnt expression have been analyses do not provide an unequivocal solution to this problem extensively investigated in the mouse, but the tissues receiving the for several reasons. signal remain largely unidentified. To define which cells respond to First, the Wnt pathway entails a complex set of molecules: in activated ␤-catenin during mammalian development, we gener- humans, for example, at least 19 Wnt factors and 10 Frizzled ated the ␤-catenin-activated transgene driving expression of nu- receptors have been identified. Ligands and receptors are distrib- clear ␤-galactosidase reporter (BAT-gal) transgenic mice, express- uted in complex, often overlapping expression patterns (for details, ing the lacZ gene under the control of ␤-catenin͞T cell factor see the Wnt web site by the Nusse laboratory, www.stanford.edu͞ responsive elements. Reporter gene activity is found in known ϳrnusse). Second, not all Wnt–Frizzled combinations end up with organizing centers, such as the midhindbrain border and the limb the activation of the canonical transduction cascade, because two apical ectodermal ridge. Moreover, BAT-gal expression identifies ␤-catenin independent pathways have been identified (4). Third, novel sites of Wnt signaling, like notochord, endothelia, and areas Wnt signaling is modulated extracellularly by a variety of secreted of the adult brain, revealing an unsuspected dynamic pattern of antagonists that bind either to the ligands or the LRP coreceptors ␤-catenin transcriptional activity. Expression of the transgene was inhibiting their activity (4). Finally, it must be stressed that the analyzed in mutant backgrounds. In lipoprotein receptor-related identification of cells with nuclear-localized ␤-catenin does not protein 6-null homozygous mice, which lack a Wnt coreceptor, imply functional signaling. Indeed, the activities of ␤-catenin are BAT-gal staining is absent in mutant tissues, indicating that BAT- constantly held in check even within the nucleus; at least in some gal mice are bona fide in vivo indicators of Wnt͞␤-catenin signal- cellular contexts, LEF͞TCF members recruit transcriptional inhib- ing. Analyses of BAT-gal expression in the adenomatous polyposis itors and maintain the promoter in an OFF state even in the .(coli (multiple intestinal neoplasia͞؉) background revealed ␤- presence of activated ␤-catenin (7, 8 catenin transcriptional activity in intestinal adenomas but surpris- Here we report the generation of mouse lines [␤-catenin- ingly not in normal crypt cells. In summary, BAT-gal mice unveil the activated transgene driving expression of nuclear ␤-galactosi- BIOLOGY entire complexity of Wnt͞␤-catenin signaling in mammals and dase reporter (BAT-gal)] that can be used as a general read-out DEVELOPMENTAL have broad application potentials for the identification of Wnt- of ␤-catenin activity. In several tissues, the expression of this responsive cell populations in development and disease. transgene mirrors known domains of Wnt signaling. Inhibition of the canonical Wnt pathway in LRP6 mutant mice results in the transgenic mice ͉ lipoprotein receptor-related protein 6 ͉ adenomatous concomitant lack of BAT-gal expression, indicating that these polyposis coli ͉ colon cancer transgenic mice are reliable reporters of Wnt͞␤-catenin activity. We then explored BAT-gal potential in revealing the status of roteins of the Wnt family are secreted factors regulating cell Wnt signaling in cancer cells. In humans, mutations of the APC proliferation, fate, and behavior in contexts ranging from tumor suppressor are associated with the formation of intestinal P ␤ early embryonic development to stem cell homeostasis (1–3). tumors (1, 9). To gain insight into the role of -catenin signaling Wnts exert many of their effects by activating the expression of in normal and neoplastic colon, we assayed BAT-gal expression ͞ϩ target genes through a pathway controlled by ␤-catenin, a in the APC [multiple intestinal neoplasia (Min) ] mouse (10), protein originally identified as a component of the cadherin cell a murine model of the human familial adenomatous syndrome ͞ϩ ͞ adhesion complex and later shown to be a key mediator for Wnt (OMIM 175100). In APC(Min ) BAT-gal crosses, we de- ␤ signaling. Wnt ligands bind a receptor complex composed of tected high levels of -catenin-induced transcription in the Frizzled and lipoprotein receptor-related protein (LRP) family epithelium of adenomas. Normal intestinal epithelium, however, ␤ members (4). This complex leads to the activation of downstream did not stain for lacZ, indicating that -catenin activity is tightly effectors ultimately promoting ␤-catenin accumulation in the controlled during normal crypt homeostasis. ␤ cytoplasm (3). In the absence of Wnt stimulation, free -catenin Materials and Methods levels are kept low by a ␤-catenin degrading complex that Generation and Analyses of Transgenic Mice. BAT-gal was con- includes the adenomatous polyposis coli (APC) and Axin tumor ͞ suppressors (1, 5). APC͞axin activities are inhibited in the structed by fusing seven TCF LEF-binding sites upstream of a presence of Wnt signaling, leading to ␤-catenin accumulation. 0.13-kb fragment containing the minimal promoter–TATA box ␤ of the gene siamois (11). Details of the synthesis of this construct Stabilized -catenin translocates into the nucleus, where it ␤ activates its target genes by binding to sequence-specific tran- and the initial controls for -catenin-specific activation of BAT- scription factors of the lymphoid enhancer factor͞T cell factor (LEF͞TCF) family (6). This paper was submitted directly (Track II) to the PNAS office. Wnt proteins are involved in cell-to-cell signaling in almost Abbreviations: APC, adenomatous polyposis coli; BAT-gal, ␤-catenin-activated transgene every process of mammalian development. To fully understand driving expression of nuclear ␤-galactosidase reporter; En, embryonic day n; LEF͞TCF, how this family of growth factors can elicit such diverse cellular lymphoid enhancer factor͞T cell factor; LRP, lipoprotein receptor-related protein; Min, responses, methods are needed to identify cells in which the Wnt multiple intestinal neoplasia; X-Gal, 5-bromo-4-chloro-3-indolyl ␤-D-galactoside. pathway is active at a given time during development and in adult §To whom correspondence should be addressed. E-mail: [email protected]. www.pnas.org͞cgi͞doi͞10.1073͞pnas.0434590100 PNAS ͉ March 18, 2003 ͉ vol. 100 ͉ no. 6 ͉ 3299–3304 Downloaded by guest on September 30, 2021 gal can be found in Supporting Text and Fig. 6, which are published as supporting information on the PNAS web site, www.pnas.org. Transgenic BAT-gal mouse lines and embryos were produced from B6D2F1 females mated with B6D2F1 males (Charles River Breeding Laboratories) by using standard pro- cedures (12). DNA was microinjected into the pronuclei of one-cell embryos, and the surviving embryos were implanted into CD1 pseudopregnant foster mothers. Transgenic mice were identified by analysis of genomic DNA from tail biopsies by PCR and Southern blot to detect lacZ. lacZ primers: forward, 5Ј- CGGTGATGGTGCTGCGTTGGA-3Ј; reverse, 5Ј-ACCAC- CGCACGATAGAGATTC-3Ј. Other experimental procedures are published as supporting information on the PNAS web site. Results and Discussion Generation of Wnt͞␤-Catenin Reporter Mice. To identify Wnt͞␤- catenin responding cells during mammalian development, we gen- erated a new transgenic mouse strain, here referred to as BAT-gal, which drives expression of nuclear ␤-galactosidase under the con- trol of multimerized LEF͞TCF-binding sites. In vitro, this construct leads to a specific and robust reporter gene activation on Wnt͞␤- catenin stimulation (see Fig. 6). To assay the efficacy and specificity of BAT-gal expression in vivo, we first performed transient analyses of mouse embryos injected with BAT-gal DNA at the one-cell stage and collected at embryonic day (E)8.5 and E10.5 for 5-bromo-4- chloro-3-indolyl ␤-D-galactoside (X-Gal) staining. At E8.5, trans- genic embryos displayed positivity at high frequency in the mid- Fig. 1. BAT-gal expression during early development. (A) First expression in the posterior epiblast of prestreak stage embryo detected by in situ hybrid- brain, a hallmark of Wnt-1 activity (13) and, at E10.5, showed strong ization with antisense ␤-galactosidase probe. (B) Diagram indicating the staining in the central nervous system, correlating with the expres- location of lacZ-positive cells (in blue) at the prestreak stage. Ant., anterior; sion pattern of several Wnts (data not shown, see Figs. 1E and 2A) Post., posterior; Extra., extraembryonic region; Embryo., embryonic region;