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1 Supporting Information Voutilainen et al. 0.07/pnas SI Materials and Methods Animals. The generation and genotyping of K-da-A (K- da) () and N-κ reporter mice () has been described earlier. oth strains were maintained in a 7L/6 background. IκαΔN mice () were maintained in a mixed 7L/6 and 9Sv background. da-deficient mice (Tabby mice) were obtained from Jackson Laboratories (stock no. 000) and were maintained in a 6A background. The X-chromosomal location of da prevented the acquisition of female da / and da +/+ littermates; therefore, WT 6A females obtained from da +/+ and da +/Y matings were used as controls for da / mice. TOP-gal (Jackson Laboratories; stock no. 006) and AT-gal () mice have been described previously. The appearance of a vaginal plug was considered embryonic day () 0, and embryos were further staged by limb morphogenesis. All analyses in mice older than.0 were performed on female mice unless stated otherwise. Male embryos were identified by Sry-specific primers or by anatomy. armine Alum and X-Gal Staining. 8 embryos and - and 6-wk-old mice were killed, and ventral skin (8) or whole mammary glands ( or 6 wk) were spread on glass slides, fixed overnight in acid alcohol (:8 mixture of glacial acetic acid and 00% ethanol), rehydrated through ethanol series, and stained in carmine alum (Sigma-Aldrich) overnight. Stained tissues were dehydrated, cleared in xylene, and mounted with epex (H). Whole-mount X-gal staining was performed on. 6. embryos, as described previously (). Samples were postfixed in % paraformaldehyde (PA) and photographed using an Olympus SZX9 stereomicroscope. or sectioning, postfixed tissues were dehydrated and embedded in paraffin. Sections were counterstained with nuclear fast red and photographed with an Olympus AX70 microscope. Recombinant Proteins. When indicated, 00 ng/ml of recombinant c-da-a protein (6), 00 ng/ml of recombinant parathyroid hormone-related protein (PTHrP; Sigma-Aldrich), 00 ng/ml of recombinant amphiregulin (R& Systems), 00 ng/ml of recombinant epigen (R& Systems), or 00 ng/ml of recombinant WntA (R& Systems) was added to the groh medium. ultured mammary glands were visualized by carmine alum staining. xplant ulture in Hanging rops and Quantitative RT-PR. mbryonic skin explants and quantitative RT-PR (qrt-pr) were performed as described previously (7, 8). In brief,. da / back skins were dissected, cut into halves along the midline, and maintained for or h in a 0-μL hanging drop of MM, 0% S, glutamine, and penicillin-streptomycin. One half was supplemented with 0 ng/ml of recombinant c-da-a protein, and the control half was supplemented with PS and 0.% SA. Mammary buds were microdissected from. da / mice. Two pools of 0 mammary buds from or embryos were collected for each sample; one pool was used as a control, and the other was exposed to c-da for h as described for skin explants. or RNA isolation, skin and mammary bud explants were harvestedin0μl of RNeasy lysis buffer (Qiagen) as specified by the manufacturer. Then 00 ng of Nase-treated RNA was reverse-transcribed with SuperScript II (Invitrogen), and qrt-pr was performed in a Lightycler 80 (Roche). Transcript number was quantified by comparison with PR product dilutions of the genes of interest using Lightycler 80 software provided by the manufacturer. The expression of each gene was normalized against Ranbp. Primer sequences are available on request. In Situ Hybridization. Whole embryos were fixed overnight in % PA at before processing either for paraffin-embedding and sectioning or for whole-mount in situ hybridization as described previously (7, 8) with InsituPro robot (Intavis). igoxigenin-labeled probes were detected by standard protocols using M Purple AP Substrate Precipitating Solution (oehringer Mannheim). The following probes were used: Wnt0b (9), PTHrP (0), amphiregulin (nucleotides of NM_00970), and epigen (nucleotides 8 of NM_0087). Radioactive in situ hybridization was performed with a S-labeled probe specific to PTHrP and da as described previously (). Immunohistochemistry and Activated aspase- Staining. Mammary tissues of. WT, da /, and K-da mice were fixed in % PA, embedded in paraffin, and sectioned at μm. ewaxed and rehydrated sections were microwaved (60 W) for 0 min in 0 mm sodium citrate buffer (ph 6.0), and unspecific staining was blocked with 0% normal donkey or goat serum. Sections were incubated overnight with primary rabbit antibodies against Lef (:,000; ell Signaling Technology) or androgen receptor (:00; Upstate iotechnology), followed by biotinylated goat anti-rabbit secondary antibody (:00; Vector Laboratories) and detected using the A Kit (Vector Laboratories) and, -diaminobenzidine (Vector Laboratories). or caspase- analysis, sections were incubated overnight with rabbit anti-activated caspase- antibody (:,000; ell Signaling Technology), followed by biotinylated goat antirabbit antibody (:00; Vector Laboratories) or Alexa luor 68- conjugated donkey anti-rabbit antiserum (:00; Jackson ImmunoResearch Laboratories). Nuclei were stained with Hoechst 8 ( μg/ml; Invitrogen/Molecular Probes). Activated caspase- positive cell profiles with a clearly visible nucleus were counted from every other section throughout the mammary buds. The percentage of apoptotic cells was calculated as the ratio of caspase- positive cell profiles to all of the nuclei in the mammary bud. du Incorporation, Whole-Mount Immunostaining, and onfocal luorescence Microscopy. or in vivo proliferation assays, pregnantdams(.) wereinjectedatadoseofmg/kgofbodyweight of -ethynyl- -deoxyuridine (du) in a solution of. mg/ml PS at h before sacrifice. In ex vivo experiments,. mammary buds were cultured for h or 8 h, followed by incubation with 0 μm du for h. du incorporation into NA was detected using the lick-it du Alexa luor Imaging Kit (Invitrogen/Molecular Probes) according to the manufacturer s protocol. or whole- mount immunofluorescence, tissues were fixed in methanol for 0 min and washed with PS. Unspecific staining was blocked by incubation in % normal donkey serum, 0.% SA, and 0.% TritonX-00 in PS for h at room temperature. Tissues were then incubated overnight at with the primary antibody rat polyclonal anti-mouse 6 (pam, :,000; Pharmingen) and detected with an Alexa luor 88 conjugated secondary antibody (Molecular Probes/Invitrogen). Tissues were mounted with Vectashield (Vector Laboratories) and visualized using a Leica SP laser scanning confocal microscope. Images were analyzed and quantitative measurements performed with Imaris 7.. software (itplane). The number of du-positive nuclei was normalized to epithelial volume as determined by 6 positivity. Mesenchymal du-positive nuclei of6

2 were quantitated within a 6-μm radius of the bud border and normalized to area. Images were processed for presentation with Adobe Photoshop S and Illustrator S software. Statistical Analyses. The Student t test was used for statistical analysis of all data except qrt-pr data, which were tested with the nonparametric Wilcoxon signed-rank test for paired samples.. Mustonen T, et al. (00) Stimulation of ectodermal organ development by ctodysplasin-a. ev iol 9: 6.. hakar AL, et al. (00) onstitutive nuclear factor-κ activity is required for central neuron survival. J Neurosci : Schmidt-Ullrich R, et al. (00) Requirement of N-κ/Rel for the development of hair follicles and other epidermal appendices. evelopment 8:8 8.. Maretto S, et al. (00) Mapping Wnt/β-catenin signaling during mouse development and in colorectal tumors. Proc Natl Acad Sci USA 00: Pispa J, Pummila M, arker PA, Thesleff I, Mikkola ML (008) dar and Troy signalling pathways act redundantly to regulate initiation of hair follicle development. Hum Mol Genet 7: Gaide O, Schneider P (00) Permanent correction of an inherited ectodermal dysplasia with recombinant A. Nat Med 9: Mustonen T, et al. (00) ctodysplasin A promotes placodal cell fate during early morphogenesis of ectodermal appendages. evelopment : liniaux I, Mikkola ML, Lefebvre S, Thesleff I (008) Identification of dkk as a target of da-a/dar pathway reveals an unexpected role of ectodysplasin as inhibitor of Wnt signalling in ectodermal placodes. ev iol 0: Wang J, Shackleford GM (996) Murine Wnt0a and Wnt0b: loning and expression in developing limbs, face and skin of embryos and in adults. Oncogene : Liu JG, et al. (998) evelopmental role of PTHrP in murine molars. ur J Oral Sci 06 (Suppl ): 6.. Laurikkala J, et al. (00) TN signaling via the ligand receptor pair ectodysplasin and edar controls the function of epithelial signaling centers and is regulated by Wnt and activin during tooth organogenesis. ev iol 9:. K-da. 7. ig. S. da is expressed in the mesenchyme of developing mammary glands. (A ) da transcripts were detected by radioactive in situ hybridization with a S-labeled probe. Low expression levels were detected in the primary (arrow) mesenchyme at. (A) and in the fat pad precursor cells (arrowhead) at 7. (). The da / specimen confirmed the specificity of the probe ( and ). In addition to the endogenous mesenchymal expression (arrow), strong ectopic da expression was detected in the epithelium (blue arrow) in K-da embryos ( and ). (Scale bars: 00 μm.).. 6. A K-da G H I G H I A ig. S. N-κ reporter activity was abolished in developing mammary glands of da-null embryos and increased in K-da embryos. N-κ reporter expression was analyzed in mammary glands of WT (A ), K-da ( ), and da / (G I) embryos by whole-mount X-gal staining at. (A,, andg),. (,, and H), and 6. (,, and I). Note the untimely ductal development in K-da embryos. (G I ) Sections of whole-mount stained fourth inguinal mammary buds of da / embryos confirmed the absence of N-κ reporter expression. (Scale bar: 00 μm.) of6

3 mg mg mg mg mg A G H K-da ig. S. Mammary gland development in WT and K-da embryos at 6.. N-κ reporter expression was analyzed in mammary glands (mg mg) of WT (A ) and K-da ( H) embryos by whole-mount X-gal staining. Note the precocious development of all K-da mammary glands. The asynchrony of development was evident in both genotypes; mammary glands and advanced the furthest, and mammary gland was least developed. (Scale bar: 00 μm.) K-da female A K-da male 6wk 8 6wk ig. S. Mammary glands and nipples develop in K-da male mice. (A and ) Nipples were readily observed in female (A) and male () K-da mice at 6 wk of age. ( and ) Whole-mount stained fourth inguinal mammary gland of female ( and ) and male ( and ) K-da mice at 8 ( and ) and at 6 wk of age ( and ). At 8, the male and female glands were highly similar. (Scale bars: 00 μm.) A male K-da male ig. S. Testis and mammary gland development in WT and K-da male embryos at.. (A and ) Testis histology of WT (A) and K-da () male embryos. ( ) Analysis of N-κ reporter expression by whole-mount staining (fourth inguinal mammary gland) revealed similar thinning (arrow) of the neck region in WT () and K-da () embryos at., which was confirmed by sectioning ( and ). (Scale bars: 00 μm.) of6

4 A I N ig. S6. N-κ reporter expression was abolished in mammary buds of IκαΔN embryos. N-κ reporter expression was analyzed at. (A and ),. ( and ), and 6. ( and ) in mammary glands of WT embryos (A,, and ), and their IκαΔN littermates (,, and) by whole-mount X-gal staining. (Scale bar: 00 μm.) Note the absence of N-κ reporter activity in mammary buds of IκαΔN embryos. ( and ) Sections of whole-mount stained IκαΔN mammary buds confirmed the absence of N-κ reporter activity. (Scale bars: 00 μm.) old of induction control h da-treated h 0 0 control h da-treated h * * skin explants ns ns PthRP Areg pgn Wnt0a Wnt0b kk ig. S7. Identification of putative target genes of da in embryonic skin. Quantitative RT-PR analysis of the indicated genes in. da / skin explants treated with c-da for h (n = ), or for h (n = ). ata are shown as mean ± S. ns, P > 0.0; *P < 0.0; P < 0.0. of6

5 K-da. A Wnt0b Wnt0b.. G PTHrP H I J PTHrP K L. M I N PTHrP N. ig. S8. xpression of PTHrP and Wnt0b in mammary buds of WT, K-da, da /, and IκαΔN embryos. xpression of Wnt0b (A ) and PTHrP (G N) was analyzed by whole-mount in situ hybridization at. (A, G I, M, and N), or by section in situ hybridization at. ( and J L) using digoxigenin-labeled (A I, M, and N) or S-labeled (J L) probes. (Scale bars: 00 μm.) xpression of both PTHrP and Wnt0b was increased in K-da embryos at.. Arrows highlight strong expression in ectopic mammary buds of K-da embryos at.. At., expression of Wnt0b was correlated with da signaling activity. xpression of PTHrP was reduced in IκαΔN mutants at.. of6

6 K-da A. TOP-Gal AT-Gal G H I J K L Lef. AT-Gal TOP-Gal M N O ig. S9. Analysis of Wnt reporters TOP-Gal and AT-Gal, and Lef protein expression in da /, control, and K-da mammary buds. TOP-Gal (A and G I) and AT-Gal ( and J L) reporter expression was analyzed in sections of whole-mount X-gal stained. (A ) and. (G L) fourth inguinal mammary glands of WT (A,, G, and J), K-da (,, H, and K), and da / (,, I, andl) embryos. oth reporters were expressed in a mosaic manner in the epithelium. In K-da embryos, reporter activity was down-regulated in the epithelium, although expression was detected in newly formed branches (arrow) and in the stalk. Note the high mesenchymal AT-Gal reporter activity and expanded mesenchymal condensate in K-da embryos at. (J and K). (M O) xpression of Lef protein (mammary bud is shown) was down-regulated in the epithelium of K-da embryos that were undergoing branching morphogenesis. (Scale bar: 00 μm.) K-da A pam du. + h wild-type,+06 * ud volume (µm),0+06 *,+06,0+06,0+0 K-da control. + 8h pam du du positive nuclei/µm K-da control *.+h. +8h Mesenchymal du positive nuclei 0,0+00.+h.+8h * K-da control 00 * 0 * h.+8h ig. S0. Analysis of cell proliferation in K-da, WT, and da / mammary buds ex vivo. The ex vivo culture system allowed simultaneous quantitative analysis of cell proliferation in da /, WT, and K-da embryos. (A and ) da /, WT, and K-da. mammary buds were cultured for h (A) or 8 h () ex vivo, followed by du incorporation for h and whole-mount analysis of du and the epithelial marker pam. (Scale bars: 00 μm.) () Quantification of the volume of. mammary buds after h and 8 h of culture. () Quantification of the number of du positive nuclei normalized to pam + cells of da / (n = 0 for. + h), WT (n = for. + h; n = for. + 8 h), and K-da (n = for. + h; n = for. + 8 h) mice. da / buds were already regressing or even completely lost after d of culture and thus were not analyzed. () Quantification (arbitrary units) of mesenchymal cell proliferation per volume in da / (n = 7 for. + h), WT (n = 7 for. + h; n = for. + 8 h), and K-da (n = 0 for. + h; n =0for. + 8 h) mammary buds. P < 0.0; *P < of6

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