Galactosidase staining of the skin of heterozygous (ColI-Cre x RBP-J loxp/wt ) (+/-) (A) and

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2 Suppl. Figure 1. ColI-Cre transgene activity in RBP-Jk loxp x R26R mice. (A and B) - Galactosidase staining of the skin of heterozygous (ColI-Cre x RBP-J loxp/wt ) (+/-) (A) and homozygous (ColI-Cre x RBP-J loxp/loxp ) (B) mice crossed into a ROSA26-LacZ (R26R) background, at 3 weeks of age. There was non-specific staining of sebaceous glands (arrow) even in control, R26R negative mice. Note the strong -Gal staining of dermal papillae in (A) and the only mesenchymal pattern of staining, surrounding the cystic epithelial structures, in (B). Bar: 100μm. (C) -Galactosidase staining of the skin of homozygous (ColI-Cre x RBP- J loxp/loxp ) mice crossed into a R26R background, at 4 days of age. Left panel: low magnification images showing exclusive ß-Gal staining in hair follicle dermal papillae (black arrows). Middle and right panel: high magnification images of a hair follicle with still normal structure with ß- Gal staining limited to dermal papilla cells and surrounding fibroblasts. Bar: left: 40μm, right: 10μm. (D) Low and high magnification images of -Galactosidase staining of the skin of heterozygous (ColI-Cre x RBP-J loxp/wt ) mice crossed into a R26R background, at 1 year of age. ß-Gal staining conditions were the same as in the previous panels. Note the substantially lower signal, still limited to the mesenchymal compartment. Bar: 100μm.

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4 Suppl. Figure 2. Mesenchymal-specific deletion of the RBP-J gene in the skin. (A) Diagram of the separation procedure of various skin cell populations. Details of the experimental procedure are given in the Materials and Methods section. Briefly, back skin of P1 mice was sequentially treated with dispase, to separate interfollicular epidermis, and collagenase, followed by hair follicles density gradient centrifugation. Hair follicle keratinocytes, depleted of c-kit positive (melanocytes) and CD133 positive cells (dermal papilla cells), were used for subsequent marker analysis and characterization. Cells from the dermal compartment after collagenase digestion were depleted of c-kit positive (melanocytes) and CD34 positive (endothelial cells) cells, followed by separation into CD133 positive and negative fractions, for enrichment of dermal papilla cells and dermal fibroblasts, respectively (Ito Y, Hamazaki TS, Ohnuma K, Tamaki K, Asashima M, Okochi H Isolation of murine hair-inducing cells using the cell surface marker prominin- 1/CD133. J Invest Dermatol 127: ). Imunofluorescence analysis with anti-cd 133 antibodies was used to confirm the dermal papilla cell specificity of expression of this marker (inset). (B) Dermal cell preparation by the above procedure was stained by double immunofluorescence with antibodies against Cre and CD133. The percentage of Cre expressing cells in the CD133 positive (dermal papilla cell enriched) and negative fractions is indicated. (C) Purified hair follicle (HF) fraction from (RBP-J loxp/loxp ) (+/+) and (ColI-Cre x RBP-J loxp/loxp ) (-/-) mice at 10 days of age (P10). Hair follicle populations from mice with the RBP-J deletion were further physically separated according to their relatively normal (RN) versus abnormal (AN) structure, i.e. loss of a straight profile and incipient cyst formation, under a stereomicroscope. Shown are also the physically purified cysts from the

5 RBP-J -/- mice at 3 weeks of age. (D) Characterization of the recovered cell populations by marker expression. Real time RT-PCR analysis of the separated interfollicular epidermis (IFE), hair follicle keratinocytes (HF), dermal papilla cells (DP) and dermal fibroblasts (DF) for expression of Cytokeratin 14 (K14), a specific marker of interfollicular and follicular keratinocytes, and of Versican, a marker of dermal papilla cells that is also expressed, to a lower extent, in dermal fibroblasts. (E) Further analysis of dermal papilla (DP) versus dermal fibroblast (DF) cell preparations by real time RT-PCR analysis of the indicated dermal papilla signature genes ( Rendl M, Lewis L, Fuchs E Molecular dissection of mesenchymal-epithelial interactions in the hair follicle. PLoS Biol 3: e331.). (F) Analysis of the RBP-J genomic region of separated dermal fibroblasts (DF), dermal papilla cells (DP), interfollicular epidermis (IFE) and hair follicle keratinocytes (HF) from mice with (-/-) and without (+/+) deletion of the RBP-J gene at P1, in parallel with hair follicles and cysts that were physically separated from the same mice at P10 and 3 weeks, respectively. Hair follicle populations from P10 mutant mice were further separated according to their relatively normal (RN) versus abnormal (AN) structure, as described above, prior to analysis. Genomic DNA was analyzed by PCR with primers specific for Exon 6 and 7 of the RBP-J gene flanked by loxp sites, and the Neomycin inserted gene ( Han H, Tanigaki K, Yamamoto N, Kuroda K, Yoshimoto M, Nakahata T, Ikuta K, Honjo T Inducible gene knockout of transcription factor recombination signal binding protein-j reveals its essential role in T versus B lineage decision. Int Immunol 14: ) in parallel with primers for Exon 2 as a control. (G) Real time PCR analysis of the same set of samples in (F) with Exon 6/7 primers, using Exon 2 primers for internal normalization.

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7 Suppl. Figure 3. Normal hair follicle morphogenesis and structure in mice with mesenchymal RBP-J deletion at early times after birth. (A) Histological images of back skin sagittal sections of mice with mesenchymal deletion of the RBP-J gene (-/-) versus littermate controls (+/+) at day 0 (P0) and 2 (P2) after birth. Bar: 40μm. (B) High magnification oil immersion images of hair follicles from the above mice. Thin histological sections (2 μm) were utilized for this analysis. For P2 hair follicles, photographs are the composite of three consecutive microscopic images. The outer root sheath (ORS) and initially differentiating layers of the inner root sheath (IRS) are indicated. Bar: 10μm.

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9 Suppl. Figure 4. Hair follicle structural alterations in mice with mesenchymal RBP- J deletion a few days after birth. (A and B) Sagittal histological section and high magnification oil immersion images of hair follicles from mice plus/minus mesenchymal RBP-J deletion at day 4 (A) and 6 (B) after birth. Outer root sheath (ORS) and various layers of inner root sheath (IRS) and shaft are indicated: Co = Cortex; Ch = hair shaft cuticle; Ci = IRS cuticle; Hu = Huxley layer; He = Henley layer. Note that, in hair follicles of P4 mutant mice, hair shaft cuticle, IRS cuticle, Huxley and Henley layers are difficult to recognize due to the disordered alignment of cells. This defect is even more pronounced in P6 hair follicles, with the hair shaft exhibiting an irregular packing of medullary cells, and IRS/ORS cells improperly organized. Bar: 60μm for left two panels, 10μm for right two panels, for each stage.

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11 Suppl. Figure 5. Cystic degeneration of back skin and wisker hair follicles of mice with mesenchymal RBP-J deletion. (A) Histological sections illustrating cystic degeneration of back skin hair follicles in mice with deletion of the RBP-J gene, at 10 days and 3 weeks after birth (left and right panels respectively). Scale bars: 40μm. (B and C) Immunofluorescence analysis of cysts in the back skin of RBP-J -/- mice with antibodies against acidic hair keratins, trichohyalin, and cytokeratins 1, 10 and 6. Scale bars: 40μm. (D) Whole view of the vibrissae of a 4 months old ColI-Cre x RBP-J loxp/loxp mouse (left two panels) and corresponding histological image (right panel). (E) histological images of vibrissa follicles of (RBP-J loxp/loxp ) (+/+) versus (ColI-Cre x RBP-J loxp/loxp ) (-/-) mice at 3 weeks after birth, in parallel with immunofluorescence analysis with antibodies against the indicated markers. Arrows indicate, in H/E panels, hair shaft and, in the immunofluorescence panels, inner root sheath. Scale bar: 40μm.

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13 Suppl. Figure 6. Hair types in mice with and without mesenchymal deletion of the RBP- J gene. (A) Illustration and quantification of different hair types in (RBP-J loxp/loxp ) (+/+) versus RBP (ColI-Cre x RBP-J loxp/loxp ) (-/-) mice at postnatal day 10. Hair was plucked from back skin of 2 mice per genotype, and 100 hair fibers were counted in each case. (B) Photomicrographs of the 4 different hair types in (RBP-J loxp/loxp ) (+/+) and (ColI-Cre x RBP- J loxp/loxp ) (-/-) mice, illustrating, for the latter mice, a large percentage of hair fibers with structural abnormalities as described in the text.

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15 Suppl. Figure 7. Comparative analysis of the skin phenotype of mice with mesenchymal versus keratinocyte-specific deletion of the RBP-J gene. (A) Low and high magnification images of back skin sagittal sections from a mouse with a Cre transgene driven by the keratin 5 promoter ( Berton TR, Wang XJ, Zhou Z, Kellendonk C, Schutz G, Tsai S, Roop DR Characterization of an inducible, epidermal-specific knockout system: differential expression of lacz in different Cre reporter mouse strains. Genesis 26: ) and homozygous for the RBP-J loxp allele (K5-Cre x RBP-J loxp/loxp ) at 10 days of age. Note hair follicle abnormalities and incipient cyst formation similar to those observed with (ColI-Cre x RBP-J loxp/loxp ) mice of the same age (as shown in Fig. 1B). (B) Immunofluorescence analysis of hair follicles of mice with deletion of the RBP-J gene driven by the Col1-Cre versus K5-Cre transgenes in parallel with Cre-negative littermate controls at 10 days of age with antibodies against the indicated markers. Images were taken under the same exposure and capture conditions, and are representative of several independent fields, a minimum of 3 mice per genotype. Bar: 40μm. (C) Expression of the K1, K10, Hes1 and Hey1 genes in the epidermis of (ColI-Cre x RBP-J loxp/loxp ) versus (K5- Cre x RBP-J loxp/loxp ) mice (upper and lower panel, respectively), in parallel with Cre-negative littermates, as assessed by real time RT-PCR. The epidermis was separated from the underlying dermis by a brief heat treatment as previously reported ( Mandinova A, Lefort K, Tommasi di Vignano A, Stonely W, Ostano P, Chiorino G, Iwaki H, Nakanishi J, Dotto GP The FoxO3a gene is a key negative target of canonical Notch signalling in the keratinocyte UVB response. Embo J 27: ), two mice per genotype. (D) Immunofluorescence analysis of hair follicles of P10 mice with Col1-Cre versus K5-Cre

16 deletion of RBP-J in parallel with controls with antibodies against versican and Sox2 as dermal papilla markers. Images are representative of several independent fields, 3 mice per genotype. Bar: 60μm.

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18 Suppl. Figure 8. Defective hair follicle differentiation and cyst formation in grafted skin of mice with Wnt5a deletion. (A) High magnification oil immersion images of hair follicles from full thickness skin grafts of E17.5 mouse embryos plus/minus homozygous disruption of the Wnt5a gene, 1 week after grafting. Lower magnification histological images and immunofluorescence analysis are shown in Fig. 5A-D. ORS = outer root sheath; IRS=inner root sheath; Co = Cortex; Ch = hair shaft cuticle; Ci = IRS cuticle; Hu = Huxley layer; He = Henley layer. (B) Full thickness skin grafts of E17.5 embryos plus/minus the Wnt5a mutation onto nude mice, at 2 and 4 weeks after grafting. Photographs are representative of 3 grafted animals, and corresponding histological sections, per time point. Bar: 160 m.

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20 Suppl. Figure 9. Involvement of the Wnt5a receptors ROR2 and Fzd6 in control of FoxN1 expression. (A) Real time RT-PCR analysis of ROR2 and Fzd6 expression in hair follicles of newborn (ColI-Cre x RBP-J loxp/loxp ) mice and E17.5 Wnt5a knockout embryos (left and right panels, respectively) in parallel with littermate controls. Hair follicles were purified as described in materials & methods. (B) Hair follicles from organ skin cultures plus/minus Wnt5a treatment (as in Fig. 6) were analyzed for ROR2 and Fzd6 expression by real time RT-PCR. (C) Hair follicles from wild type P0 mice (purified and cultured as described in materials & methods) were placed in culture and transfected with two sets of anti- ROR2 sirnas in parallel with scrambled controls. Expression of the ROR2 and FoxN1 genes was determined by real time RT-PCR. (D) Cultured hair follicles from wild type P0 mice were transfected with two sets of anti-ror2 sirnas and corresponding scrambled controls as in the previous panel. Hair follicles were treated with Wnt5a (200ng/ml) or BSA only for the last 24 hours of culture (for a total culture time of 144 hours). Expression of the indicated genes was determined by real time RT-PCR. Plcd1: Phospholipase C delta-1; Dsc2: desmocollin 2. (E) Hair follicles from P0 mice were transfected with two sets of anti-fzd6 sirnas in parallel with scrambled controls, followed by real time RT-PCR analysis of the indicated genes. (F) Hair follicles transfected with two sets of anti-fzd6 sirnas or scrambled control as in the previous panel were cultured in the presence of Wnt5a or BSA as in the experiment of panel D, followed by real time RT-PCR analysis of the indicated genes.

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22 Suppl. Figure 10. Decreased FoxN1 and Tricohyalin expression in full thickness skin grafts from ROR2 knockout mice. (A) Full thickness skin of E18.5 mouse embryos with homozygous disruption of the ROR2 gene ( Takeuchi S, Takeda K, Oishi I, Nomi M, Ikeya M, Itoh K, Tamura S, Ueda T, Hatta T, Otani H et al Mouse Ror2 receptor tyrosine kinase is required for the heart development and limb formation. Genes Cells 5: ), was grafted in parallel with skin of wild type littermates onto the back of nude mice. 4 grafts per genotype were performed. Macroscopic as well as histological analysis at 12 days after grafting revealed no consistent alterations in hair follicle density and structure between mutant and wild type skin grafts. Shown are the results of immunofluorescence analysis with antibodies against the Trichohyalin and FoxN1 proteins. Images are representative of several independent fields, 3 grafts per genotype. Bar: 40 m.. (B) Quantification of the immunofluorescence signal for FoxN1 expression. 2μm sections, stained with anti-foxn1 antibody and Alexa 488 conjugated secondary and counterstained with DAPI, were scanned with a Zeiss Axiovision fluorescence microscope linked with Axiovision Software. Only hair follicles with well distinguished layers were used for counting. Scanned images were opened with ImageJ 1.38x software and nuclei of the hair cortex, visualized by DAPI staining, were outlined using the freehand selection function. FoxN1 staining intensity values for each outlined nucleus were then collected individually using the ROI manager. Shown is a scatter plot of FoxN1 fluorescence staining levels in nuclei of ROR2 mutant hair follicles (red) versus wild type controls (blue). The average intensity values for the two groups are shown on the side, together with the statistical significance of the results calculated by Student t-test.

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24 Suppl. Figure 11. Altered pigment localization in hair follicles as a result of mesenchymal RBP-J deletion or Wnt5a mutation. (A) Melanin staining of hair follicles from P10 mice plus/minus mesenchymal RBP-J deletion (left panels) or hair follicle reconstitution assays with RBP-J mutant versus wild type dermal papilla cells (right panels). Shown are high magnification oil immersion images of 2 μm paraffin sections stained with Fontana-Masson silver method for melanin visualization, with nuclear-fast red for counterstaining. Note the differential deposition of pigment in the cortex of wild type versus mutant hair follicles as indicated by arrows. Bar: 10 m. (B) Similar melanin staining analysis of hair follicles from full thickness grafts of embryonic skin plus/minus Wnt5a mutation (as in Fig. 5A) or hair follicle reconstitution assays with Wnt5a mutant versus wild type dermal papilla cells (as in Fig. 5E). Bar: 10 m.