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First published online March 30, 2004
doi: 10.1242/10.1242/dev.01052

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Transient activation of ß-catenin signalling in adult mouse epidermis is sufficient to induce new hair follicles but continuous activation is required to maintain hair follicle tumours

Cristina Lo Celso^*, David M. Prowse^*, and Fiona M. Watt

Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK

View larger version (62K): [in a new window]   Fig. 1. Characterisation of the Nß-cateninER fusion protein and generation of transgenic mice. (A) Schematic diagram showing the K14 expression cassette and Nß-cateninER transgene. (B) Anti-ER immunofluorescence staining of 3T3 cells expressing Nß-cateninER following treatment with ethanol or 4OHT for 24 hours. (C) The induction of luciferase activity after 4OHT treatment. 3T3 cells were transduced with GFP or Nß-cateninER and transiently transfected with the luciferase reporters FOPFLASH or TOPFLASH. The luciferase activity of each reporter was measured in triplicate and the s.d. is shown. (D) Anti-ER immunofluorescence staining (green) of wild-type and transgenic K14Nß-cateninER (line D4) mouse back skin, untreated and after 7 days treatment with 4OHT. Nuclei were stained with propidium iodide (red). Insets show higher magnification views of boxed areas. (E) Western blot of primary keratinocytes cultured from wild-type (WT) and transgenic (lines 3953 and D4) mice, probed with anti-ER (top panel) or, as a loading control, anti-Erk MAPK (bottom panel) antibodies. (F) Gross phenotype of wild-type and Nß-cateninER mice (line D2) treated daily for 14 days with 4OHT, showing dramatic stimulation of hair growth in the transgenic mouse. Scale bars: (B) 50 µm, (D) 100 µm (main pictures), 25 µm (inserts).

View larger version (144K): [in a new window]   Fig. 2. Histological analysis of the effects of sustained ß-catenin activation in back skin of transgenic mice (lines D2 and D4). Arrows indicate outgrowths from the permanent portion of hair follicles (days 7 and 14, line D2; day 3, line D4). Arrowheads indicate small empty cysts (days 14, 21, line D2). Bracket indicates a region of hyperproliferative IFE (line D4, day 9). Scale bar: 100 µm.

View larger version (94K): [in a new window]   Fig. 3. De novo hair follicle formation in interfollicular epidermis. (A-F) Histology of the paw skin of (A,C,E) wild-type and (B,D,F) transgenic (line D4) mice treated daily with 4OHT for 1 week. A,B are montages reconstructed from images of sections covering the entire circumference of the paw. The boxed regions in A,B are shown at higher magnification in C,E and D,F respectively. Arrows in F indicate new follicles arising from interfollicular epidermis. (G-J) Visualisation of alkaline phosphatase activity (blue) as a dermal papilla marker in back skin (G) and paw (H-J) of transgenic (line D4) mice 8 days after a single application of 4OHT. Arrows in G show alkaline phosphatase-positive cells associated with the original dermal papilla (lowest arrow) and with new follicles arising from interfollicular epidermis (top arrow) and outer root sheath (middle arrow). The boxed regions in H are shown at higher magnification in I,J. (I) Original dermal papilla; (J) Two new rudimentary follicles. Scale bars: 500 µm (A,B) 100 µm (C-J).

View larger version (115K): [in a new window]   Fig. 4. Shh and Ptc expression. In situ hybridisation was performed on the dorsal skin of (A-D) untreated wild-type mouse with anagen follicles, (E-H) D2 or (I-L) D4 transgenic mice treated with 4OHT for 7 days, and (M-P) on the dorsal paw skin of D4 transgenic mice treated with 4OHT for 7 days. (B,F,J,N) Dark-field views of Shh in situ hybridisations in A,E,I,M, respectively. (D,H,L,P) Dark-field views of Ptc in situ hybridisations in C,G,K,O, respectively. Arrows in B indicate asymmetric Shh expression in the bulb of the hair follicle, while the arrow in D indicates more uniform Ptc expression in the bulb. Arrows in F,J show Shh expression in the outgrowths arising from the permanent portion of the follicles. Arrowheads in J show abnormal symmetrical expression of Shh in the bulb of the hair follicle and the asterisk in N indicates Shh expression in epithelial downgrowths in the interfollicular epidermis. Scale bars: 100 µm.

View larger version (96K): [in a new window]   Fig. 5. Analysis of proliferation in back skin of (A) wild-type or (C,E) D2 transgenic mice and in (B,D,F) paw skin of D4 transgenic mice. (A-D) BrdU labelling (green) with propidium iodide nuclear staining (red). Green fluorescence of hair shafts is non specific. (E,F) Cyclin D1 immunohistochemistry (brown) with Haematoxylin counterstain (blue). Mice were treated with 4OHT for the periods indicated. Arrows in C and E indicate outgrowths arising from the hair follicles that are positive for BrdU and Cyclin D1, respectively. Scale bars: 100 µm.

View larger version (93K): [in a new window]   Fig. 6. Expression of hair follicle differentiation markers. (A-D) Untreated wild-type anagen follicles. (E-H) Dorsal back skin of D2 transgenic mice treated with 4OHT for the periods indicated. (I-L) Dorsal paw skin of D4 transgenic mice treated with 4OHT for 5 days. Paraffin sections were immunostained with (A,E,I) anti-keratin 17, (B,F,J) anti-CDP, (C,G,K) anti-trichohyalin, or (D,H,L) anti-Lef1 antibodies and counterstained with Haematoxylin. Arrows in F and H indicate positive staining in the epithelial outgrowths from pre-existing follicles. Scale bar: 100 µm.

View larger version (135K): [in a new window]   Fig. 7. Effect of ß-catenin activation on sebocyte differentiation. Immunolocalisation of fatty acid synthase in dorsal back skin of (B) untreated wild-type mouse skin, (C) C5 transgenic treated with 4OHT for 15 days (arrows highlight duplication of sebaceous gland), (D) D2 transgenic treated with 4OHT for 28 days and (E) D4 transgenic treated with 4OHT for 9 days. A is wild-type follicle stained with secondary antibody only (bracket indicates the sebaceous gland). Scale bars, 100 µm.

View larger version (78K): [in a new window]   Fig. 8. Effects of transient ß-catenin activation in K14Nß-cateninER transgenic mice. (A) Schematic representation of experiments performed with the D2 and D4 transgenic lines. The arrow represents time in days (not to scale). Dots are time points analysed. Continuous lines connecting dots represent periods of daily 4OHT treatment and dashed lines indicate no 4OHT treatment. (B-G) Mice received the number of doses (d) of 4OHT indicated and were kept without 4OHT for number of days shown (+). (B,C,E-G) Back skin, (D) paw skin. Arrows in B and C indicate epithelial outgrowths from the permanent portion of the hair follicles and from sebaceous glands. Arrows in D indicate new hair buds arising from interfollicular epidermis. (H-Q) expression of cyclin D1 (H), Lef1 (I,L), keratin 17 (J,M), keratin 1 (K) and ß-catenin (green, N-Q; red fluorescence is propidium iodide counterstain) in D2 tumours, after 28 days of 4OHT treatment (H-J,N,O) or 28 days of treatment followed by 28 (P,Q) or 106 days (K-M) without 4OHT. Arrows in I highlight Lef1 expression. O,P are high magnification views of boxed regions in N, Q respectively. Scale bars: 100 µm (A-M), 50 µm (N-Q).

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