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First published online 6 February 2008
doi: 10.1242/dev.016550

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Sustained epithelial β-catenin activity induces precocious hair development but disrupts hair follicle down-growth and hair shaft formation

Katja Närhi¹, Elina Järvinen¹, Walter Birchmeier², Makoto M. Taketo³, Marja L. Mikkola^1,* and Irma Thesleff^1,*,

¹ Developmental Biology Program, Institute of Biotechnology, Viikki Biocenter, PO Box 56, University of Helsinki, FIN-00014, Helsinki, Finland.
² Max-Delbrück Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
³ Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan.

View larger version (103K): [in this window] [in a new window]   Fig. 1. Catnbex3K14/+ mice show a dramatic skin phenotype. (A,B) Macroscopic examination at E18 revealed severely disturbed skin morphology in Catnbex3K14/+ mice. Mutant skin was covered by pigmented dots (arrows) and white granules. Eyes were open and external ears were poorly developed. (C,D) Scanning electron microscopic analysis of the skin of a Catnbex3K14/+ mutant embryo (D) and its control littermate (C) at E14.5 revealed the random and dense pattern of epithelial evaginations on the surface of the mutant skin when compared with the regular array of hair placodes in the control embryo. Insets in B-D are higher magnifications of the boxed regions of head (B) and back skin (C,D). Scale bars: 1 mm in C,D; 0.2 mm in inset in C.

View larger version (108K): [in this window] [in a new window]   Fig. 2. Forced expression of epithelial β-catenin leads to precocious hair follicle induction. (A-L) Detection of placodes in control (A-C,G-I) and Catnbex3K14/+ skin (D-F,J-L) by in situ hybridization at E12.5 (A,D,G,J), E13 (B,E,H,K) and E13.5 (C,F,I,L) using Wnt10b (A-F) and Edar (G-L) as markers. In the Catnbex3K14/+ mutants, first hair placode-like structures have already appeared at E12.5 (insets in D and J). Insets (A,D,G,J) are higher magnifications of the boxed areas in the shoulder region of E12.5 skin. (M-O) Whole-mount X-gal staining for E11, E12.5 and E13 Rosa26;K14Cre mice showing the Cre recombinase activity. Arrowheads indicate mammary placodes. L, limb. Arrows indicate Cre activity in shoulder region. Scale bars: 2 mm.

View larger version (118K): [in this window] [in a new window]   Fig. 3. The prenatal morphogenesis of Catnbex3K14/+ mutant hair follicles is disturbed. (A-L) Histological sections of embryonic skin showed that the first morphological signs of hair placodes were evident in the controls at E14 (C). They reached germ stage by E15.5 (G) and stage 4 at E17 (I,K). Placodes of second-wave hair follicles had also appeared at E17 (I). In Catnbex3K14/+ mutant skin, epithelial thickenings were already present at E13.5 (B). New placodes and buds of enlarged size and irregular shape were induced continuously during advancing development (D,F,H,J). Mutant placodes were already observed in dorsal midline at E14.5 (bracket, F). In addition, epithelial budding was initiated from the outer root sheath of follicles (arrowheads in J and L). The layer of condensed mesenchyme was thicker in the mutants both under the placode and under interfollicular epithelium (C-E, curly bracket in F). In addition, the epidermis was thicker in the mutants when compared with control skin (curly brackets in F,K,L). Broken lines show the epithelial-mesenchymal border (A-H,L). Asterisks indicate keratin deposits within the mutant epithelium (L). Arrows indicate placodes and buds. Scale bars: 200 µm.

View larger version (76K): [in this window] [in a new window]   Fig. 4. Hair shaft production is severely impaired in Catnbex3K14/+ skin. (A,B) Advancing hair development was examined by transplanting back skin from E17 embryos to nude mice. After 5 weeks, dense hair was present in control graft, whereas the Catnbex3K14/+ mutant graft had produced only a few hairs. (C-F) Light microscopic examination revealed no major differences in hair structure between control and mutant grafts. Arrows indicate zig-zags and arrowheads indicate awl hairs (C,E). High-magnification pictures (D,F) show internal structure of awl hairs. (G-N) Histological analysis of control (G,I) and mutant (H-N) grafts after 3 (G,H,K,L) or 5 weeks (I,J,M,N). Control skin showed regular row of catagen (G) and anagen-stage hair follicles (I), but in the mutant graft only few well-developed follicles were observed (arrows in H and J). In the mutant skin grafts, the down-growth of hair follicles was dramatically impaired and a variety of abnormalities were obvious (K-N): extra sebocytes in large clusters (arrows in K and L), epithelial budding from the outer root sheath of aberrant follicles (arrows in M) and large epithelial cysts filled with keratin-like material (arrowheads in J,L,N). Scale bars: 2 mm in A; 200 µm in C,E,G,I; 100 µm in D,F,L-N; 50 µm in K.

View larger version (75K): [in this window] [in a new window]   Fig. 5. Catnbex3K14/+ hair placodes form throughout the skin in a random and dense pattern. (A-J) Detection of placodes by expression of Sostdc1 (A-D,G,J), Wnt10b (E,H) and Shh (F,I) in E14.5 control (A,C,E-G) and Catnbex3K14/+ (B,D,H-J) skin showed dramatic spreading of mutant hair placodes throughout the skin, even in regions that, in control embryos, still lacked placodes: dorsal paw skin (arrow in B); the immediate surroundings of eye, ear lobe and vibrissa pads (arrows in D); and dorsal midline and tail skin (arrows in B''). Mutant placodes were also observed in ventral paw skin (B'), which in adult wild-type mice remain largely hairless. (E-J) High magnifications of dorsal skin revealed the regularly spaced control placodes (E-G), whereas the distances varied greatly between adjacent mutant placodes (H-J). Arrows indicate fusions of mutant placodes (H-J). (K) Placode numbers calculated from E14.5 Sostdc1 wholemounts indicated a 1.6-fold increase in Catnbex3K14/+ mice when compared with control. Data are represented as mean±s.d. (L) The distribution of placodes according to diameter as measured from E14.5 Sostdc1 wholemounts. Mutant placodes showed larger variation in size and were, on average, smaller than control placodes. The placode was determined as the unstained area surrounded by the positive Sostdc1 expression. Number of placodes measured: mutant, n=218; control, n=137. Scale bars: 2 mm in A,A'',C; 0.5 mm in A'; 200 µm in E.

View larger version (95K): [in this window] [in a new window]   Fig. 6. Forced β-catenin expression causes increased epithelial and mesenchymal Wnt activity and upregulation of placode marker genes. (A-D) Vibratome sections of X-gal stained E14 BAT-gal and BAT-gal; Catnbex3K14/+ mice indicated increased Wnt activity in mutant skin both in placode epithelium and underlying mesenchyme (B,D) compared with control (A,C). (E-J) By radioactive in situ hybridization, Wnt10b was localized to placode epithelium. (E,F) Sostdc1 was restricted to epithelium flanking the placodes but was absent from the placode, and its expression was more intense in the mutant (G,H). Dkk1 was expressed in mesenchyme underlying the placodes, and was more intense and widespread in the mutant skin (I,J). Arrows indicate hair placodes. Broken lines indicate epithelial-mesenchymal border. Scale bars: 100 µm. (K) Quantitative RT-PCR reveals doubling of Dkk1, Sostdc1 and Shh expression in E13.75 Catnbex3K14/+ skin compared with control. Edar expression levels were unaltered in the mutant skin. Data are represented as mean±s.d.

View larger version (71K): [in this window] [in a new window]   Fig. 7. Forced activation of epithelial β-catenin increases BMP signaling and compensates for the absence of Eda. (A-D) Immunohistological detection of phosphorylated Smad1/5/8 in E14 control (A,C) and Catnbex3K14/+ (B,D) skin, indicating stimulation of BMP pathway activity in the mutant skin. (E-H) Whole-mount in situ hybridization analysis of E14 embryos showed stimulated expression of Bmp2 (E,F) and Bmp4 (G,H) in Catnbex3K14/+ skin when compared with control. High magnifications of mutant wholemounts revealed prominent epithelial evaginations with Bmp2 and Bmp4 expression in the tips (inserts in F and H). (I-L) Vibratome sections of wholemounts (E-H) indicated more intense expression of Bmp2 and Bmp4 in Catnbex3K14/+ skin compared with controls (I-L). Ectopic expression of Bmp4 had been induced in mutant placode epithelium (L). (M) Quantitative RT-PCR showed 1.3-fold upregulation of Bmp2 and Bmp4 in E14.5 Catnbex3K14/+ skin compared with controls. No difference was seen in Bmp7 expression. Data are represented as mean±s.d. (N-Q) Expression of Sostdc1 in E13 and E14.5 Eda-deficient mice (Eda-/-) and Eda-/-; Catnbex3K14/+ mice. Sostdc1 was absent from E13 and E14.5 Eda-/- skin (N,P) owing to lack of primary hair placodes. When Eda-/- mutants were crossed to Catnbex3K14/+ mice, placode formation was rescued and the placode phenotypes at E13 and E14.5 were indistinguishable from the Catnbex3K14/+ mutant phenotype (O,Q). Broken lines indicate epithelial-mesenchymal borders. Scale bars: 200 µm in A; 100 µm in C and inset in F,I; 2 mm in E,N. (R) A model of the interactions between Wnts and BMPs in hair placode formation, suggesting roles for BMPs as reaction-diffusion inhibitors of placode patterning. Placodal cells display Wnt activity. Wnts induce the expression of their feedback inhibitors Dkk1 and Dkk4, as well as of Bmp2 and Bmp4, which in turn downregulate Wnt activation at least through inhibition of Lef1. In interplacode region BMPs induce the expression of Sostdc1 which may act by suppressing Wnt activity in interplacodal cells. Black lines indicate transcriptional regulation, and red lines show protein-protein interaction. Green area, placode; yellow area, condensed mesenchyme; white area, interfollicular epithelium.