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First published online 17 March 2004
doi: 10.1242/dev.01079

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BMPR1A signaling is necessary for hair follicle cycling and hair shaft differentiation in mice

¹ Laboratory for Cell Culture Development, RIKEN Brain Science Institute, Saitama 351-0198, Japan
² Molecular Neuropathology Group, RIKEN Brain Science Institute, Saitama 351-0198, Japan
³ Laboratory for Behavioral Genetics, RIKEN Brain Science Institute, Saitama 351-0198, Japan
⁴ Division of Gene Function in Animals, Nara Institute of Science and Technology, Nara 630-0192, Japan
⁵ Immune System Development Group, RIKEN Research Center for Allergy and Immunology, Kanagawa 230-0045, Japan
⁶ Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA

View larger version (54K): [in a new window]   Fig. 1. Analysis of mice expressing Cre-recombinase under control of the Emx1 locus. Whole-mount tissue from E13.5 Emx1-Cre/LacZ mice was stained with X-gal (A). Whole-mount tissue from P0 Emx1-Cre/lacZ mice was stained with X-gal (B) and a frozen section from P0 Emx1-Cre/lacZ mice was stained with X-gal, and counterstained with Hematoxylin-Eosin (C,D). Section from P14 Emx1-Cre/lacZ mice was stained with X-gal (E). c, cortex; dp, dermal papilla; ds, dermal sheath; irs, inner root sheath; m, matrix; ors, outer root sheath. Scale bars: 500 µm in A-C; 50 µm in D; 20 µm in E.

View larger version (109K): [in a new window]   Fig. 2. Hair-specific ablation of the Bmpr1a gene in mice. Hair-Bmpr1a KO mice (right) lack external hairs at the limbs (arrowheads) at P30 (A). (B-D) The right hindlimbs of control mice at P30: (B) whole limb; (C) instep; (D) outstep. (E-G) Hindlimbs of P30 Hair-Bmpr1a KO mice: (E) whole limb shows an abnormal spreading of first digit and malformed claws in hindlimbs; (F) instep; (G) outstep. (H,I) HE stained section of forelimb from control mice (H) and Hair-Bmpr1a KO mice (I) at E15-16. TUNEL analysis in adjacent section of control mice (J) and Hair-Bmpr1a KO mice (K) (see boxed areas in H and I). The number of TUNEL-labeled nuclei in first digit of Hair-Bmpr1a KO mice limb at E15-16 was reduced (K) compared with control mice (J). In situ hybridization analysis of Bmpr1a using an exon 2-specific probe in a section from P30 control (L,M). The expression of Bmpr1a is efficiently abolished in the P30 Hair-Bmpr1a KO mice (N). c, cortex; irs, inner root sheath; m, matrix; me, medulla; ors, outer root sheath. Scale bars: 20 mm in A; 2 mm in B-G; 100 µm in H,I; 50 µm in J-N.

View larger version (90K): [in a new window]   Fig. 3. Abnormal hair follicle formation in Hair-Bmpr1a KO mice. Hematoxylin-Eosin (HE)-stained sections of hindlimbs from Hair-Bmpr1a KO mice are shown in the developmental stage of HF. First anagen phase of P14 control mice (A). Hair follicles in the control skin retract into dermis, maintaining close contact between the dermal papilla and the epithelial hair shaft in P14 control mice (arrow). Abnormally differentiated hair shafts were observed in P14 Hair-Bmpr1a KO mice (B-D). Retracting epithelial hair shafts that separate from the dermal papilla and form cysts in hair canals were observed in P14 Hair-Bmpr1a KO mice (arrow, B). Section from P30 control mice shows second anagen phase of HF (E). In P30 Hair-Bmpr1a KO mice (F-H), nearly all of the canals have small cysts (arrowhead, H) in the HF. Section from older (10-month-old) control mice was processed with HE staining (I). The hair canals were wider and large cysts (arrowhead) were observed in hair canals in older Hair-Bmpr1a KO mice (J-L). The number of HFs per mm2 was counted in P14, P30 and mature mice (n=7 per group) (M). Data are given as mean±s.e.m.; * P<0.05; **P<0.01; Student's t-test. d, dermis; s, subcutis; pc, panniculus carnosus. Scale bars: 100 µm in A,B,E,F,I,J; 50 µm in C,D,G,H,K,L.

View larger version (98K): [in a new window]   Fig. 4. Altered proliferation of hair follicle cells in Hair-Bmpr1a KO mice. Phosphorylated histone (PH) immunostaining signals exhibit dividing epithelial cells of HFs from P30 control mice (A). P30 Hair-Bmpr1a KO mice showed that PH-positive cells were located abnormally in hair canals of HF (B). Older (10-month-old) Hair-Bmpr1a KO mice showed a significantly reduced index of PH immunosignals (D) than did control mice (C). BrdU incorporation in Hair-Bmpr1a KO mice (F) was severely reduced compared with control mice (E). The number of PH-positive cells per HF was counted in P14, P30 and older mice (n=24 per group) (G). Data are given as mean±s.e.m.; *P<0.05; Student's t-test. Scale bars: 100 µm in A (left panel), B (left panel) and C-F; 50 µm in A (right panel) and B (right panel).

View larger version (136K): [in a new window]   Fig. 5. Formation of the IRS in Hair-Bmpr1a KO mice and control mice. Frozen sections of hindlimb skin were prepared from control mice (A-D) and Hair-Bmpr1a KO mice (E-H) at P14. Expression of keratin 6 (A,B,E,F) in the IRS, and expression of keratin 5 (C,D,G,H) in the ORS were detected by immunofluorescence staining. B,D,F and H are merged with Hoechst (blue) for nuclear staining. Formation of IRS characterized by the expression of IRS-keratin 6 was severely reduced in Hair-Bmpr1a KO mice (E,F). (I,J) Ultrastructure of hair follicle in control mice (I) and Hair-Bmpr1a KO mice (J) at P30. Insets show keratin 5 (red) and Hoechst (blue) signals at P30. Absence of IRS cells resulted in proliferation of keratin-5-positive ORS cells. c, cortex; ci, cuticle of IRS; he, Henle's layer; hu, Huxley's layer; irs, inner root sheath; me, medulla; ors, outer root sheath; th, trichohyalin granule. Scale bars: 100 µm in A-H and insets in I,J; 5 µm in I,J.

View larger version (120K): [in a new window]   Fig. 6. The expression of growth regulators in Hair-Bmpr1a KO mice and control mice. Expression of EGFR, notch, TRAF6, PDGFR and Met in HFs from P30 Hair-Bmpr1a KO mice and P30 control mice (as indicated) was analyzed by immunohistochemistry (shown in green for antibodies, Hoechst blue for EGFR, notch and TRAF6; brown for antibodies for PDGFR and Met). The expression levels of all of these regulators in Hair-Bmpr1a KO mice were comparable with those in control mice. No signal was detected in negative controls (normal goat serum was the control for anti-EGFR, notch, TRAF6, and Met goat polyclonal antibodies; no primary antibody was used as a control for anti-PDGFR monoclonal antibody). Scale bars: 100 µm.

View larger version (62K): [in a new window]   Fig. 7. Expression of Shh and ß-catenin in Hair-Bmpr1a KO mice and control mice. (A-F) A digoxigenin-labeled Shh riboprobe was used on sections of skin from hindlimb of P14 (A,B), P30 (C,D) and older (10-month-old; E,F) mice. (G-J) The expression area of ß-catenin was wider in P30 Hair-Bmpr1a KO mice (I,J) than the P30 control mice (G,H). Scale bars: 100 µm in A-G,I; 50 µm in H,J.

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