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First published online 21 April 2004
doi: 10.1242/dev.01125

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Epithelial Bmpr1a regulates differentiation and proliferation in postnatal hair follicles and is essential for tooth development

¹ Department of Dermatology, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA
² Department of Cell and Developmental Biology, University of Pennsylvania Medical School, Philadelphia, PA 19104, USA
³ Center for Childhood Communication, Abramson Research Center, The Children's Hospital of Philadelphia, Philadelphia, PA 19104, USA
⁴ Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, Collège de France, BP 163, 67404 Illkirch Cedex, France
⁵ Departments of Molecular, Cell and Developmental Biology, Orthopaedic Surgery, and Biological Chemistry, David Geffen School of Medicine at UCLA, Los Angeles, CA 90095, USA
⁶ Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, NC 27709, USA

View larger version (127K): [in a new window]   Fig. 1. Anti-phospho-SMAD1/5/8 immunofluorescence (red) of paraffin sectioned mid-dorsal skin at P5 (early anagen; A,B), P13 (full anagen; C,D), P16 (anagen-catagen transition; E), P18 (catagen; F), P20 (telogen; G) and P28 (first postnatal anagen; H). Nuclei are counterstained with Hoechst 33258 and appear blue. Presence of nuclear phospho-SMAD is indicated by yellow arrows. (B,D) Higher magnification photographs of the follicles shown in A and C, respectively. Diffuse red staining in the center of some follicles is due to autofluorescence of the hair shaft. ORS, outer root sheath; IRS, inner root sheath; DP, dermal papilla; SG, sebaceous gland.

View larger version (76K): [in a new window]   Fig. 2. Activity of Cre recombinase in Cre transgenic lines revealed by X-gal staining. (A,B) Wholemounts of E14.5 ROSA26R (A) and K14-Cre43; ROSA26R (B) embryos. Cre activity is detected globally in the epidermis of the K14-Cre43 embryo. (C) Frozen section of K14-Cre43; ROSA26R dorsal skin at P1. (D) Transverse section of E15 bcre-32; ROSA26R embryo torso after whole-mount X-gal staining. Arrows mark the limits of staining in ventral epidermis. HF, hair follicle; NT, neural tube.

View larger version (103K): [in a new window]   Fig. 3. Phenotypes of mice carrying epidermal-specific deletions of Bmpr1a. (A) K14-Cre43; Bmpr1acl/cl (right) and K14-Cre43; Bmpr1acl/+ control (left) newborns. The homozygous mutant lacks hindlimbs (blue arrow) and has open eyes (black arrow). (B) Ventral view of P13 bcre-32; Bmpr1acl/null (right) and bcre-32; Bmpr1acl/+ (left) littermates. The mutant lacks mid-ventral hair (arrow). (C) P20 K14-Cre52; Bmpr1acl/cl (right) and K14-Cre52; Bmpr1acl/+ (left) littermates. (D,E) Absence of incisor teeth (arrows) in a K14-Cre52; Bmpr1acl/cl mouse at P20 (E), compared with a heterozygous littermate control (D). (F) K14-Cre40; Bmpr1acl/cl (right) and K14-Cre40; Bmpr1acl/+ (left) littermates at P34. (G) K14-Cre40; Bmpr1acl/cl mouse at P34. (H) K14-Cre40; Bmpr1acl/cl mouse at 5 months. Arrows indicate pigment accumulations. (I) Uninduced K14-Cre-ERT2; Bmpr1acl/cl mouse at 5 months. (J) Front paws from uninduced 5-month-old K14-Cre-ERT2; Bmpr1acl/cl mouse showing growths under the nails (arrows). (K,L) Anti-BMPR1A immunofluorescence (red) of newborn K14-Cre43; Bmpr1acl/+ control (K) and K14-Cre43; Bmpr1acl/cl (L) skin cryosections. BMPR1A is present in control epidermis and hair follicle (HF), but only background basal lamina staining is detected in mutant skin. Nuclei are counterstained with Hoechst 33258. (M) Left side: PCR amplification of genomic DNA from epidermis, and dermis containing HF germs, from a newborn K14-Cre43; Bmpr1acl/cl mouse and a Bmprcl/+ littermate control. Primers detect the unrecombined floxed (FL), wild-type (WT) and recombined (lacking exon 2; X) alleles, and produce amplification products of 230 bp, 150 bp and 180 bp, respectively. Right side: PCR amplification of genomic DNA from organs of a 5-month-old uninduced K14-Cre-ERT2; Bmpr1acl/cl mouse using the same primers.

View larger version (119K): [in a new window]   Fig. 4. Embryonic and newborn hair follicle, mammary and tooth phenotypes of mice carrying epidermal-specific deletions of Bmpr1a. (A,B) Histology of E16.5 dorsal skin from K14-Cre43; Bmpr1acl/+ control (A) and K14-Cre43; Bmpr1acl/cl (B) embryos. (C,D) Anti-K17 immunofluorescence (red) of skin sections from the embryos shown in A and B, respectively. (E,F) Mid-dorsal skin from K14-Cre43; Bmpr1acl/+ control (E) and K14-Cre43; Bmpr1acl/cl (F) newborns. (G-I) Mid-ventral skin from bcre-32; Bmpr1acl/+ control (G), bcre-32; Bmpr1acl/null (H) and bcre-32; Bmpr1acl/null; Bmpr1bnull/null (I) newborns. (J,K) Sagittally sectioned mammary buds from E13.5 K14-Cre43; Bmpr1acl/+ control (J) and K14-Cre43; Bmpr1acl/cl (K) embryos. (L,M) Whole-mount in situ hybridization of E13.5 K14-Cre43; Bmpr1acl/+ control (L) and K14-Cre43; Bmpr1acl/cl (M) embryos with a Wnt10b probe. Mammary buds are indicated (arrows). (N,O) Sagittally sectioned inguinal mammary gland (arrows) from newborn K14-Cre43; Bmpr1acl/+ control (N) and K14-Cre43; Bmpr1acl/cl (O) mice. (P,Q) Transverse sections of oral cavity from E13.5 K14-Cre43; Bmpr1acl/+ control (P) and K14-Cre43; Bmpr1acl/cl (Q) embryos showing molar tooth buds. (R,S) Sagittally sectioned E16.5 K14-Cre43; Bmpr1acl/+ control (R) and K14-Cre43; Bmpr1acl/cl (S) heads showing absence of developing incisor teeth in the mutant (red arrows). (T,U) Sagittally sectioned K14-Cre43; Bmpr1acl/+ control (T) and K14-Cre43; Bmpr1acl/cl (U) newborn heads. Incisor teeth (red arrows) and molars (green arrows) are indicated in T and are absent at the corresponding locations in U. Vibrissa follicles (blue arrows) are present in the mutant.

View larger version (67K): [in a new window]   Fig. 5. Effects of epidermal-specific deletion of Bmpr1a on the histology of postnatal anagen hair follicles, localization of phospho-SMAD1/5/8, and expression of hair follicle differentiation markers. (A,B) Histology of dorsal skin from K14-Cre40; Bmpr1acl/+ control littermate (A) and K14-Cre40; Bmpr1acl/cl mutant (B) at P8. Nuclei in the center of a mutant follicle are indicated by an arrow (B). (C,D) Histology of mid-ventral skin from bcre-32; Bmpr1acl/+ control littermate (C) and bcre-32; Bmpr1acl/null mutant (D) at P8. (E,F) Anti-phospho-SMAD1/5/8 immmunofluorescence of P8 dorsal skin from K14-Cre40; Bmpr1acl/+ control littermate (E) and K14-Cre40; Bmpr1acl/cl mutant (F). Staining is present in nuclei of IRS and hair-shaft precursors in control follicles (yellow arrows; E) but is much reduced in mutant follicle epithelium (F). Weak staining is present in mutant dermal papillae and in a few epithelial cells close to the epidermis (yellow arrows). (G-L) Immunofluorescence of P8 dorsal skin from K14-Cre40; Bmpr1acl/+ control littermate (G,I,K) and K14-Cre40; Bmpr1acl/cl mutant (H,J,L) using AE13 (G,H), AE15 (I,J) and anti-keratin 17 (K,L) antibodies. (M,N) Immunofluorescence of P8 mid-ventral skin from bcre-32; Bmpr1acl/+ control littermate (M) and bcre-32; Bmpr1acl/null mutant (N) using anti-keratin 6 antibody. Yellow arrows indicate the hair shaft (G), IRS and medulla (I), and outer root sheath (K-N). White arrow indicates epidermal staining (N). Immunofluorescence signals are red in E-L and green in M,N. Nuclei are counterstained with Hoechst 33258. Scale bars: in D applies to A-D; in F applies to E,F; in N applies to G-N.

View larger version (100K): [in a new window]   Fig. 6. Effects of epidermal-specific deletion of Bmpr1a on expression of regulators of hair shaft and IRS differentiation. (A-F) In situ hybridization of mid-dorsal skin from K14-Cre40; Bmpr1acl/+ control littermate (A,C,E) and K14-Cre40; Bmpr1acl/cl mutant (B,D,F) at P8, using the digoxigenin-labeled antisense probes indicated. Hybridization signals (purple-brown) are indicated by arrows. Dark brown cells (A,E,F) are pigmented. (G,H) Immunofluorescence of P12 dorsal K14-Cre40; Bmpr1acl/+ control littermate (G) and K14-Cre40; Bmpr1acl/cl mutant (H) skin with anti-GATA3. (I,J) In situ hybridization of ventral head skin from K14-Cre40; Bmpr1acl/+ control littermate (I) and K14-Cre40; Bmpr1acl/cl mutant (J) at P8 using 35S-labeled antisense probe for Msx1. (K,L) In situ hybridization of mid-ventral P8 skin from bcre-32; Bmpr1acl/+ control littermate (K) and bcre-32; Bmpr1acl/null mutant (L) using 35S-labeled antisense probe for Lef1. Bright red regions (K; white arrows) are due to reflection of light by the hair shaft. (M,N) Immunofluorescence for ß-catenin (red) in P12 dorsal skin from K14-Cre40; Bmpr1acl/+ control littermate (M) and K14-Cre40; Bmpr1acl/cl mutant (N). Arrows (A,C,E,F,I-L) indicate hybridization signals. Arrows (G,M,N) indicate nuclear localized GATA3 and ß-catenin, respectively. Nuclei are counterstained with Hoechst 33258 (I-N). Scale bar in N applies to A-N. DP, dermal papilla.

View larger version (67K): [in a new window]   Fig. 7. Abnormal follicular cycling and proliferation in epithelial Bmpr1a mutants. (A-F,G,H) Histology of dorsal skin from K14-Cre40; Bmpr1acl/+ control littermates (A,E,G) and K14-Cre40; Bmpr1acl/cl mutants (B,F,H), and ventral skin from bcre-32; Bmpr1acl/+ control littermate (C) and bcre-32; Bmpr1acl/null mutant (D) at P12 (A,B), P17 (C,D), P21 (E,F) and P34 (G,H). Control skin progresses through anagen (A), catagen (C), telogen (E) and first postnatal anagen (G), but mutant skin remains in an abnormal anagen-like state. Arrow in B indicates misshapen, expanded dermal papilla; arrow in F indicates abnormal, undifferentiated cells in the center of a mutant follicle. (E',F') Alkaline phosphatase staining of skin sections from the samples shown in E and F, revealing locations of dermal papillae (DP; blue arrows). Red arrow (F') indicates a pigmented cast being extruded to the epidermis. (E'',F'') Anti-BrdU staining of skin sections from the samples shown in E and F, revealing continued proliferation of mutant follicles (arrow in F''). (G',H') Anti-BrdU staining of sections from the samples shown in G and H, showing proliferation in the bulbs of control anagen follicles (G'; yellow arrow), and in the bulbs and outer root sheaths (yellow arrows), and epidermis (white arrow) of mutant skin (H'). Scale bars: in F' applies to A-F'; in F'' applies to E'',F''; in H applies to G,H; in H' applies to G',H'. SG, sebaceous gland.

View larger version (78K): [in a new window]   Fig. 8. Development of follicular cysts and matricomas, mesenchymal BMP signaling, and paw phenotype in 5-month-old K14-Cre-ERT2; Bmpr1acl/cl Bmpr1a mutant mice. (A-F) Histology of dorsal skin from uninduced (A,B,D-F) and tamoxifen-treated (C) mutants. Abnormal hair follicles are shown in A,D; follicular cysts in B,C; and proliferations of undifferentiated matrix-like cells, histologically similar to human matricomas, in E,F. (G-I) Anti-BrdU immunofluorescence of dorsal skin from uninduced mutants. (J-U) Immunofluorescence of dorsal skin from uninduced mutants with antibodies to the proteins indicated (red). (V) Anti-phospho-SMAD immunofluorescence of abnormal hair follicles showing nuclear phospho-SMAD in dermal cells (arrow). A dashed white line indicates the dermal-epithelial boundary. (W,X) Sagittal sections of the nail region from uninduced control K14-Cre-ERT2; Bmpr1acl/+ mouse (W) and uninduced K14-Cre-ERT2; Bmpr1acl/cl mutant (X), showing expanded mesenchyme in the mutant. Arrow indicates a calcifying osteoid.