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. 2007 Jun 12;104(24):10063-8.
doi: 10.1073/pnas.0703004104. Epub 2007 Jun 6.

Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling

Krzysztof Kobielak  1 , Nicole Stokes, June de la Cruz, Lisa Polak, Elaine Fuchs
Affiliations

Loss of a quiescent niche but not follicle stem cells in the absence of bone morphogenetic protein signaling

Krzysztof Kobielak et al. Proc Natl Acad Sci U S A. .

Abstract

During the hair cycle, follicle stem cells (SCs) residing in a specialized niche called the "bulge" undergo bouts of quiescence and activation to cyclically regenerate new hairs. Developmental studies have long implicated the canonical bone morphogenetic protein (BMP) pathway in hair follicle (HF) determination and differentiation, but how BMP signaling functions in the hair follicle SC niche remains unknown. Here, we use loss and gain of function studies to manipulate BMP signaling in the SC niche. We show that when the Bmpr1a gene is conditionally ablated, otherwise quiescent SCs are activated to proliferate, causing an expansion of the niche and loss of slow-cycling cells. Surprisingly, follicle SCs are not lost, however, but rather, they generate long-lived, tumor-like branches that express Sox4, Lhx2, and Sonic Hedgehog but fail to terminally differentiate to make hair. A key component of BMPR1A-deficient SCs is their elevated levels of both Lef1 and beta-catenin, which form a bipartite transcription complex required for initiation of the hair cycle. Although beta-catenin can be stabilized by Wnt signaling, we show that BMPR1A deficiency enhances beta-catenin stabilization in the niche through a pathway involving PTEN inhibition and PI3K/AKT activation. Conversely, sustained BMP signaling in the SC niche blocks activation and promotes premature hair follicle differentiation. Together, these studies reveal the importance of balancing BMP signaling in the SC niche.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.
Fig. 1.
Postnatal ablation of Bmpr1a affects homeostasis of the SC niche in adult hair follicles. (A) Chart illustrating the temporal progression through the first and second postnatal hair cycles and the regiment for administering TM and BrdU. (B) Phenotypes of CONTM and Bmpr1a cKOTM mice, after shaving back skins during telogen, targeting Bmpr1a and monitoring after initiation of the next hair cycle. cKOTM mice did not regrow hair. (C and D) Skin sections stained with H&E or with antibodies that are color-coded according to the fluorescent tags of the secondary antibodies. Brackets denote bulge (Bu), and arrowheads denote follicle cells outside the niche. Note that the CD34 monoclonal antibody gives high dermal background in older animals; the hair shafts typically display autofluorescence. FACS analyses are of SC populations sorted by α6-integrin and high CD34. R3, basal SCs; R2, i.e., the large box including R3, total bulge SCs. Note that both CD34-positive bulge pools are diminished 2 weeks after Bmpr1a targeting in cKOTM skin. (E and F) Skin sections as in C and D, but in this case, depicting analysis of bulge SCs after pulse–chase and pulse experiments with BrdU (see A for time schedule). FACS analyses are based on α6-integrin and incorporation of BrdU during a 4-h pulse. Cells that incorporated BrdU above limits of detection by immunofluorescence microscopy are boxed as R5. Note that the pool of BrdU-incorporating cells is substantially increased in cKOTM skin.
Fig. 2.
Fig. 2.
Expansion of the proliferating populations and their ability to repair epidermal wounds when BMP signaling is ablated in hair follicles postnatally. cKOTM and CONTM mice were targeted for Bmpr1a ablation at P44 and analyzed at P77 (A–D) or P90-P230 (E and F). Frozen skin sections (10 μm) were subjected to indirect immunofluorescence for Sox9, P-cadherin, and Lhx2 (A, B, and E), or to in situ hybridizations (ISH) with digoxygenin cRNA probes specific for Sox4 and Shh (C, D, and F). Note that Sox9hiLhx2hi- and P-cadherinhiLhx2(+)-positive cells mark two distinct populations emerging from the bulge lacking BMPR1A. These populations developed into tumor masses over time. (G) A 4-h pulse at P120 shows that the tumor borders are highly proliferative. (H) Ablation was performed at P44, and a 3-day pulse was administered at P67. Skin was analyzed after a 3-week chase. Note the absence of label-retaining cells in tumor masses. (I) Wound healing. cKOTM mice were mated to Rosa26-floxed-lacZ mice; after Bmpr1a ablation (LacZ+), skins were scraped to remove the epidermis, and wound repair was monitored for the periods indicated. Note the reepithelialization (arrows) by BMPR1A-deficient follicle cells. Arrowhead denotes border between scab/blood clot and the epidermis, dotted lines denote dermo–epithelial borders, and brackets denote bulge (Bu). hg, hair germ; SG, sebaceous gland.
Fig. 3.
Fig. 3.
Precocious and sustained activation of Lef1/β-catenin and P-PTEN/PI3K/AKT signaling in hair follicles lacking BMPR1A. (A–E) cKOTM and CONTM mice were targeted for Bmpr1a ablation at P44 and analyzed at P59. Frozen skin sections (10 μm) were subjected to indirect immunofluorescence or immunohistochemistry by using the antibodies indicated in the lower right of each frame. Color coding is according to the fluorescent tags of the secondary antibodies. Arrowheads denote aberrant expression. (F) Immunoblot analyses of protein extracts isolated from P59 skin preparations as described (26). Antibodies used are indicated at the right. (G and H) TOPFlash assays. MKs were transfected with vector encoding the reporter gene TOPFlash or the control FOPFlash containing mutations in the multimerized Lef1 DNA binding sites. MKs were then treated with the recombinant factors or with the PI3K inhibitor Ly as indicated, and luciferase assays were conducted as described (13). Experiments were conducted in triplicate. Lam5, laminin5; β-cat, β-catenin; β4, β4-integrin; hg, hair germs; DP, dermal papilla; Nogg, Noggin; Wnt3, Wnt3a.
Fig. 4.
Fig. 4.
Mice expressing a dominant active BMPR1A transgene display precocious hair follicle differentiation. Bitransgenic mice harboring a Dox-regulatable, superactive BMPR1A (dTg), and noninducible CON mice were treated with Dox at P19, after shaving the lower back skin areas (brackets). Analyses of mice were performed at P35, following a 4-h pulse with BrdU. (A) Transgene schematics. (B) Phenotypic abnormalities in the regrowth of the hair coat of dTgDox-treated mice. (C–I) P35 skin sections subjected to either H&E staining or immunofluorescence with the antibodies indicated. Color-coding is according to the fluorescent tag of the secondary antibodies. AE15 recognizes trichohyalin, an IRS marker. Note that following BMPR1A superactivation, dTgDox hair follicles develop cysts of terminally differentiated cells located just below the bulge (Bu, bracket). Dotted lines demarcate dermo–epidermal boundaries. Mx, matrix cells. Boxed areas are magnified in Insets.
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