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First published online 11 February 2009
doi: 10.1242/dev.030700

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Notch signaling in bulge stem cells is not required for selection of hair follicle fate

Department of Developmental Biology, Division of Dermatology, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, St Louis, MO 63110, USA.

View larger version (113K): [in this window] [in a new window]   Fig. 1. Hair follicle stem cells lacking total Notch signaling are capable of choosing a follicular fate. (A) Diagram of the timeline/cohorts used to delete Rbpj (or Notch genes) in adult hair follicle stem cells, to induce the hair cycle, and to examine the behavior of anagen hair follicles 14 days after hair cycle induction. Note that the topical application of RU486 started when the mice were at P50 and the following analyses are performed at the end of the experimental period (i.e. 28 days after the last RU486 treatment). (B) X-Gal staining shows Rbpj-deleted bulge cells (blue) in Krt1-15-CrePR1; Rbpjflox/flox; Rosa26R (K15-RBP-jCKO) and control hair follicles that are not depilated and hence are still in the telogen phase of the hair cycle. Depilated hairs, however, have entered anagen. Blue Rbpj-deleted cells in K15-RBP-jCKO hair follicles have migrated down to the bulb and are detectable in all layers of the anagen hair follicle. (C) AE13 (cortex and cuticle marker) staining confirms that blue Rbpj-deleted keratinocytes in K15-RBP-jCKO undergo the follicular differentiation program. The inset shows the normal pattern of AE13 staining in an anagen hair follicle (counterstained with Hematoxylin). (D) Immunohistochemistry for Rbpj demonstrates that blue cells in K15-RBP-jCKO anagen hair follicles are depleted of Rbpj protein. (E) Bulge stem cells deleted for all Notch receptors can differentiate into hair follicle keratinocytes and are detectable in anagen hair by X-Gal staining on Krt1-15-CrePR1; Notch1flox/flox; Notch2flox/flox; Notch3–/–; Rosa26R (K15-N1N2N3CKO) skin after Cre/hair induction as outlined in A. (F) Back skin of K15-RBP-jCKO and control mice highlighting the aberrant hair shafts produced by keratinocytes derived from Rbpj-deficient stem cells. The genotype of control mice is shown in A. Scale bars: 25 µm.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Stepwise deletion of Notch pathway components in hair follicle keratinocytes results in progressive hair follicle deterioration culminating in transformation into epidermal keratin cysts. (A) H&E-stained skin sections from a comprehensive allelic series of Notch-deficient mice at P9 demonstrate progressive hair follicle destruction, which is most evident in mice lacking total Notch signaling (N1N2N3CKO and PSDCKO), which show densely packed keratin cysts beneath the epidermis. Note that Rbpj-deficient hair follicles have a milder phenotype than those lacking total Notch signaling. (B) A scoring system devised to permit a quantitative measure of hair follicle destruction (e.g. 0, intact hair follicle; 10, keratin cyst) confirms the inverse correlation between Notch dosage and the level of hair follicle deterioration. The destruction level of RBP-jCKO hair follicles (asterisk) is similar to that of N1N2hN3CKO. Hair follicles in three randomly selected 100x microscope fields are scored and the average score ± s.d. is presented in the bar chart. (C) Immunofluorescence staining for keratin 14 and loricrin (an epidermis-specific marker) shows the transformation of Msx2-Cre/+; Notch1flox/flox; Notch2flox/+; Notch3+/– (N1N2hN3hCKO) hair follicles to fully formed epidermal keratin cysts (asterisks) in the second anagen (P35). Msx2-Cre/+; Notch1flox/flox (N1CKO), Msx2-Cre/+; Notch1flox/flox; Notch2flox/+ (N1N2hCKO), Msx2-Cre/+; Notch1flox/flox; Notch2flox/+; Notch3–/– (N1N2hN3CKO), Msx2-Cre/+; Notch1flox/flox; Notch2flox/flox (N1N2CKO), Msx2-Cre/+; Notch1flox/flox; Notch2flox/flox; Notch3–/– (N1N2N3CKO), Msx2-Cre/+; Rbpjflox/flox (RBP-jCKO), and Msx2-Cre/+; PS1flox/flox; PS2–/– (PSDCKO). Scale bars: 100 µm in A; 50 µm in C.

View larger version (65K): [in this window] [in a new window]   Fig. 3. Hair follicles that are derived from Notch/Rbpj-deficient stem cells transform into epidermal keratin cysts. (A) Diagram outlining the experimental approach used to delete Rbpj or Notch genes in adult hair follicle stem cells during the second telogen (P50) and to examine the status of hair follicles in the subsequent anagen phase. Note that there is no depilation step. (B) X-Gal-stained skin sections collected 50 days after the last RU486 treatment demonstrate the replacement of hair follicles by keratin cysts in K15-RBP-jCKO skin. Note that Rbpj-expressing (pink) cells also contribute to this epidermal structure. The same transformation happens with K15-N1N2N3CKO hair follicles (data not shown). The genotype of control mice is shown in A. (C) Expression of keratin 10 (suprabasal epidermal marker) and loricrin (granular layer marker within a lacZ-positive cyst; inset) confirms that K15-RBP-jCKO keratinocytes forming the keratin cysts have undergone the epidermal differentiation program. (D) Ten weeks after RU486 treatment, K15-N1N2N3CKO mice show severe alopecia with numerous keratin cysts visible on the back skin. Keratin cyst formation in K15-N1N2N3CKO and/or barrier defects associated with the loss of Notch proteins in bulge-derived epidermal keratinocytes lead to an intense dermal inflammation in these mice (see Fig. S4 in the supplementary material). RU486-treated Krt1-15-CrePR1; Notch1flox/flox; Rosa26R (K15-N1CKO) mice have a mild hair loss phenotype compared with similarly treated age-matched controls (Krt1-15-CrePR1; Notch1flox/+; Notch2flox/+; Notch3–/–; Rosa26R). Scale bar: 25 µm.

View larger version (63K): [in this window] [in a new window]   Fig. 4. Hair follicle stem cells lacking Notch signaling contribute to epidermal homeostasis under normal conditions. (A) Diagram of the approach used to delete Rbpj in adult hair follicle stem cells during the second telogen (P50) and to determine their fate 35 days later without depilation. (B) X-Gal staining of the skin shows that blue cells migrate up from the bulge to form epidermis in K15-RBP-jCKO mice. Note that no blue clones emanating from hair follicles are found in the RU486-treated control animals (counterstain is Nuclear Fast Red). (C) Rbpj antibody staining confirms that most (>90%) blue cells in K15-RBP-jCKO are Rbpj-deficient. The few Rbpj-positive blue cells in K15-RBP-jCKO epidermis either responded to a signal produced by Rbpj-deficient cells or followed deleted cells in their migration towards the epidermis (a group effect). (D) Immunohistochemical staining for keratin 6 (ORS marker) and keratin 10 (epidermal marker) demonstrate that blue Rbpj-deficient bulge stem cells have differentiated into epidermal keratinocytes. (E) The leukocyte content of K15-RBP-jCKO dermis is comparable to that of control dermis 35 days after Cre induction. The CD45 pan-leukocyte marker is used to demonstrate the absence of inflammation in K15-RBP-jCKO skin at the location where Rbpj-deficient bulge cells have migrated into epidermis. In C-E, no counterstaining was performed; the genotype of control mice is shown in A. Scale bars: 25 µm.

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