Figure 5: Lgr5+ HFSCs are indispensable for WIH-A and important for WIHN.
(a) IF analysis of skin tissue sections indicated that tamoxifen treatment for Lgr5-Cre:Ptenflox/flox mice induced marked p-AKT and the division of Lgr5-EGFP+ cells in the HF. Scale bars, 50 μm. (b) Subcutaneous injection of tamoxifen induced HF TAT at the injection site in Lgr5-Cre:Ptenflox/flox mice (n=6). (c) Intraperitoneal administration of tamoxifen induced widespread HF TAT in back skin. Lgr5-Cre:Ptenflox/flox mice, n=9; WT mice, n=12. (d,e) Deletion of Lgr5+ cells in Lgr5-Cre:R26iDTR/+ mice completely attenuated the WIH-A (d), and the number of anagen HFs was quantified (n=7) (e). (f,g) Wound-induced anagen HFs were markedly decreased in mice that received Lgr5-mTNFR1-ShRNA (ShRNA) compared with mice that received a mock sequence. (h,i) WIH-A analysis in β-catenin knockout and the control mice at PWD-15 (n=7). (j,k) Depletion of Lgr5+ stem cells result in extensive reduction of neogenic HFs. n=5 for TM-treated mice, and n=6 for TM- and DT-treated mice. (l) Lgr5+ cells (mTomato+/mGFP+, mT+/mG+) were mainly located in the ORS of the anagen hair follicle (Anagen V), and the Lgr5+ cell lineage cells (mT−/mG+) contributed to all the cellular components of the hair bulb. Lgr5+, Lgr5 expressing cells. Lgr5-L, Lgr5+ cell lineage tracing. Scale bars, 20 μm. (m) Lgr5+ progeny cells (mT−/mG+) migrated from the HF toward the wound area at PWD-7. (n) Lgr5+ progeny cells (mT−/mG+) formed clones in newly formed epithelium at PWD-30. (o,p) Lgr5+ progeny cells participated in the neogenic HF in wounds (o), and the number of Lgr5+ progeny cells in different neogenic HFs varied greatly as follows: the neogenic HFs without or with less than 3% of Lgr5+ lineage cells comprised 53% of all analysed neogenic HFs (50 in 94), and those with 3–30%, 30–60%, 60–90% and over 90% of Lgr5+ lineage cells comprised 16% (15 in 94), 19% (18 in 94), 8% (7 in 94) and 4% (4 in 94) of all analysed neogenic HFs, respectively (p). All 94 neogenic HFs were analysed from 5 different Lgr5+ cell lineage tracing mice. Scale bars, 50 μm. Data are expressed as the mean±s.e.m. **P<0.01, ***P<0.005, unpaired t-test, two-tailed.
