Alteration in hair cycling of the hairpoor mouse
The hairpoor mouse exhibited increased proliferation and differentiation of epidermal cells  and premature catagen followed by the early telogen at the first hair cycle , indicating aberrant HF development. Because the hairpoor mice display abnormal hair loss with aging similarly to MUHH patients, we hypothesized that HFSC may be affected in this disorder leading to abnormal HF cycle, resulting in acceleration of hair loss.
To investigate this hypothesis, we first observed and compared the hair cycle between the hairpoor and the littermate wildtype mice, including the second telogen stage (Fig. S1 and Fig. 1a). H&E staining showed that a much shortened anagen stage following the premature catagen and earlier onset of the 1st telogen in the hairpoor mouse compared to that of the wild type mouse. At P19-20, the HF of +/HrHp was at the 1st telogen stage, while HF of the wild type mouse was still at the 1st catagen, showing the earlier onset of telogen in the hairpoor mouse. Due to the shortened cycle, the onset of the 2nd telogen was much earlier and furthermore its duration was remarkably shorter in the hairpoor mouse compared to the wild type mouse. The 2nd telogen started at P35 and lasted for approximately one day (P35-P36) in the hairpoor HF, while it started at P42 and lasted for more than 2 weeks in the wild type HF (Fig 1a).
Among the telogen phased HFs of the hairpoor mouse, the clear morphological abnormality was not observed compared to the wild type HF as shown (Fig. 1b). However, HFs’ stages in the hairpoor mouse were much differed from that of the wild type mouse. At the 1st telogen, the proportion of telogen phased HF was 91.5% in the wild type mouse whereas it was only 58.3% in the hairpoor mouse. In the case of the 2nd telogen, all the wild type HF was at telogen phase, but only about 30% of the hairpoor HF were at telogen phase and 70% were already into the anagen phase (Fig. 1c). Following the short 2nd telogen, the HFs were a mixture of mostly anagen, a few catagen, and telogen phased HFs in the hairpoor mouse (data not shown).
Interestingly, the number of HF did not differ significantly between the wild type and the hairpoor mouse until P21. However, at P28, the HF number in +/HrHp was found to be drastically lower than that of the wild type mouse and the difference was maintained throughout the observed period (Fig. 1d), suggesting that the HFs’ regeneration capability seemed to have been affected by the first telogen and those defects persisted in the hairpoor mouse. These results clearly indicated that the transition of the stages of HF cycle was abnormal in the hairpoor mouse and raised a possibility of the impairment of HFSC that controls the HF cycle.
Decrease in the number of CD34+ HFSC n hairpoor mice
In order to dissect the abnormal hair cycle in the hairpoor mouse, we investigated whether there was any changes in HFSCs. First, the number of bulges expressing K15, a bulge marker, and CD34, a HFSC marker, , was determined at telogen stages using immunohistochemistry. At both telogen stages, all K15+ bulges were shown to have similar intensities for both the wild type and the hairpoor mice (Fig. 2a). In contrast to K15, CD34 expression displayed a different pattern between two lines. Almost all the K15+ bulges were CD34+ at the first telogen (7.25/7.5) and the second telogen (6.8/6.8) stages of the wild type HFs. In contrast, on average, only 45 % (3.5/7.75) and 70 % (3.8/5.4) of the K15+ bulges showed CD34+ expression at the first and second telogen of the hairpoor HFs, respectively. When the number of CD34+ bulges were determined, the bulge showing detectable expression level of CD34 was considered as CD34+ bulge. (Fig. 2b). In addition to reduced number of CD34+ bulges, there was much weaker expression in the CD34+ bulges, of the hairpoor HFs compared to those of the wild type HFs (Fig. 2a, b). Interestingly, the proportion of CD34+/K15+ bulges was higher at 2nd telogen than at the 1st telogen of the hairpoor HFs, although the expression level was much lower than that of the wild type HF,
To assess the extent of the reduced expression of CD34 in the hairpoor HFs, Cd34 mRNA expression level was determined using qRT-PCR. Relative expression level of Cd34 mRNA was significantly reduced in the hairpoor epidermis compared to that of the wild type (Fig. 2c), with less than 20% of that of the wild type mouse for both telogen stages.
We next determined the proportion of HFSCs (ITGA6+/CD34+) in the bulge at both telogen stages using FACS analysis (Fig. 2d, e). In wild type mice, ITGA6+/CD34+ cells composed of 5.2 % and 9.6 % of the bulge cells at the 1st and 2nd telogen stages, respectively. In contrast, the bulges in hairpoor mice contained only 0.2 % and 1.8 % ITGA6+/CD34+ cells of the bulge cells at the 1st and the 2nd telogen stages, respectively. This result clearly indicates that the number of HFSCs expressing CD34 was significantly reduced in the hairpoor mouse. Taken together, the hairpoor mouse contained much fewer HFSCs present in the HFs compared to the wild type, suggesting aberrance in the maintenance of HFSC in the hairpoor mouse.
Loss of HFSC quiescence and increased cell proliferation in the hairpoor HF
In order to investigate whether the quiescence of HFSC was disturbed in the hairpoor mouse, we first determined the number of label-retaining cells (LRC) in HF using 5-bromodeoxy-uridine (BrdU) incorporation method at 60 days post an injection of BrdU at P10. There was less than one BrdU+ LRC detected in the bulge of hairpoor HF while the wild type HF contained three BrdU+ LRCs on average (Fig. 3a, b). We then determined cell proliferation in HFs to delineate whether HFSCs lost quiescence. Cell proliferation was assessed by measurement of the BrdU incorporation in cells 1 hr after BrdU injection. The experiments were carried out at anagen I-II period for both wild type and hairpoor mice and revealed that the hairpoor mouse exhibited 15 BrdU+ cells/HF on average while the wild type mouse showed 0.48 BrdU+ cells/HF (Fig. 3c, d), showing excessive cell proliferation in the hairpoor HFs.
To further characterize HFSCs in the hairpoor mouse, the histological study was conducted on skin sections prepared after synchronization of HF cycling via depilation at the 2nd telogen phase. As shown in the Fig. 4a, HF cycle of the hairpoor mouse was much shorter than that of the wild type mouse. At 14 days post depilation, while the wild type HFs were still in the full anagen phase, the hairpoor HFs were into the catagen phase (Fig. 4a). This deviation in the hair cycle of the hairpoor mouse was more prominent in subsequent days. Specifically, at 21 days post depilation, the hairpoor HFs were in the anagen phase again whereas the wild type HFs were at the late catagen/early telogen phase, indicating that the telogen stage of the hairpoor HFs passed between 14 and 21 days post depilation. In contrast, the wild type HFs began the telogen stage at 21 days remain in the same stage at 28 days post depilation while the hairpoor HFs were in anagen stage again..
Cell proliferation in HFs was examined at 7 days after depilation when both the wild type and the hairpoor HFs were in anagen phase. Immunohistochemical detection of Ki67 indicated higher proliferation of the cells in the bulge and bulb of the hairpoor HFs compared to those of the wild type HFs (Fig. 4b). In addition, expression of Lymphoid enhancer-binding factor 1 (LEF1), transcription activator in the presence of ß-catenin, was expressed at both the bulges of 1st and 2nd telogen staged HFs of the hairpoor mouse (Fig. 4c). Taken together, the HFSCs in the hairpoor mouse were not able to maintain the quiescence, resulting in proliferation of the cells in HF.
Expression of HFSC markers in the hairpoor mice
To dissect the status of the HFSC of the hairpoor mice, expression of various HFSC markers were observed including LIM homeobox 2 (LHX2), SRY-box 9 (SOX9), Nuclear factor of activated T cell 1 (NFATc1), and Transcription factor 4 (TCF4) [29-33] by immunohistochemstry. LHX2, expressing in the bulge, plays an essential role in HF morphogenesis as well as in maintenance of HFSC quiescence. SOX9 is known to be associated with Shh signaling and expressed in the bulge and outer root sheath. As shown in Fig 5a and 5b, expressions of LHX2 and SOX9 were not significantly different in the 1st telogen and 2nd telogen phases of both the wild type and the hairpoor mouse HFs. Expression of other bulge markers, NFATc1 and TCF4, were also detected in the junctional zone and interfollicular epidermis, and the bulges, respectively. TCF4, shown to suppress HFSC quiescence and thus inhibit transition of HFs from telogen to anagen, was also expressed in the bulge. As with LHX2 and SOX9, no noticeable difference was observed in NFATc1 and TCF4 expressions between the hairpoor and the wild type HFs. Thus, expression of all the HFSC markers tested in this study with exception of CD34 did not show differences between the hairpoor and the wild type.
Disruption of the HFSC quiescence signaling in the hairpoor mouse
Next, we investigated whether the HFSC niche signaling was involved in the abnormal hair cycling of the hairpoor mouse. We observed the expression of K6, a HFSCs niche marker, by IHC and found that all the K15+ bulges showed K6+ in the hairpoor HFs as the same as in the wild type (Fig. S2). We also observed expression of Bmp6 and Fgf18 in the epidermis of mice, since K6+ was shown to regulate the quiescence of HFSC through expression of Bmp6 and Fgf18 . Expression of Bmp6 and Fgf18 displayed a different pattern from each other. Bmp6 expression was significantly decreased only in the 2nd telogen phase in the hairpoor HFs compared to that of the wild type HFs. In contrast to Bmp6, Fgf18 expression was significantly decreased in both 1st and 2nd telogen phases in the hairpoor HFs compared to the wild type ones (Fig. 6a, b and c). These findings demonstrate that reduced expression of Fgf18 and Bmp6 concurred with disturbance of the HFSC quiescence in the hairpoor mice, suggesting that the lack of expression of the quiescence maintaining regulators may cause disarrayed HF cycling. Next, we dissected whether the HF in the telogen phase of the hairpoor mouse was in the competent telogen state or the telogen-anagen transition state  by determining the expression of the Wnt signaling molecules in the skin. Expression of Sfrp1, a Wnt inhibitor, was reduced in the 2nd telogen of the hairpoor HFs to 40% of that of the wild type (Fig6 d) and expression of Wnt7b was increased in both 1st and 2nd telogen in the hairpoor compared to the wild type (Fig6 e). In addition, expression of Axin2 and Wnt10b mRNA was remarkably increased in the 2nd telogen of the hairpoor mouse compared to the wild type mouse (Fig. 6f, g). These observations suggest that overexpression of HR in the hairpoor mouse led to the reduction of HFSC quiescence signaling molecules in HFSC niche, and concomitantly activated the Wnt signaling pathway, resulting in transition of the hairpoor HFs from telogen to telogen-anagen transition state.