**5. Dysregulation of HFSC marker expression**

The hair follicle at telogen is composed of several compartments, including the interfollicular epidermis, infundibulum, isthmus, bulge, and hair germ. Recent studies have identified multiple new stem cell and progenitor populations in each compartment, which exhibits unique marker expression profiles (**Figure 3**) [31, 32].

### **5.1. Bulge**

**Figure 2.** Hair loss phenotype of mutant mice. Macroscopic presentation of a one-year-old control (left) and a K5-cKO

During postnatal morphogenesis stages (P0–P16), the hair cycling of mutant hair follicles appeared to proceed normally, although the mutant integument became histologically noticeable with a thickened interfollicular epidermis (IFE) and enlarged sebaceous glands [22, 23]. However, entry into the first postnatal telogen (resting phase, normally starts around P18) was delayed in the mutant hair follicles. At P28, when control hair follicles entered into the first anagen (growth phase), they still had a long epithelial strand, a characteristic structure of catagen (regression phase) [27], and were positive for placental cadherin (P-cadherin), a marker for the epithelial strand. As a result, the start of the first anagen was delayed until P37 in the K5-cKO mice. In the K14-KO mice, the percentages of hair follicles that properly entered into

Moreover, mutant hair follicles were morphologically abnormal. They exhibited hyperkeratotic plugs and cyst-like structures with an expanded infundibulum and isthmus. Strikingly, these severely deformed mutant hair follicles regrew and entered into the second anagen much later than the control hair follicles. However, the mutant hair follicles in anagen did not proceed further into the second catagen or telogen. Instead, they started to degenerate, as revealed by the shrinking hair bulbs and reduced expressions of Ki67 and Lef1 [23]. In one-year-old mutant

Fibroblast growth factor 18 (Fgf18) is expressed in hair follicles and colocalized with keratin 15 (K15) and CD34 [28, 29], both of which are expressed in the bulge region at telogen. Fgf18 shows a cyclic expression pattern in hair follicles; its levels are low in anagen and high throughout telogen [29]. In mutant mice with epidermis-specific loss of Fgf18, the length of telogen was short, resulting in rapid succession of hair cycling [29]. Interestingly, the expres-

mouse (right). Note that the vibrissae were lost in the mutant mouse (arrows).

catagen, telogen, and anagen were significantly reduced.

mice, the number of hair follicles was severely diminished.

**3. Abnormal hair cycling**

32 Hair and Scalp Disorders

In the skin, label-retaining cells (LRCs) having a highly proliferative activity reside in the bulge region of the hair follicle [33], and LRCs in the bulge are multipotent [34, 35]. Since the first identification of K15 as a bulge marker [36], several factors have been demonstrated to be expressed in the bulge region at telogen, such as CD34 [37], leucine-rich repeat-containing G protein-coupled receptor 5 (Lgr5) [38], and transcriptional factors Sox9 [39, 40], Tcf3 [41], Lhx2 [42, 43], and nuclear factor of activated T cells c1 (Nfatc1) [44]. CD34, a hematopoietic stem and progenitor cell marker, colocalizes with LRCs and K15 expression in the bulge region [37]. Lgr5 marks the lower bulge and secondary hair germ during telogen, and contributes to all hair lineages, but not to the epidermis and sebaceous glands [38]. Sox9 expressing cells also contribute to all skin epithelial lineages [39, 40]. Tcf3 and Tcf4 are downstream targets of Wnt signaling, and in Tcf3/Tcf4-null mice, hair follicle formation was initiated, but further development was severely impaired [45]. Lhx2 maintains the HFSC character downstream of Wnt and Shh signaling [43]. Nfatc1 colocalizes with other bulge stem cell markers, including CD34, Sox9, Tcf3, and Lhx2 [44]. Nfatc1 mediates HFSC quiescence by transcriptionally suppressing cyclin-dependent kinase 4 (CDK4) expression upstream of BMP4 signaling [44].

**Figure 3.** Distinct stem cell populations in the hair follicle. The hair follicle is divided into the interfollicular epidermis (IFE), infundibulum, junctional zone (JZ)/isthmus, sebaceous gland, bulge, and hair germ. The expressions of proposed stem cell markers at telogen are indicated. Note that the expression domains of these markers dynamically change during hair cycling. A murine hair follicle in telogen is immunostained for keratin 10 (a marker for suprabasal cells, green) and keratin 15 (a marker for bulge stem cells, red). Nuclei are counterstained with 4′, 6-diamidino-2-phenylindole. The dotted area indicates the sebaceous gland.

Comparison of the expression profiles of these bulge stem cell markers between the early and late stages in mutant mice demonstrated that the expressions of these markers diminished at later stages or were mislocalized to areas outside the bulge [22, 23]. These results indicate that aPKCλ regulates the expression and localization of HFSCs.

#### **5.2. Junctional zone/isthmus**

Markers that recognize distinct cell populations of the upper (junctional zone) and lower bulge regions have been identified. As mentioned above, Lrig1 is a marker for the junctional zone/ isthmus, which is located between the sebaceous gland and the bulge [30]. Lrig1-expressing cells have a bipotent activity in the steady state, and give rise to the IFE and sebaceous glands [30]. The cell-surface glycoprotein MTS24 is another marker for the isthmus/junctional zone [46]. MTS24-expressing cells do not express CD34 or keratin 15, and LRCs are infrequently observed among them. Lgr6 is expressed in a distinct population between the upper K5- and CD34-expressing cells and lower MTS24- and Lrig1-expressing cells in the bulge region [47]. Although prenatal Lgr6-positive cells contribute to the IFE, sebaceous glands, and hair follicles, the contribution to the hair follicles diminishes with age. In mutant hair follicles, the expression domains for Lrig1 and MTS24 were considerably expanded.
