**7. Loss of HFSC quiescence**

Mutant mice analyses have identified intrinsic and paracrine mechanisms to clarify the loss of HFSC quiescence caused by epidermis-specific inactivation of aPKCλ.

#### **7.1. Intrinsic mechanism: oriented cell division**

Oriented cell division is crucial for tissue morphogenesis and homeostasis [49, 50]. Basal cells in the epidermis show the following two types of cell division: symmetric cell division (SCD) and asymmetric cell division (ACD). SCD, in which alignment of the mitotic spindle is parallel to the basement membrane, results in two equivalent daughter cells, whereas ACD, in which alignment of the mitotic spindle is perpendicular to the basement membrane, results in two daughter cells with different fates (one basal cell and one more differentiation-committed suprabasal cell) (**Figure 4**).

In the epidermis, a balance between SCD and ACD is important for coordinated proliferation and differentiation. A shift from SCD to ACD in basal cells of the developing epidermis coincides with the onset of stratification [51]. At embryonic day 12.5 (E12.5), most of the murine epidermis is single-layered, and the majority of cell divisions (>90%) are symmetric, whereas at E14.5–18.5, more than 70% of cell divisions are perpendicular to the basement membrane [51]. ACD promotes Notch signaling, leading to epidermal differentiation [52]. During SCD and ACD, the aPKC-Par complex localizes at the apical surface in a β1-integrin- and α-catenindependent manner [51]. Thus, in the β1-integrin KO and α-catenin KO epidermis, apical localization of the aPKC and Par3-LGN-inscuteable complex is abolished. Niessen et al. demonstrate that epidermal loss of aPKCλ induced a shift toward ACD not only in the IFE but also in the bulge stem cells and the junctional zone/isthmus region, leading to an expansion of progenitor cell populations committed to epidermal cell fate [22].

**Figure 4.** Localization of aPKC in the simple epithelium and multilayered epidermis. (A) In the simple epithelium, aPKC forms a ternary complex with Par3 and Par6 and is localized to the apical surface of the cell and tight junctions (yellow circles). (B) In the stratified epidermis, tight junctions are only present in the granular layer. During symmetric cell division, in which alignment of the mitotic spindle is parallel to the basement membrane (gray), and during asymmetric cell division, in which alignment of the mitotic spindle is perpendicular to the basement membrane, aPKC (pink) is localized to the apical surface of the cell.

Lineage tracing analysis using Lgr5-Cre mice confirmed that epidermal loss of aPKCλ changes the fate of a bulge stem cell to an epidermal lineage [22]. In telogen, Lgr5-positive cells resided in the lower bulge and hair germ, as described above. During anagen, Lgr5 progeny exclusively contributed to down-growing hair follicles, whereas upon loss of aPKCλ, Lgr5-positive cells contributed to the upper junctional zone/isthmus and the IFE, as well as the lower-growing hair follicles. Consistent with this the expression domains for Lig1 and MTS24 increased. These findings indicate that aPKCλ regulates oriented cell division and thereby controls epidermal stem cell behavior and cell fate decisions.

#### **7.2. Paracrine mechanism**

HFSCs remain quiescent during telogen. Near the end of telogen, the HFSCs become activated to elicit the growth phase of the hair cycle. Basically, Bmp signaling induces quiescence and Wnt signaling activates HFSCs. However, the molecular mechanism underlying the cyclic inhibition and activation of HFSCs has recently started to be elucidated. Three types of Bmps from different sources induce quiescence in HFSCs: Bmp2 from subcutaneous adipocytes, Bmp4 from dermal fibroblasts [53], and Bmp6 from the inner layer of the bulge (**Figure 5**) [54]. Bmp antagonism is one of the key concepts to understand morphogenesis. In the telogen to anagen transition, the dermal papilla secretes HFSC-activating factors, Fgf7, Fgf10, Wnts, and Tgfβ2 [55], and Bmp inhibitors, such as noggin [53, 56], overcome the inhibitory effects of Bmps to activate HFSCs (**Figure 5**) [57].

and ACD, the aPKC-Par complex localizes at the apical surface in a β1-integrin- and α-catenindependent manner [51]. Thus, in the β1-integrin KO and α-catenin KO epidermis, apical localization of the aPKC and Par3-LGN-inscuteable complex is abolished. Niessen et al. demonstrate that epidermal loss of aPKCλ induced a shift toward ACD not only in the IFE but also in the bulge stem cells and the junctional zone/isthmus region, leading to an expansion of

**Figure 4.** Localization of aPKC in the simple epithelium and multilayered epidermis. (A) In the simple epithelium, aPKC forms a ternary complex with Par3 and Par6 and is localized to the apical surface of the cell and tight junctions (yellow circles). (B) In the stratified epidermis, tight junctions are only present in the granular layer. During symmetric cell division, in which alignment of the mitotic spindle is parallel to the basement membrane (gray), and during asymmetric cell division, in which alignment of the mitotic spindle is perpendicular to the basement membrane, aPKC

Lineage tracing analysis using Lgr5-Cre mice confirmed that epidermal loss of aPKCλ changes the fate of a bulge stem cell to an epidermal lineage [22]. In telogen, Lgr5-positive cells resided in the lower bulge and hair germ, as described above. During anagen, Lgr5 progeny exclusively contributed to down-growing hair follicles, whereas upon loss of aPKCλ, Lgr5-positive cells contributed to the upper junctional zone/isthmus and the IFE, as well as the lower-growing hair follicles. Consistent with this the expression domains for Lig1 and MTS24 increased. These findings indicate that aPKCλ regulates oriented cell division and thereby controls epidermal

HFSCs remain quiescent during telogen. Near the end of telogen, the HFSCs become activated to elicit the growth phase of the hair cycle. Basically, Bmp signaling induces quiescence and Wnt signaling activates HFSCs. However, the molecular mechanism underlying the cyclic inhibition and activation of HFSCs has recently started to be elucidated. Three types of Bmps from different sources induce quiescence in HFSCs: Bmp2 from subcutaneous adipocytes, Bmp4 from dermal fibroblasts [53], and Bmp6 from the inner layer of the bulge (**Figure 5**) [54]. Bmp antagonism is one of the key concepts to understand morphogenesis. In the telogen to anagen transition, the dermal papilla secretes HFSC-activating factors, Fgf7, Fgf10, Wnts, and

progenitor cell populations committed to epidermal cell fate [22].

(pink) is localized to the apical surface of the cell.

36 Hair and Scalp Disorders

stem cell behavior and cell fate decisions.

**7.2. Paracrine mechanism**

**Figure 5.** Current model for the maintenance of HFSC quiescence. A hair follicle at telogen is presented. K6-positive cells mark the inner layer of the bulge, from which quiescence-inducing Bmp6 and Fgf18 are produced. The HFSCs are kept quiescent by Bmp2 from subcutaneous adipocytes and Bmp4 from dermal fibroblasts. At the telogen to anagen transition, HFSC-activating factors, such as Fgf7, Fgf10, Wnts, Noggin (Bmp antagonist), and Tgfβ2, antagonize the inhibitory effects of quiescence-inducing factors.

The inner layer of the bulge has attracted attention as a source of the quiescence-inducing factors Bmp6 and Fgf18 [54]. Although induction of K6 expression is closely associated with hyperproliferative conditions [58, 59], such as psoriasis and squamous cell carcinoma, a recent study has clarified that during telogen, K6 is strongly expressed in the inner layer of the bulge and K6-positive bulge cells secrete Bmp4 and Fgf18 to inhibit proliferation of the CD34-positive outer bulge stem cells [54]. Thus, upon the ablation of K6-positive bulge cells, HFSCs become prematurely activated and enter a new cycle of hair growth [54]. In the K5-cKO mice, K6 expression in the inner bulge cells was abolished, and the expressions of Bmp6 and Fgf18 were suppressed at the mRNA and protein levels [23], suggesting that aPKCλ regulates HFSC quiescence upstream of Bmp6 and Fgf18. Moreover, because K6-positive bulge cells are also involved in intercellular junctions that anchor the old hair shaft [54], a decrease in the expression of K6 in the inner layer of the mutant bulge may be involved in the falling-off of hair shafts in mutant hair follicles.
