K14-cre–Driven Apc Loss Induced Aberrant Hair Follicles throughout the Epidermis
To understand the basis for delayed and abnormal hair development in the KA mutants, we conducted a histological and immunohistochemical examination (Figure 4). The hair follicle is an epidermal appendage that consists of an upper permanent portion, and a lower cycling portion that produces the hair [22,23]. The outer root sheath (ORS) is contiguous with and biochemically similar to the basal layer of the epidermis. The inner layers of the hair follicle include three concentric layers of inner root sheath and three concentric layers of hair-producing cells. At the base of the hair follicle is the germinative hair follicle bulb, which contains rapidly proliferating “matrix” cells that differentiate to populate all of the layers of the inner root sheath and the hair shaft itself [22]. During the anagen phase of the hair cycle (until P15), hair follicles of phenotypically normal mice grew deeply into the subcutaneous fat and were uniformly spaced and aligned in parallel arrays at a specific angle relative to the skin surface (Figure 4A). In contrast, KA mutant follicles were irregularly spaced and often seen as disoriented and clamped invaginations at P3 that became even more remarkable at P12 when the mutant mice were covered by fur coat (Figure 4F). Bulbs were often bent in addition to being irregularly angled to one another and their sizes and locations were often variable. Clusters of multiple invaginations or dysplastic follicular structures were frequently observed throughout the epidermis, whereas other regions showed gaps with no follicles. Serial sectioning indicated that some of the hair follicles in the P12 mutant skin were not properly formed or shorter than normal. Taken together, these features could account for the apparently delayed, followed by outgrowth, of the short and shaggy-looking fur coat of these mutant mice.
Figure 4  Histological and Immunochemical Examination of P12 Skin and Teeth
(A–E) P12 normal skin. (F–J) P12 mutant skin. (K–N) P12 normal oral cavity. (O–R) P12 mutant oral cavity. Stained with H&E for histology (A, F, K–L, O–P), Ki67 (B, G), β-catenin (C, H, M, Q), K14 (D, I, N, R), and K6 (E, J). Aberrant follicular morphogenesis, characterized by formation of irregularly spaced, nonpolarized hair follicles, in mutant skin is evident. Despite the abnormal histology, proliferation seems to be confined to hair bulb-like structures (arrows in [G], inset [G′] at higher magnification), but in mutant skin (arrows in [H], inset [H′] at higher magnification) and oral cavity (arrows in insets [Q′] at higher magnification) elevated cytosolic localization of β-catenin is detected in some cells. Scale bars: 50 μm for (A–F), (H–J); 250 μm for (K) and (O); 100 μm for (G), (L–N), (P–R); 20 μm for (Q′). Apc is a regulator of β-catenin that is important for Wnt signaling. We examined the patterns of expression of β-catenin in the affected tissues. In the normal skin, β-catenin, a member of the adherens junction complex, was found in the ORS of hair follicles and basal layer of epidermis, where K14 expression is also observed (Figure 4C and 4D), whereas the expression of K1, involucrin, and loricrin (markers for spinous and granular layers of epidermis) was only observed in the nonbasal epidermis (unpublished data). The patterns of expression of K14, K1, involucrin, and loricrin, in skin from mutant and normal littermate mice at P3–P17, showed no significant differences in the terminal differentiation (Figure 4A–4D, 4F–4I). Similarly, the pattern of expression of K6, which is normally only expressed in the suprabasal or inner layer of the ORS of the hair follicle but not in the epidermis (Figure 4E), did not change. Due to the abnormal and disorganized structure of hair follicles themselves, K6 localization highlighted the histological abnormality (Figure 4J). Yet as in the normal skin, K6 was principally seen only in the suprabasal layer of the ORS that did not colocalize with the basal markers, K14 or K5 (Figure 4I and 4J).
In normal skin, proliferating cells were detected in either the basal layer of epidermis or in germinative hair follicle bulbs at the base (Figure 4B). In the mutant skin, either BrdU incorporation or Ki67 expression was observed not only in cells in bulbs at the base of the hair follicle but also in bulb-like structures that were budding out from the ORS of the existing hair follicles (Figure 4G and 4G′). Each budding tip was becoming like a hair follicle bulb containing proliferating cells. Hence, despite the abnormal histology in the mutant skin, proliferation seems to be confined to bulb-like structures as in the normal skin (Figure 4G and 4G′). The exact locations of hair follicle bulbs were not as easy to define for some mutant follicles due to their disorganized structures. Interestingly, in the mutant skin, in addition to diffuse membrane-bound localization as in the normal skin, cells with strong cytosolic β-catenin localization were also observed frequently (Figure 4H and 4H′). These elevated β-catenin–expressing cells were usually surrounded by proliferating cells, forming bulb-like structures. Comparison of immunochemically stained serial sections showed that these intense cytosolic β-catenin stainings were usually found in either K14-positive K1-negative basal epidermis or K14-positive K6-negative basal ORS cells, and are surrounded by proliferating cells.
To determine the initiation of hair follicle morphogenesis in these mutants, we examined the expression pattern of Sonic hedgehog (Shh), a factor expressed in hair bulbs in embryonic skin (Figure 5). The aberrant hair follicle morphogenesis is evident as early as E14.5 in mutant embryonic skin, by multiple apolarized expression of Shh throughout the epidermis (Figure 5B), whereas that of control embryos was well polarized and regularly spaced (Figure 5A). With development, control mouse hair follicles invaginate downward in a polarized manner (Figure 5C), whereas those of mutant embryos were completely irregular and apolarized (Figure 5D). It was also noted that the size of each “budding” follicle, as detected by Shh expression, was variable (Figure 5D). The intensity of Shh staining was generally stronger in mutant skin than in the normal skin. The aberrant initiation of multiple hair placodes during early hair follicle morphogenesis was also evident by the whole-mount in situ hybridization (ISH) of E15.5 mutant embryos for β-catenin (Figure 5F and 5F′). The expression pattern of β-catenin in embryos clearly demonstrated the formation of regular arrays of hair placodes in the normal embryonic skin (Figure 5E and 5E′), but such regular patterning was lost, and often tightly clustered abnormal hair placodes were initiated in mutant embryonic skin (Figure 5F′). Aberrant hair placodes were also evident throughout the skin surface of limbs in E15.5 mutants (Figure 5F), whereas those of the control embryos had not yet formed (Figure 5E). Most interestingly, in the mutant footpads, where hair placodes do not normally form (Figure 5G), we also found ectopic irregularly sized and spaced hair placodes, indicating that the footpads still have the potential to form hair placodes in the absence of the Apc gene (Figure 5H).
Figure 5  Expression of Shh and β-catenin Transcripts in Normal (ApcCKO/CKO) and Mutant (K14-cre; ApcCKO/CKO) Embryonic Skin
(A–D) Section ISH with Shh probe in E14.5 normal (A), E14.5 mutant (B), E16.5 normal (C), and E16.5 mutant (D) skin. Broken lines indicate the interface between epithelium and mesenchyme. Scale bars: 50 μm. Whole mount in situ detection of β-catenin in E15.5 normal (E, G), mutant (F, H) embryos. Aberrant initiation of multiple hair placodes is evident at E14.5. Loss of K14-driven Apc loss caused aberrant pattern formation (F′) and formed ectopic hair placodes in normally hairless foot pads (H, arrows) which are absent in normal (G). These results collectively suggest that the terminal differentiation does take place normally in the mutant skin, but initiation of embryonic hair follicle morphogenesis is severely disrupted, accompanied by a continuous ectopic hair follicle morphogenesis in postnatal mutant skin.