Epidermal Stem Cells in the Isthmus/Infundibulum Influence Hair Shaft Differentiation: Evidence from Targeted DLX3 Deletion

In the skin, distinct stem cell populations are present in the epidermis and the pilosebaceous unit (hair follicle and sebaceous gland; ; ). Recent studies have highlighted the strong heterogeneity and compartmentalization of stem cell niches in the skin (; ). During telogen (resting phase), hair follicle stem cells are located in the lower part of the hair follicle, in the bulge and hair germ areas, and express markers such as Lgr5, K15, and CD34 (; ). Upon reentry into anagen (growing phase), components of the new hair are derived from these hair follicle stem cells. Even though they do not contribute to the homeostasis of the epidermis, these cells do participate in regeneration in the context of wound healing (; ; ). Recently, epidermal stem cells were identified in the upper part of the hair follicle: in the isthmus that is located near the attachment of the sebaceous gland and in the infundibulum area, which spans the isthmus and the epidermis. These cells express specific markers, such as Lrig1 and MST24, and contribute to the formation of the sebaceous gland and to epidermal differentiation in response to injury (; ). During anagen, the cells from the isthmus/infundibulum area do not contribute to the formation of the new hair. However, here we show that the deletion of the transcription factor DLX3 in the epidermis and isthmus/infundibulum area, but not in the bulge region, leads to altered hair shaft differentiation without affecting hair growth.

We previously showed that during hair morphogenesis DLX3 is expressed in the hair matrix at the beginning of hair shaft differentiation and subsequently in most layers of the hair follicle except for the outer root sheath. Epithelial deletion of DLX3 during embryogenesis (DLX3^K14Cre-cKO) results in impaired expression of hair keratins and leads to alopecia (). Absence of DLX3 in the epidermis also results in an IL-17-dependent inflammatory response in the skin (). During telogen, DLX3 expression is found in the bulge, which generates the new hair shaft in the subsequent anagen stage, as well as in the isthmus/infundibulum area (Figure 1a). DLX3 expression near the collar of the sebaceous gland persists during anagen (Figure 1a). However, DLX3 was not detected in the sebaceous gland (Figure 1b). Co-staining with Lrig1 demonstrated that DLX3 expression overlaps with the expression of Lrig1 in epidermal stem cells in the infundibulum (Figure 1c).

In order to address the function of DLX3 in this subpopulation of isthmus/infundibulum stem cells, we used the inducible K14CreERT mouse line. Using topical tamoxifen-treatment conditions (suboptimal dose for 5 consecutive days, Figure 1d; see Supplementary Material online for detailed experimental procedures) established by tracing the cells undergoing cre recombination after tamoxifen treatment in K14CreERT;R26R^YFP line, we obtained cre recombination in the epidermis and isthmus/infundibulum area, but not in the bulge (P56; Figure 1e, left panel). To confirm that the bulge cells rarely underwent cre recombination in these conditions, we induced anagen by depilation at P56 and analyzed the distribution of yellow fluorescent protein (YFP)-positive cells in fully grown hair 14 days after depilation (PD14). At this stage, YFP-positive cells were largely detected in the epidermis and isthmus/infundibulum area, but very few hair follicles exhibited YFP-positive cells in the newly formed bulb derived from the bulge (Figure 1e, right panel). Therefore, these conditions were used to delete DLX3 in the epidermis and isthmus/infundibulum (Figure 1f) without affecting its expression in the bulge in the majority of hair follicles.

DLX3^{K14CreERT-cKO} mice were generated and treated as described above for K14CreERT;R26R^YFP mice, and specimens were collected 6 days (PD6) and 14 days (PD14) after depilation (Figure 2a). The gross appearance showed that at PD6 there was similar progression of hair growth between DLX3^{K14CreERT-cKO} and control mice (Figure 2a). However, whereas the newly grown coat appeared smooth in control mice at PD14, it appeared rough in the depilated area of DLX3^{K14CreERT-cKO} mice (Figure 2a). The overall histology of the newly formed hair follicles was not significantly affected at PD6 and PD14 (Figure 2a). Consistent with the lineage analysis using K14CreERT;R26R^YFP mice, DLX3 was deleted in the epidermis and isthmus/infundibulum (at PD6 and PD14), whereas the expression in the bulb of the newly formed hair follicle was not affected in the vast majority of hair follicles (Figure 2b). In addition, the expression of hair keratins that are known targets of DLX3 () was not affected (Figure 2c). However, scanning electron microscopy analysis of the hair shaft at PD14 revealed major structural defects in the cuticle in DLX3^{K14CreERT-cKO} mice (Figure 2d). Although newly grown hair shafts in the depilated area of control mice exhibited a regular parallel distribution, hairs in the depilated area of DLX3^{K14CreERT-cKO} grew in a disorganized pattern, which was consistent with the rough appearance of the coat (Figure 2d). Moreover, higher resolution images revealed that most of these hairs showed different degrees of cuticle defects such as the detachment of cuticle scales, the formation of globular structures at the surface of the cuticle, or the complete absence of cuticle scale pattern that appeared normal in all hairs in control mice (Figure 2d). These results demonstrate that the absence of DLX3 in the isthmus/infundibulum has a deleterious effect on hair shaft formation, although the pattern of sebum distribution is comparable between wild-type and DLX3^{K14CreERT-cKO} mice (Figure 2e).

In this study we show that even though DLX3 is still expressed in the hair matrix, its deletion in the isthmus/infundibulum affects the integrity of the hair shaft. Therefore, the DLX3-expressing cells of the isthmus/infundibulum that form a collar surrounding the developing hair shaft have an important role in maintaining the structural integrity of the growing hair. On the basis of these observations, we propose a model in which the differentiation of hair shaft cells is initiated in the hair matrix, but as the cells move to the surface of the epidermis, the surrounding cells in the isthmus/infundibulum signal to the underlying hair shaft and contribute to the final cuticle structure formation. These signals may be relayed by the adjacent inner root sheath where altered TACE-TGFα-EGFR signaling was recently shown to cause cuticular abnormalities (). Although the signals from the isthmus/infundibulum to the developing hair shaft remain to be elucidated, our results identify DLX3 as a regulator of these signals in the isthmus/infundibulum.

All animal studies have been approved by the Animal Use and Care Committee at the National Institute of Arthritis and Musculoskeletal and Skin Diseases.

We thank members of the NIAMS Light Imaging Core Facility. We thank S Yuspa for comments and suggestions. This research was supported by the Intramural Research Program of the National Institute of Arthritis and Musculoskeletal and Skin Diseases of the National Institutes of Health.

Supplementary material is linked to the online version of the paper at http://www.nature.com/jid