Patterns of hairless (hr) gene expression in mouse hair follicle morphogenesis and cycling

Abstract

The hr (hairless) gene encodes a putative transcription factor with restricted expression in the skin and brain. Mutations in the hr locus cause papular atrichia in humans and complete hair loss in mice and other mammals. To further elucidate the role of hr in skin biology, and to identify potential target cells for hr regulation, we studied hr mRNA localization during hair follicle (HF) morphogenesis and cycling in normal C57BL/6J mice. In situ hybridization revealed that hr expression was present in the suprabasal cell layers of the epidermis, whereas the basal and highly differentiated keratinocytes of the granular layer were hr-negative. During the early stages of HF morphogenesis, hr mRNA was detected in the developing hair peg. Later, it became concentrated in the HF infundibulum, in the HF matrix, and in the inner root sheath (IRS), whereas the dermal papilla (DP) and outer root sheath were consistently hr mRNA-negative. During catagen, hr gene expression gradually declined in the regressing IRS, shortly but dramatically increased in the zone of developing club hair, and became up-regulated in the epithelial cells adjacent to the DP. The co-localization of hr mRNA with the site of the morphological defects in mutant skin implicates hr as a key factor in regulating basic cellular processes during catagen, including club hair formation, maintenance of DP-epithelial integrity, IRS disintegration, and keratinocyte apoptosis in the HF matrix.

Figure 1.

In situ hybridization with hr mRNA probe in C57BL mouse skin during HF morphogenesis (days 1 to 8 postpartum; A–E) and telogen-anagen stages of depilation-induced HF cycling (days 1 to 12 after depilation; F–J). A: Stage 2 of morphogenesis: the hr mRNA is absent in the basal keratinocytes (arrowheads) but present in the suprabasal cell layer and in the innermost keratinocytes (arrow) of the HF peg thus forming the upper zone of hr expression in the HF (zone 1). B: Stage 4: the downward growing portion of the HF peg below zone 1 remains hr-negative, whereas another zone (zone 2) of hr expression occurs in the bulbar HF portion. C: Stage 5: the cone-shaped IRS (zone 3) is highly positive. D: Stage 7: strong hr-mRNA positivity is observed in IRS keratinocytes, whereas the ORS remains hr-negative. E: Stages 7 to 8: the DP (arrowheads) remains hr-negative. F: Telogen: high hr mRNA-positivity is seen in the secondary HF germ (arrowhead). G: Anagen II: the keratinocytes of the downward growing HF are hr mRNA-negative, except the small cell cluster above the DP (arrowhead). H: Mid-anagen: this small zone of hr expression spreads over the HF matrix (hm) and IRS. I: in the middle portion of the HF, hr expression in Huxley’s layer (hul) of the IRS gradually decreases, along with its gradual cornification. The ORS and Henley’s layer remain hr mRNA-negative. J–Ja: In the upper portion of the anagen VI HF, hr mRNA reactivity is localized to the HF infundibulum (inf) and to the innermost cell layer of the restricted portion of the ORS (arrowhead). The sebaceous gland (sg) and the bulge (bg) area of the ORS remain negative. (apm, arrector pili muscle; ch, club hair; ep, interfollicular epidermis; hel, Henley’s layer; hul, Huxley’s layer; hm, HF matrix; hs, hair shaft; inf, HF infundibulum; k-irs, keratinized IRS). Scale bar, 23 μm.

Figure 2.

In situ hybridization with hr mRNA probe in C57BL mouse skin during catagen progression of depilation-induced HF cycling (days 17 to 21 after depilation). A: At the transition from anagen to catagen, the decline of hr expression in the HF matrix (hm) is noted (compared to Figure 3C ▶ ). B: Catagen III: the middle portion of the IRS remains hr mRNA-positive. C: Catagen V, hr expression in the upper IRS continues to decline, while it begins in the zone of club hair formation (black arrowhead). The epithelial strand (es) between the DP and the zone of club hair remains hr-negative. D: Catagen VI: hr expression in the zone of club hair formation (ch) is increased. In the IRS, it persists in the restricted lowermost still unkeratinized portion. Some faint hr mRNA positivity occurs in the epithelial strand keratinocytes adjacent to the DP (white arrowhead). E: Catagen VII: with the completion of club hair formation, hr expression in this zone decreased (black arrowhead), but slowly increased in DP-associated keratinocytes (white arrowhead). F: Catagen VII to VIII: hr expression is still present in cell cluster above the DP (white arrowhead). In situ hybridization with sense hr mRNA probe (negative control) (G ) and antisense MK1 mRNA probe (positive control, H) in C57BL mouse skin during HF morphogenesis. Basal cell layer is marked with black arrowheads. (apm, arrector pili muscle; ch, club hair; es, epithelial strand; hm, HF matrix; mc, melanocytes). Scale bars: 23 μm (A–F); 46 μm (G–H).

Figure 3.

Schematic representation of hr gene expression patterns (two different intensities, low and high, are shown in gray and black, respectively) during late anagen-catagen-telogen progression of depilation-induced HF cycling. (apm, arrector pili muscle; ch, club hair; cs, perifollicular connective tissue sheath; es, epithelial strand; hm, HF matrix; irs-r, remnants of inner root sheath; mc, melanocytes; sg, sebaceous gland; tk, zone of trichilemmal keratinization).

Figure 4.

Proposed scenario of reorganization of specific HF structures during the formation of the club hair in normal (C57BL or wild-type) and hairless (HRS/J hr/hr) mouse skin. During catagen in wild-type mouse skin, the degradation and shortening of IRS starts before the cessation of activity in the precortex. As a result of early IRS shortening, the lower end of the hair shaft comes into direct contact with ORS cells inducing trichilemmal keratinization of the club hair. In hairless mutant skin, dysregulation of apoptosis in HF matrix (A) results in early termination of hair shaft production and in delay of IRS disintegration. As a result, the IRS coalesces around the end of hair shaft (B) and the normal serrated club hair never forms. Note the loss of contact between the DP and HF in hairless skin (C). Legends as in Figures 1 to 3 ▶ ▶ ▶ . IRS is marked with black.