1. ^*Department of Dermatology, Charite, Humboldt Universitat zu Berlin, Berlin, Germany
2. ^‡Department of Neurobiology, Millenium Pharmaceuticals, Cambridge, Massachusetts, U.S.A.
3. ^†Department of Dermatology, University of Hamburg, Hamburg, Germany

Correspondence: Prof R. Paus, Department of Dermatology, University Hospital Eppendorf, University of Hamburg, Martinistr. 52, D-20246 Hamburg, Germany E-mail: paus@uke.uni-hamburg.de

Received 3 May 1999; Accepted 28 May 1999.

To the Editor:

Increasing evidence suggests that neurotrophins not only control neuronal development, plasticity, and maintenance (^{Lewin & Barde 1996;Bothwell 1997}), but also are critically involved in the control of hair follicle (HF) development and growth (^{Holbrook et al.1993;Crowley et al.1994;Botchkarev et al.1998a,1999a}).

Specifically, we have recently shown that neurotrophin-3 (NT-3) is functionally important for HF morphogenesis, as its overexpression or partial deletion in mice leads to a significant acceleration or retardation of HF development, respectively (^{Botchkarev et al.1998a}). As a member of the neurotrophin family, NT-3 shows multiple interactions with all types of neurotrophin receptors: NT-3 binds with high affinity to the tyrosine kinase C (TrkC) receptor, as well as with low affinity to the tyrosine kinase A (TrkA), tyrosine kinase B (TrkB), and p75 kDa neurotrophin receptor (p75NTR) (^{Lewin & Barde 1996;Bothwell 1997;Dechant & Barde 1997}).

Because all four receptors are expressed in the HF epithelium or mesenchyme during defined stages of HF morphogenesis (^{Botchkarev et al.1998a}),¹,² the target receptor(s) that mediate the stimulatory effects of NT-3 on HF development are still unclear. TrkC, the high-affinity receptor for NT-3, is expressed by the hair placode epithelium during the initial steps of HF development, whereas in the fully developed HF TrkC expression appears in the dermal papilla, outer root sheath, and hair matrix (^{Botchkarev et al.1998a}). In order to explore the relative contribution of TrkC signaling in the control of HF, we have studied HF morphogenesis in the back skin of TrkC knockout (–/–) mice, generated in a mixed background as described previously (^{Klein et al.1994}).

At day 5 of postnatal developments (P5), back skin of TrkC knockout (n = 3) and corresponding age-matched wild-type (n = 3) mice was dissected at the level of subcutis, immediately transferred to liquid nitrogen and embedded as described (^{Paus et al.1994}). Eight micrometer cryostat sections were prepared from frozen skin samples, and histochemical detection of endogenous alkaline phosphatase activity was performed to identify precisely the defined stages of HF morphogenesis (^{Handjiski et al.1994}). The percentage of HF in different stages of morphogenesis was assessed and calculated on the basis of accepted morphologic criteria (^{Paus et al.1997;Philpott & Paus 1998}). Only every tenth cryosection was used for analysis in order to exclude the repetitive evaluation of the same HF, and 2–3 cryosections were assessed from each animal. A total of 150–350 follicles in 50–60 microscopic fields (approximately 50–60 follicles per animal) were analyzed and compared with that of a corresponding number of HF from the appropriate, age-matched wild-type mice. Thickness of skin was assessed as described before (^{Botchkarev et al.1998b,1999b}). All sections were analyzed at 200 magnification, and means and SEM were calculated from pooled data. Differences were judged as significant if the p value was < 0.05, as determined by independent Student's t test for unpaired samples. Photo-documentation was performed with the help of a digital image analysis system (ISIS METASYSTEMS, Altlussheim, Germany).

Comparative, quantitative histomorphometry (^{Botchkarev et al.1998a}) showed considerable, statistically significant differences in HF development between TrkC null mice and age-matched wild-type control. In contrast to wild-type mice, where all HF were in the latest stages of morphogenesis (6–8), TrkC mutants showed a dramatic retardation of HF development. This was evident by the predominance of stages 4–7 HF (p < 0.005), and the marked reduction in the number of stage 8 HF in TrkC null mice (p < 0.005) (Figure 1a,c,d). Furthermore, as an important indirect indicator for a retarded HF development (^{Botchkarev et al.1998a;Philpott & Paus 1998}), skin thickness in TrkC mutants was significantly lower (p < 0.005) than in wild-type mice (Figure 1b–d).

Figure 1.

Retardation of hair follicle morphogenesis in TrkC knockout mice. The percentage of HF in defined stages of morphogenesis was evaluated by quantitative histomorphometry using established morphologic criteria in cryostat sections of the skin of TrkC knockout and wild-type mice at P5. (A) Dynamics of HF morphogenesis in TrkC knockout and wild-type mice show a presence of HF in stages 4–6 in TrkC knockout mice, a significant increase in the percentage of HF in stage 7 of HF morphogenesis, and a decline of HF at stage 8 in TrkC knockout mice, compared with wild-type animals. (B) Skin thickness in TrkC mutants is significantly declined at P5, compared with wild-type mice. (C, D) Representative skin examples of wild-type (C) and TrkC knockout mice (D) at P5. HF at different stages of development are indicated by arabic numbers. EP, epidermis; PCM, panniculus carnosus muscle. Scale bars: 200 m.

Full figure and legend (27K)

Although the precise roles for other neurotrophin receptors (TrkA, TrkB, p75NTR) in the mediation of the effects of NT-3 on HF development remain to be elucidated, the current data fully corroborate our findings in NT-3 mutants (^{Botchkarev et al.1998a}), and suggest that NT-3 most likely stimulates HF morphogenesis via its high-affinity TrkC receptor. Recent observations in other models suggest that expression of NT-3 and TrkC are upregulated by Wnt- and BMP-family members (^{Kobayashi et al.1988;Zhang et al.1998;Patapoutian et al.1999}), which are shown to be critical for HF development (^{Philpott & Paus 1998;Millar et al.1999;Botchkarev et al.1999c}). Therefore, serving as a potential target for Wnt- and/or BMP-regulation, NT-3/TrkC signaling represents an important stimulatory component in the inductive signaling cascade driving HF morphogenesis.