MCs originate from embryonic epidermal progenitors. (A–C) Confocal analysis of K8 and YFP immunostaining performed on vibrissae (A), paw (B), and skin (C) sections from K14-CRE/Rosa-YFP mice. (D–F) Immunofluorescence of YFP and Rab3c performed on skin sections from K14-CRE/Rosa-YFP mice. Arrowheads point to MCs. The inset in D is magnified (right). (G) Immunofluorescence of YFP performed on HF section from K14-CRE/Rosa-YFP mice (left) and bright field microscopy (right). (H) Immunofluorescence of YFP and NF200 performed on whisker section from K14-CRE/Rosa-YFP mice. Arrowheads point to nerves. DP, dermal papilla; Me, melanocytes; Ne, nerve. Bars, 20 µm.

MC turnover during adult homeostasis is ensured by epidermal progenitors. (A) Immunostaining of K8 performed on vibrissa sections from K18-CREER/Rosa-YFP treated with 15 mg TAM and analyzed 1 and 3 wk after the last injection. (B) Quantification of YFP-positive cells in MCs (n = 2 mice per time point; error bars = SEM; p-value is from the paired Student's t test). (C) BrdU immunostaining in K14 (left)- and in K8 (right)-positive cells. (D) Immunostaining of K15 and K8 in the vibrissa follicle. (E) Immunostaining of K15 in vibrissa sections from K18-CREER/Rosa-YFP analyzed 1 wk after 15 mg TAM injection. (F) Immunostaining of K8 and YFP in vibrissa sections from K15-CREPR/Rosa-YFP analyzed 5 (left) and 21 d (right) after the administration of 2.5 mg RU486 per day. (G) BrdU immunostaining in K14 (left)- and in K8 (right)-positive cells. (H) Immunostaining of K8 performed on paw sections from K14-CREER/Rosa-YFP treated with TAM for 1 mo. (A and C–G) Dashed lines delineate the basal layer of the epidermis. (A and D–G) Arrowheads point to MCs (D, E, and G) and K8– and YFP–double positive MCs (A and F). WT, wild type. Bars, 20 µm.

MCs express epithelial markers during embryogenesis. (A) Immunostaining of K8 and K14 performed on skin sections shows the presence of MCs in E17 mice. (B) Immunostaining of K8 and K20 performed on skin sections of 1-d-old mice. (C) Confocal microscopy analysis of K8 and K14 immunostaining on skin sections of E17 embryo shows that some MCs coexpress K8 and the epidermal marker K14. (D) Immunostaining of K8 and P-cadherin on back skin sections at E17 shows the expression of P-cadherin, an epidermal progenitor marker in MCs. The inset is magnified (middle and right). Arrowheads point to MCs. (A, B, and D) Dashed lines delineate the basal layer of the epidermis. IFE, interfollicular epidermis. Bars, 20 µm.

MCs do not originate from the neural crest progenitors. (A) Immunostaining of K8 and YFP performed on skin sections from Pax3-CRE/Rosa-YFP mice. (B) Immunofluorescence of K14 and YFP performed on skin sections from Pax3-CRE/Rosa-YFP mice showed that epidermal cells from the paw and back skin do not express YFP, whereas a mosaic expression of YFP could be seen in epidermal cells of the whisker. (C) Immunofluorescence of NF200 and YFP performed on skin sections from Pax3-CRE/Rosa-YFP mice. (D) Immunostaining of K8 and YFP performed on skin sections from Wnt1-CRE/Rosa-YFP mice. (E) Immunofluorescence of YFP from Wnt1-CRE/Rosa-YFP. (F and G) Immunofluorescence of K8 and YFP from Wnt1-CRE/Rosa-YFP (F) and Pax3-CRE/Rosa-YFP mice (G) showed that the percentage of MCs positive for YFP in vibrissae correlates with the degree of YFP chimerism in keratinocytes. Arrowheads point to K8– and YFP–double positive MCs. (A–E) Dashed lines delineate the basal layer (BL) of the epidermis. Ne, nerve; Vi, vibrissa. Bars, 20 µm.

Conditional deletion of Atoh1 in embryonic epidermal progenitors results in the absence of MC specification. (A and B) Immunofluorescence of K14 and K8 (A) and K14 and K20 (B) performed in newborn wild-type (WT) and Atoh1 cKO mice shows the complete loss of MCs in Atoh1 cKO mice. Arrowheads point to MCs. (C) Immunofluorescence of K8 and Rab3c in 2-mo-old wild-type and Atoh1 cKO mice. Arrowheads point to K8– and Rab3c–double positive MCs. (D) FM1-43x administration to 1-mo-old mice resulted in the uptake of the fluorescent dye in MCs of wild-type mice but not in Atoh1 cKO mice. FM1-43x labeling is still observed in peripheral nerve endings in Atoh1 cKO mice. Arrowheads point to K8– and FM1-43x–double positive MCs. (E) Electron microscopy showed the absence of cells with electron-dense neurosecretory granules (Gr) contacting with nerve endings (Ne) in Atoh1 cKO. The inset is magnified (middle). (A–E) Dashed lines delineate the basal layer of the epidermis (A–D) or the MC (E). Bars, 20 µm.