Mice
The Animal Research Committee of Tokyo University of Agriculture and Technology approved all animal experiments (Approval No. #25–70).
The Nes-Cre/CAG-CAT-EGFP mouse line [23] was generated by crossing CAG-CAT-EGFP mice (courtesy of Junichi Miyazaki, Osaka University, Japan), in which the chloramphenicol acetyltransferase (CAT) gene is flanked by two loxP sites [14], with Nes-Cre mice (courtesy of Ryoichiro Kageyama, Kyoto University, Japan) [9]; both mouse strains have a C57BL/6 background. The Nes-CreERT2/CAG-CAT-EGFP mouse line was generated by crossing CAG-CAT-EGFP mice with Nes-CreERT2 mice (courtesies of Itaru Imayoshi and Ryoichiro Kageyama, Kyoto University, Japan), which harbor the CreERT2 gene downstream of the nestin promoter and also have a C57BL/6 background. CAG-CAT-EGFP and Nes-CreERT2 mice were provided by the Center for Animal Resources and Development at Kumamoto University. Nes-CreERT2 mice were provided by RIKEN BioResource Research Center through the National Bio-Resource Project of MEXT (Japan).
Administration of 4-hydroxy-tamoxifen (OHT) to Nes-CreERT2/CAG-CAT-EGFP mice
OHT (10 mg; Cayman Chemical; Ann Arbor, MI, USA) was dissolved in ethanol to obtain a 100 mg/mL OHT suspension, which was further dissolved in corn oil (Merck, Darmstadt, Germany) to obtain a 10 mg/mL OHT solution. Finally, a volume of solution containing 1 mg of OHT was injected intraperitoneally for 5 consecutive days into Nes-CreERT2/CAG-CAT-EGFP mice aged 4 weeks or 7 weeks.
Tissue collection
Embryonic skin was harvested from Nes-Cre/CAG-CAT-EGFP mice at various gestational ages (embryonic days 15.5, 16.5, and 18.5) when early or advanced hair germs and hair pegs were recognized [25]. Trunk skin was also sampled from this mouse line at postnatal days 0 and 33 (P0 and P33), when lanugo and anagen HFs were recognized [21]. In addition, the dorsal skin of 4-week-old Nes-CreERT2/CAG-CAT-EGFP mice administered OHT for 5 consective days was sampled on the sixth day after initiation of OHT administration (Fig. 3a). Finally, 7-week-old Nes-CreERT2/CAG-CAT-EGFP mice were administered for OHT for 5 consective days, their dorsal hairs were depilated to introduce anagen HF on the sixth day after initiation of OHT administration, and their dorsal skin was sampled on the seventh day after depilation (Fig. 3a). Skin samples were fixed with 10% neutral buffered formalin, embedded in paraffin, and subjected to immunofluorescence analysis.
Immunofluorescence analysis
Paraffin-embedded formalin-fixed skin samples were sectioned, deparaffinized, and pretreated with 10 mM citrate buffer (pH 6.8) for antigen retrieval. Sections were then incubated with blocking buffer (5% goat serum, 3% skim milk, and 0.2% Tween 20 in phosphate-buffered saline) before incubation with the following primary antibodies: monoclonal mouse anti-GFP (clone 6AT316; Abcam, Cambridge, UK), polyclonal rabbit anti-GFP (Medical & Biological Laboratories, Nagoya, Japan), polyclonal rabbit anti-laminin (Abcam), monoclonal rabbit anti-K5 (Abcam), monoclonal mouse anti-K14 (clone LL002; Abcam), monoclonal mouse anti-K15 (clone LHK15; Abcam), monoclonal rabbit anti-vimentin (VIM) (clone EPR3776; Abcam), monoclonal rabbit anti-SOX2 (clone SP76; Abcam), monoclonal rabbit anti-SOX10 (clone EPR4007-104; Abcam), monoclonal rabbit anti-S100 alpha 6 (S100A6) (clone EPR13084-69; Abcam), and monoclonal mouse anti-trichohyalin (clone AE15). Slides were then labeled with either Alexa Fluor™ 488- or 546-conjugated secondary antibodies (Life Technologies, Carlsbad, CA, USA) or combinations thereof for double immunofluorescence. Nuclei were counterstained with Hoechst 33258 (Life Technologies). Slides were examined under a confocal laser-scanning microscope (LSM710NLO 2 photon, Carl Zeiss, Jena, Germany; Nikon AX, Nikon, Tokyo, Japan) and image data were captured using imaging software (ZEN, Carl Zeiss; Nikon AX R, Nikon).
Statistical analysis
A Mann-Whitney U test was performed to compare frequencies of EGFP+ cells in epithelial cells at peg and bulbous peg stages. Furthermore, the same test was performed to compare ratios of EGFP+K14+ cells to K14+ cells of anagen HFs between Nes-Cre/CAG-CAT-EGFP and OHT-administered Nes-CreERT2/CAG-CAT-EGFP mice using Graph Pad Prism8 software (GraphPad Software, San Diego, CA, USA). A p-value of less than 0.05 was considered statistically significant.