Nestin is a marker of unipotent embryonic and adult progenitors differentiating into an epithelial cell lineage of the hair follicles

Abstract

Nestin is an intermediate filament protein transiently expressed in neural stem/progenitor cells. We previously demonstrated that outer root sheath (ORS) keratinocytes of adult hair follicles (HFs) in mice descend from nestin-expressing cells, despite being an epithelial cell lineage. This study determined the exact stage when nestin-expressing ORS stem/precursor cells or their descendants appear during HF morphogenesis, and whether they are present in adult HFs. Using Nes-Cre/CAG-CAT-EGFP mice, in which enhanced green fluorescent protein (EGFP) is expressed following Cre-based recombination driven by the nestin promoter, we found that EGFP⁺ cells appeared in the epithelial layer of embryonic HFs as early as the peg stage. EGFP⁺ cells in hair pegs were positive for keratin 14 (K14) and K5, but not vimentin, SOX2, SOX10, or S100 alpha 6. Tracing of tamoxifen-induced EGFP⁺ cells in postnatal Nes-CreERT2/CAG-CAT-EGFP mice revealed labeling of some isthmus HF epithelial cells in the first anagen stage. EGFP⁺ cells in adult HFs were not immunolabeled for K15, an HF multipotent stem cell marker. However, when hairs were depilated in Nes-CreERT2/CAG-CAT-EGFP mice to induce the anagen stage after tamoxifen injection, the majority of ORS keratinocytes in depilation-induced anagen HFs were labeled for EGFP. Our findings indicate that nestin-expressing unipotent progenitor cells capable of differentiating into ORS keratinocytes are present in HF primordia and adult HFs.

Figure 1

Presence of EGFP⁺ cells in the HFs of neonatal Nes-Cre/CAG-CAT-EGFP mice. Truncal skin of Nes-Cre/CAG-CAT-EGFP (A, C, D, E) and CAG-CAT-EGFP (B) mice was collected at P0 and immunolabeled for EGFP by monoclonal anti-GFP antibody (green). Double-immunolabeling for EGFP (green) and (C) K14 (red), (D) K5 (red), or (E) trichohyalin (red) in the outer layers of lanugo HFs of Nes-Cre/CAG-CAT-EGFP mice at P0. Nuclei were counterstained by Hoechst 33258 (blue). Scale bars, 20 μm.

Figure 2

EGFP⁺ cells with characteristics of epithelial cells in Nes-Cre/CAG-CAT-EGFP mouse embryos during the hair peg stage. (a) Time-course analysis of EGFP expression in epithelial cells of embryonic HFs in Nes-Cre/CAG-CAT-EGFP mice. Skin collected at germ (A), advanced germ (B), peg (C, D), and bulbous peg (E) stages was subjected to immunolabeling for EGFP (green) and laminin (white). Scale bars, 10 μm (A, B, C, E), 5 μm (D). (b) Immunolabeling for EGFP (A, E, I, M, R), K5 (B), VIM (F), SOX2 (J), SOX10 (N), and S100A6 (S) in hair peg epithelia. Nuclei were counterstained by Hoechst 33258 (C, G, K, O, T). A subset of cells in anagen hair bulbs, presumably melanocytes, were immunolabeled for SOX10 (Q). Scale bars, 10 μm (D, H, L, P, U), 20 μm (Q).

Figure 3

EGFP⁺ cells are present in postnatal HF epithelia and differentiate into ORS keratinocytes. (a) Scheme of OHT induction to express EGFP under the nestin promotor, depilation of truncal hairs, and sample collection. (b) Immunolabeling of EGFP (A, B, F, J, N), K14 (C, K), and K15 (G, O) in first-anagen HFs (A–I) and depilation-induced anagen HFs (J–Q) of Nes-CreERT2/CAG-CAT-EGFP mice. EGFP⁺ cells in the isthmus region of first-anagen HFs (A, B, F) were immunolabeled for K14 (C) but not K15 (G). The majority of EGFP⁺ cells in depilation-induced anagen HFs were immunolabeled for K14 (K), but EGFP⁺ cells in the isthmus region were not immunolabeled with K15 (O) as in the first-anagen HFs. Nuclei were counterstained by Hoechst 33258 (D, H, L, P). Scale bars, 20 μm (A, M), 10 μm (E, I, Q). (c) Comparison of frequencies of EGFP⁺K14⁺ cells in K14⁺ cells in first-anagen HFs with depilation-induced anagen HFs of OHT-administered Nes-CreERT2/CAG-CAT-EGFP mice (Student’s t-test). ****, p < 0.0001.