Notch signaling via Hes1 transcription factor maintains survival of melanoblasts and melanocyte stem cells

Abstract

Melanoblasts (Mbs) are thought to be strictly regulated by cell-cell interactions with epidermal keratinocytes, although the precise molecular mechanism of the regulation has been elusive. Notch signaling, whose activation is mediated by cell-cell interactions, is implicated in a broad range of developmental processes. We demonstrate the vital role of Notch signaling in the maintenance of Mbs, as well as melanocyte stem cells (MSCs). Conditional ablation of Notch signaling in the melanocyte lineage leads to a severe defect in hair pigmentation, followed by intensive hair graying. The defect is caused by a dramatic elimination of Mbs and MSCs. Furthermore, targeted overexpression of Hes1 is sufficient to protect Mbs from the elimination by apoptosis. Thus, these data provide evidence that Notch signaling, acting through Hes1, plays a crucial role in the survival of immature Mbs by preventing initiation of apoptosis.

Figure 1.

Activation of Notch signaling in Mbs in the E16.5 epidermis. (A and B) Activation of Notch1 in Mbs. (A) Immunofluorescence staining showing antibodies for NICD1 (green) and Dct (red). Arrowheads indicate Mbs in the epidermis. (B) Negative control staining for NICD1. (C and D) In situ hybridization staining of Jagged2. (C) Jagged2 (red) and anti-Keratin5 (K5) antibody (green). (D) Negative control staining using Jagged2 sense probe. (E and F) Expression of Hes family genes in Mbs. RT-PCR (E) and Q-PCR (F) analyses were performed using purified Mbs from the E16.5 epidermis. (F) Expression value of each transcript relative to Hes1 was calculated. Data represent the mean of three data points. Error bars indicate SD. Note that Hes1 expression is the most prominent among Hes family genes, whereas expression of Hes5 and Hey1 is much less intensive and might represent transcripts present in contaminating cells (Fig. S1, available at http://www.jcb.org/cgi/content/full/jcb.200509084/DC1). (G) Activation of Hes1 promoter in Mbs. Immunofluorescent staining of E16.5 pHes1-d2EGFP epidermis is shown.

Figure 2.

The number of MC lineage cells is dramatically reduced by MC lineage–specific knock out of RBP-J. (A–F) Coat-color phenotype of a Tyr-Cre;RBP-J^f/f mouse. P4 (A), P18 (B), and P32 (C) Tyr-Cre;RBP-J^f/f mice with their control littermates are shown. Hairs from a P18 Tyr-Cre;RBP-J ^+/f mouse (D) are fully pigmented, whereas hairs from a P18 Tyr-Cre; RBP-J^f/f mouse (E) are a mixture of pigmented and unpigmented. Hairs grown in the next hair cycle from a P32 Tyr-Cre;RBP-J^f/f mouse (C, shaved area; and F) are almost all unpigmented compared with the mixture of the primary and secondary hairs (C, unshaved area). (G–I) Immunofluorescent staining of P4 skin using anti-Dct (green) antibody. Skin from wild-type (WT; G), Tyr-Cre;RBP-J ^+/f (H), and Tyr-Cre; RBP-J^f/f (I) mice are shown. Arrowheads indicate MCs in the HF. (J–M) Whole-mount staining of dorsal skin epidermis from E16.5 (J and K) and P0 (L and M) mice using anti– c-Kit antibody. Epidermis obtained from a Tyr-Cre;RBP-J ^+/f (J and L) or a Tyr-Cre;RBP-J^f/f mouse (K and M) is shown.

Figure 3.

Inhibition of Notch signaling in skin organ culture induces apoptosis of Mbs. (A–D) Elimination of Mbs from DAPT-treated skin. The E13.5 dorsal skin was cultured with (A and B, right) or without (A and B, left) 1 μM DAPT for 4 d and transplanted onto nude mice (A and B) or processed for immunofluorescent staining using antibodies against the Mb-specific markers Pax3 and Dct (C and D). (E–M) Induction of apoptosis of Mbs in embryonic skin treated with DAPT. E15.5 dorsal skin was cultured in the presence or absence of DAPT for 24 h. After organ culture, skin sections were processed for either TUNEL staining (E–G) or anti–cleaved caspase 3 immunostaining (H–M). TUNEL-positive cells are shown in green, Dct-positive Mbs in red, and nuclear staining in blue (E and F). (G) Plotted percentages of TUNEL-positive Mbs, on average, from four sections of either control or DAPT-treated skin show a significant difference (**, P < 0.01). Cleaved caspase 3–positive cells are shown in green (H and K), Dct-positive Mbs in red (I and L), and nuclear staining in blue. Merged images are shown in J and M. Arrowheads indicate apoptotic Mbs in the epidermis. (N) Down-regulation of Hes1 expression in Mbs after DAPT treatment for 24 h. Deference in Hes1 expression in between DAPT-treated and control Mbs was calculated based on the ΔΔCt method by Q-PCR. Error bars indicate SD.

Figure 4.

Constitutive expression of Hes1 promotes survival of Mbs in Dct-Hes1 Tg mice. (A) Specific expression of Hes1 transgene in Mbs in Dct-Hes1 Tg mice. RT-PCR analysis for either Hes1 transgene or β-actin is shown. Mbs and non-Mbs (see supplemental text, available at http://www.jcb.org/cgi/content/full/jcb.200509084/DC1) were isolated from either wild-type (WT) or Dct-Hes1 Tg mice. RT-PCR analysis was performed in the presence (RT+) or absence (RT−) of reverse transcriptase. (B–E) Attenuation of DAPT-induced apoptosis in Dct-Hes1 skin. Dorsal skin from E13.5 wild-type mice (B and D) or Dct-Hes1 Tg (C and E) was organ cultured without (B and C) or with (D and E) DAPT for 4 d and stained with anti-Pax3 and anti-Dct antibodies. (F) Numbers of Dct- and Pax3-positive Mbs were counted in each skin section and then plotted. Data represent the mean of three sections with SD. (G) E13.5 dorsal skin obtained from either wild-type (left) or Dct-Hes1 Tg mice (right) was cultured in the presence of 1 μM DAPT and grafted onto a nude mouse.

Figure 5.

Critical role of Notch signaling in the maintenance of MSCs. (A and B) Activation of Notch signaling in MSC. (A) P4 skin section was stained with anti-Dct (green) and anti-NICD1 (red). (B) Skin section from a P4 pHes1-d2EGFP Tg mouse was stained with anti-GFP (green) and anti-Dct (red). Arrowheads indicate Mbs at the LPP. (C–N) Loss of MSCs in Tyr-Cre;RBP-J^f/f mice. Skin sections from P4 (C–F), P12 (G–J), and P32 (K–N) Tyr-Cre;RBP-J ^+/f mice (C, D, G, H, K, and L) and Tyr-Cre;RBP-J^f/f mice (E, F, I, J, M, and N) were immunostained using anti-Dct antibody (green). Whole HF images (C, E, G, and I), LPP of HF images (D, F, H, J, L, and N), and HM images (K and M) are shown. Note that Dct⁺ Mbs at the LPP of a HF exist at P4 (E and F) but have essentially disappeared at P12 (I and J), whereas mature MCs at the HM retain in Tyr-Cre;RBP-J^f/f mice (I). Arrowheads indicate Mbs at the LPP of the HF, and arrows indicate MCs at the HM.