LPP melanocytes are reduced in <i>Tg(DctSox10)</i> homozygotes during hair morphogenesis.

<p>(A) Brightfield images of hairs in <i>Tg(DctSox10)</i> and <i>+/+</i> littermates at P2. (B) Number of DCT⁺ melanocytes within the LPP of hairs at P2 (stage 6 hairs) and P7/8. At both time points, LPP melanocytes per hair are reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> compared to <i>Tg(DctSox10)/+</i> and <i>+/+</i> mice (*p<0.017). (C, D) Quantitative immunohistochemical analysis of stage 6 hairs from P2 skins for DCT and TRP1, or DCT and KIT. The population of DCT⁺/TRP1⁺ cells is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> in comparison to <i>Tg(DctSox10)/+</i> and +/+ mice (*p<0.008). <i>Tg(DctSox10)</i> also causes a switch in KIT intensity from KIT^hi in wild type to KIT^low in <i>Tg(DctSox10)</i> animals (*KIT^lo and **KIT^hi comparisons made between +/+ and <i>Tg(DctSox10)/+</i> or <i>+/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i>; p<0.005).</p

<i>Sox10</i> is required by LPP melanocytes postnatally.

<p>(A) Number of KIT⁺ LPP melanocytes within hairs from <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) mice. P0–3/P10 indicates skins harvested from pups on P10 that were maintained by lactating mothers that were IP injected with TAM on P0–3. 0–3dpp/7dpp indicates skins harvested from adult mice on 7dpp after IP injections of TAM on 0–3dpp. (B) White hairs remain visible in adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice that were treated with TAM by IP injection on 0–3dpp, allowed for complete hair regeneration, replucked and allowed for a second round of hair regrowth (brackets indicate plucked/replucked region, lower image is a magnification of plucked region; mouse in 2E and 3B are the same, imaged prior to and after replucking). (C) Number of PAX3⁺ bulb melanocytes within hairs from <i>Sox10^fl/fl</i> and <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated as described in B but harvested on 7dpp after replucking (0–3dpp/7dpp repluck). (D) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 3) mice treated as described in B but harvested on 7dpp after replucking (*p<0.002). (E) Persistent hair graying is visible in <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with IP TAM for pulse of five days beginning at five weeks old and imaged at one and two years old.</p

<i>Sox10</i> is required by bulb melanocytes postnatally.

<p>(A–B) <i>Sox10^fl/fl</i> (<i>fl/fl; +/+</i>) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (<i>fl/fl; Cre/+</i>) pups treated with TAM by IP injection to the lactating mother on P0–3 display variegated hypopigmentation on the belly and back and exhibit a white head spot upon the emergence of the morphogenetic coat (P10 shown here, n>5). (C) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from these mice at P10 are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to similarly-treated <i>Sox10^fl/fl</i> animals (*p = 0.002). (D–E) Adult <i>Sox10^fl/fl; Tyr::CreERT2</i> mice treated with TAM by IP injection on 0–3dpp exhibit white hairs within the plucked region upon hair regrowth that is not visible in similarly treated <i>Sox10^fl/fl</i> mice (brackets indicate plucked region, lower image is a magnification of plucked region). (F) Number of PAX3⁺ melanocytes per hair bulb in skins harvested from similarly-treated mice at 7dpp are significantly decreased in <i>Sox10^fl/fl; Tyr::CreERT2</i> animals compared to <i>Sox10^fl/fl</i> animals (*p = 0.001). (G–H) Fluorescent and corresponding brightfield images of hair bulbs from mice described in D–E. Arrows and arrowheads indicate PAX3⁺/SOX10⁺ and PAX3⁺/SOX10⁻ melanocytes, respectively. (I) Distribution of melanocytes double-labeled for PAX3 and SOX10 within pigmented (gray) and non-pigmented (white) hair bulbs in skins from <i>Sox10^fl/fl</i> (n = 3) and <i>Sox10^fl/fl; Tyr::CreERT2</i> (n = 4) harvested on 7dpp from mice treated with TAM on 0–3dpp (*p<0.006).</p

<i>Tg(DctSox10)</i> results in congenital white spotting and premature hair graying.

<p>(A, B) Ventral and dorsal views demonstrating variable hypopigmentation in <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> mice during hair morphogenesis and adult hair cycling. (C) Frequency of pigmented (pig+) and non-pigmented (pig−) anagen III/IV (7dpp) hairs that contain (DCT+ LPP cells) or do not contain (no LPP cells) LPP melanocytes within <i>Tg(DctSox10)</i> or <i>+/+</i> mice. The ages of mice analyzed ranged between 9–22 weeks at harvest. Significance determined by chi-square analysis (p<<0.0001) and evaluation of standardized residuals (*, z = −8.84; **, z = 12.24).</p

Alteration of the <i>Tg(DctSox10)</i> phenotype through the reduction of <i>Mitf</i>.

<p>(A–B) Comparison of <i>Tg(DctSox10)/Tg(DctSox10)</i> and <i>Tg(DctSox10)/Tg(DctSox10); Mitf^vga9/+</i> animals at P70. Addition of the <i>Mitf^vga9/+</i> allele reduces the congenital hypopigmentation seen in <i>Tg(DctSox10)/Tg(DctSox10)</i> animals, and is evident in dorsal views (loss of back spotting) and in ventral views (reduction in belly spot size). Premature hair graying of <i>Tg(DctSox10)</i> homozygotes seen at p70 is retained with <i>Mitf^vga9</i> (n = 6). (C) <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003644#s1" target="_blank">Introduction</a> of <i>Sox10^lacZ</i> into <i>Tg(DctSox10)/Tg(DctSox10)</i> homozygotes partially rescues both congenital white spotting and premature hair graying (n = 2). (D) At 40 weeks of age, <i>Tg(DctSox10)/+; Mitf^vga9/+</i> double heterozygotes exhibit visibly increased hair graying in comparison to <i>Tg(DctSox10)/+</i>. (E) Hair graying severity was determined in animals 6–10 weeks of age by quantitating the number of non-pigmented anagen III/IV hair bulbs in +/+, <i>Tg(DctSox10)/+, Tg(DctSox10)/+; Mitf^vga9/+</i>, and <i>Mitf^vga9/+</i> skins after plucking and harvesting at 7dpp. <i>Tg(DctSox10)/+; Mitf^vga9/+</i> mice exhibit a significant increase in non-pigmented hair bulbs in comparison to the single heterozygotes or +/+ animals (**p<0.0015). <i>Tg(DctSox10)/+</i> animals also produce more non-pigmented hair bulbs in comparison to +/+ and <i>Mitf^vga9/+</i> animals (*p<0.002). (F) <i>Tg(DctSox10)/+; Mitf^vga9/+</i> animals (from E) display extensive ectopic pigmentation within the LPP of their hair follicles beyond what is normally observed in <i>Tg(DctSox10)/+</i> animals (arrows, n = 4). (G) Number of LPP melanocytes per anagen III/IV hair follicle in <i>Tg(DctSox10)/+, Tg(DctSox10)/+; Mitf^vga9/+, Mitf^vga9/+</i> animals (from E) that double label for DCT, TRP1, and produce ectopic pigmentation. Hairs from <i>Tg(DctSox10)/+; Mitf^vga9/+</i> animals exhibit significantly more TRP1⁺/PIG⁺ LPP melanocytes than in either single heterozygote (*p<0.0125).</p

Overexpression of <i>Sox10</i> results in premature differentiation of LPP melanocytes in anagen hairs.

<p>(A) Number of DCT⁺ LPP melanocytes per anagen III/IV hair follicle (independent of the presence or absence of hair pigmentation) is significantly reduced in <i>Tg(DctSox10)/Tg(DctSox10)</i> mice when compared to wild type and <i>Tg(DctSox10)/+</i> mice (*p<0.0003). The ages of mice analyzed ranged between 9–22 weeks at harvest. (B) Eosin-stained skin sections of these hairs demonstrate the presence of ectopic pigmentation in the LPP of <i>Tg(DctSox10)/+</i> and <i>Tg(DctSox10)/Tg(DctSox10)</i> hairs (arrows) that is not see in wild type hairs. In <i>Tg(DctSox10)/+</i> LPP regions, this pigmentation often appeared in cells that were highly dendritic. (C, D) Brightfield and corresponding fluorescent images of anagen III/IV hair follicles double labeled for DCT and TRP1 (C) or KIT (D) in wild type and <i>Tg(DctSox10)/+</i> animals. The intensity of KIT fluorescence expression was variable, and categorized as KIT^lo (arrows) or KIT^hi (arrowheads), and did not appear to correlate with the presence or absence of pigmentation. (E,F) Comparison of the number LPP melanocytes per anagen III/IV hair follicle in <i>+/+</i> and <i>Tg(DctSox10)/+</i> animals that express DCT, and TRP1 or KIT, and produce ectopic pigmentation (*p<0.008).</p

SOX10 knockdown results in elevated SOX9 expression in mouse and human melanocytes.

<p><b>A</b>, Experimental design used to investigate the level of SOX9 and SOX10 expression <i>in vitro</i>. Cultured human keratinocytes, melanocytes, cells derived from biopsies of patients with giant congential naevi and melanoma cells (M010817 cell line) were subjected to RNA isolation and subsequent Q-RT-PCR analysis. Keratinocytes were used as a control. <b>B, C</b>, Quantitative real-time PCR analysis showing the decline of SOX9 expression (<b>C</b>) and increase of SOX10 expression (<b>B</b>) that correlate with the acquisition of malignant state by human NRAS^Q61K-mutated cells. Data are presented as the mean fold change and are normalized over levels found in melanocytes. <b>D, E</b>, SOX10 and SOX9 expression in a large set of proliferative and invasive cell lines analysed by gene expression using microarrays (<b>D</b>) and Western blot (<b>E</b>) techniques. <b>F</b>, Experimental design used to deregulate SOX10 expression in human cells derived from the biopsy of a patient with NRAS^Q61K-mutated giant congenital naevus. <b>G, H</b>, Quantitative real-time PCR analysis of SOX10 (<b>G</b>) and SOX9 (<b>H</b>) expression after the knockdown of SOX10. <b>I</b>, Experimental design used to analyze the expression of Sox9 in the melanocytic lineage from <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice. <b>K, L</b>, Cells were isolated from the trunk skin of <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61K</i> <i>Sox10^LacZ/+</i> mice and stained for Melan-a and c-Kit antibodies. FACS-sorted cells were subsequently used for the RNA isolation and quantitative real-time PCR with primers specific for the coding regions of <i>Sox9</i> gene. Data are presented as the mean fold change and are normalized to the control. Kerat, keratinocytes; M, melanocytes; Nev, naevus cells; Mel, melanoma cells; KD, knock down.</p

Differential expression of SOX10 and SOX9 in human melanocytes, human giant congenital naevi and human melanoma samples.

<p><b>A</b>, Scheme showing the localization of epidermal melanocytes (in red) in the human skin. <b>B, C</b>, Immunostaining for MITF (green, left panel) and SOX9 (red, right panel) in the human skin demonstrating the lack of SOX9 expression in the epidermal melanocytes. Inserts show higher magnification images of MITF and SOX9 immunostainings. Scale bars, 25 μm. <b>D</b>, Scheme showing the localization of melanocytes (in red) within the hair follicle. <b>E</b>, Immunostaining for MITF (green) and SOX9 (red) in the human skin reveals the expression of SOX9 in the cells of outer root sheath but not in the MITF-positive melanoblasts/melanocytes. Scale bar 100 μm. <b>F, G</b>, High magnification images of immunostaining for MITF and SOX9 in the upper part of human hair follicle (<b>F</b>) and the follicular bulb (<b>G</b>). <b>H</b>, Analysis of SOX9 (red, left panel) and SOX10 (red, right panel) expression in the patients with human giant congenital naevi demonstrates the lack of SOX9 expression in the SOX10-positive giant congenital naevi cells. Inserts show higher magnification. <b>I</b>, Representative examples of immunostaining for SOX9 (green) and SOX10 (red) in a tissue microarray of primary melanoma samples are shown. <b>J-K</b>, Distribution of SOX10 vs. SOX9 expression in human melanoma (based on TCGA database). 334 melanoma patients were divided in two groups, namely SOX10 High/ SOX9 Low and SOX10 Low / SOX9 high based on SOX10 and SOX9 expression levels. DP, dermal papilla; HF, hair follicle; M, melanocytes; ORS, outer root sheath. Scale bars, 25 μm.</p

Mouse giant congenital naevi and melanoma reveal no expression of Sox9.

<p><b>A-D</b>, Immunostaining for Sox9 (<b>A, C</b>) and Sox10 (<b>B, D</b>) in the skin sections of <i>Tyr::Nras^Q61K</i> and <i>Tyr::Nras^Q61KINK4a^−/−</i> mice. <b>E-H</b>, Experimental strategy used to abrogate the expression of Sox9 (E) and Sox10 (G) in the mouse melanocytic lineage. Pictures of two representative mice 1 year after tamoxifen injections reveal no reduction in the skin hyperpigmentation in <i>Tyr::Nras^Q61KSox9^fl/+Tyr-CreERT2</i> mice as compared to their <i>Tyr::Nras^Q61K</i> littermates (F) in contrast to a pronounced skin whitening observed upon Sox10 loss (H). BF, bright field; HF, hair follicle; mo, months; P0, postnatal day 0. Scale bars, 25 μm.</p

Experimental suppression of SOX9 expression rescues the effects of SOX10 deregulation in human melanoma cells.

<p><b>A</b>, SOX9 overexpression in human melanoma cells closely resembles the gene expression signature of SOX10 knockdown as revealed by unsupervised hierarchical clustering of control M010817 melanoma cells, SOX9 overexpressing M010817 cells and SOX10 knock down M010817 cells. Microarray gene expression accession number: GSE37059. <b>B</b>, Western blot analysis showing that SOX10 expression is downregulated upon overexpression of SOX9 in two independent human melanoma cell lines (A375 and M010817). <b>C</b>, Chromatin immunoprecipitation assay demonstrating the binding of SOX9 to the promoter of SOX10 in human melanoma M010817 cells. <b>D, E</b>, Quantitative real-time PCR analysis of SOX10 (<b>E</b>) and SOX9 (<b>F</b>) expression after the knockdown of SOX10 and after the double knockdown of SOX10 and SOX9 in M010817 cell line. <b>F</b>, Quantification of number of Annexin V-positive cells based on the FACS analysis in the melanoma M010817 cells upon SOX9 KD, SOX10 KD or double SOX9/SOX10 KD. OE, overexpression; KD, knock down; ChIP, chromatin immunoprecipitation; prom, promoter.</p