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>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

<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

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

<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

Human melanoma lymph node metastases (entirely embedded).

<p>(A) Box plot comparison of lymphatic vessels (number of D2–40 positive vessels per core) in primary versus metastatic melanoma. * One case, * two cases. (B) Absence of D2–40 staining within metastatic tissue showing no lymphatic vessels (×30). (C) D2–40 positivity shows dilated lymphatic vessels present at the tumor/lymphatic parenchyma interface (arrow, ×150). (D) CD34 staining shows important blood micro-neovascularisation within metastases. (×50). (E) CD34-positive blood microvessels (arrowhead) associated with CD34–negative cells showing vascular mimicry (arrow, ×400). (F) and (G) Cells of vascular mimicries embedded in a PAS (F) and collagen IV (G) positive laminina (arrow, ×320)).</p

<i>In vivo</i> characterization of neural crest derived cells.

<p>To characterize neural crest-derived clones <i>in vivo</i>, we stereotaxically injected 50,000 cells of each NCSC clones (separetly) in mice striatum (<b>A</b>). <i>Asclepios</i> induced massive tumors after 4 weeks as attested by the beta-galactosidase expression of the tumor cells. (<b>B</b>). Immunological characterization of those tumors revealed that they were GFAP (<b>c</b>), beta-III-tubulin (<b>D</b>), nestin (<b>E</b>), N-cadherin (<b>F</b>) and NrCAM-positive (G). Lectin labeling (<b>H</b>) confirmed the presence of blood vessels in the tumors. Finally, no vimentin (<b>I</b>) or Sox2 (<b>J</b>) expressions were observed. Nuclei were counterstained with Dapi (blue). Scale bars = 50 µm.</p

Human melanoma lymph node metastases (entirely embedded).

<p>(A) and (B) Vascular mimicries (arrow): (A) Melanoma cells with endothelial-like morphology, condensed nuclei, minimal Masson Fontana positive (inset) pigmentation (H&E, ×500), (B) mostly negative for PanMelanoma (red) (×150).</p

Melanoma xenograft tissues.

<p>(A) Mouse vessel-specific MECA32 staining shows micro-neovascularisation within melanoma xenografts (arrowhead, ×100) and positive vessels of the adjacent soft tissue (arrow). (B) Vascular mimicries negative for MECA32 (arrow) associated with and connecting MECA32-positive vessels (arrowhead, ×175). (C) Interphase FISH for human chromosome 17 and HER-2 combined with immunofluorescence staining for the mouse-specific vessel marker MECA32: Tumor capillaries with endothelial cells positive for MECA32 (yellow) and negative for HER-2 (arrow) as well as endothelial-like cell with negativity for MECA32 and positivity for human chromosome 17 and HER-2 (arrowhead, green; ×500). (D) and (G) Melanoma tumor cell-derived endothelial-like cells: (D) Capillary endothelial-like cell positive for human chromosome 17 and HER-2 (arrow) and positive internal control of tumor cells (arrowhead, ×500. (E) and (F) Negative internal controls of slide D and G. Melanoma xenograft adjacent soft tissue with mouse capillaries and fibroblasts negative for human chromosome 17_HER-2 probe (E, ×500) and negative for FISH y probe and CD31 (F, ×700). (G) Interphase FISH for human y-probe combined with immunofluorescence staining for the human-specific vessel marker CD31: Vessel endothelial-like cell positive for the Y chromosome (red) and CD31 (arrowhead, green; ×1000). (H) Vascular mimicries (arrow): Endothelial-like cells positive for human chromosome 17 and HER-2 (arrowhead, ×300). (K) and (L) Green fluorescent protein labelled melanoma cell lines: Melanoma cells (arrowhead, green) and endothelial cells (arrow) contributing to complex branching neo-vascularisation. (K ×200; L ×500).</p