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First published online November 28, 2005
doi: 10.1242/10.1242/dev.02161

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Molecular characterization of melanocyte stem cells in their niche

¹ Laboratory for Stem Cell Biology, RIKEN Center for Developmental Biology, 2-2-3 Minatojima Minami-machi, Kobe, Hyogo 650-0047, Japan
² Molecular Oncology, Swiss Institute for Experimental Cancer Research, National Center of Competence in Research, 1066 Epalinges, Switzerland

View larger version (94K): [in a new window]   Fig. 1. Immunohistochemical characterization of the MSCs. Phenotypical characterization of MSCs in the lower permanent portion of the hair follicle (top) and MCs in the hair matrix region (bottom) in stage 8 guard hairs. Skin sections were stained with antibodies against Dct, Tyr, Si, Tyrp1, Kit, Pax3, Mitf, Sox10, Lef1 or Mki67 (green), and with anti-ß-gal (red). Nuclear staining (blue) was with TO-PRO3. Arrowheads indicate MSCs in the lower permanent portion of the hair follicle. Scale bars: 10 µm.

View larger version (45K): [in a new window]   Fig. 2. Sox10 expression during melanocyte development. Skin sections obtained from (A) an E16.5 embryo, (B) an E18.5 embryo and (C) a P2 mouse (Dct-lacZ) were stained with anti-Sox10 (green) and anti-ß-gal (red). Sox10 expression was detected in all ß-gal+ melanoblasts in the epidermis (A) and in the developing hair follicle (B). Downregulation of Sox10 was observed in some melanocytes located in the bulge area (C). Arrowheads indicate ß-gal+ melanoblasts. Scale bars: 50 µm. (D) Frequency of Sox10+ melanocytes located in various regions of developing skin, shown as percentages of Sox10+ cells in ß-gal+ melanoblasts located in the epidermis of the E18.5 embryo, or the bulge/sub-bulge region or hair matrix region of the P2 or P4 mice. (E,F) Downregulation of Sox10 mRNA in bulge melanocytes. Expression of Sox10 mRNA was examined by in situ hybridization/immunohistochemistry double staining. (E) ß-gal+ (red) melanoblasts in a developing P2 hair follicle. All melanoblasts expressed Sox10 mRNA (green). (F) Melanocytes in a P4 hair follicle. Some Sox10-negative melanocytes were observed in the bulge region. Scale bars: 50 µm.

View larger version (78K): [in a new window]   Fig. 3. System for in vivo labeling of the cells in the melanocyte lineage with GFP. Existence of MSCs in an isolated single hair follicle. Arrowheads indicate that MSCs reside in the lower permanent portion of the hair follicle. (B) Strategy for marking melanocytes with GFP. (C,D) GFP expression in a hair follicle and the embryonic epidermis. (E) FACS analysis of a single-cell suspension obtained from the epidermis of a melanocyte-specific GFP transgenic mouse at E16.5. The single-cell suspension was stained with PE-anti-CD45 and APC-anti-c-Kit, and processed for FACS analysis. The FACS profile of the cells in CD45-negative population is shown. Scale bars: 50 µm.

View larger version (31K): [in a new window]   Fig. 4. Quantitative comparison of amplified cDNA with unamplified cDNA. (A) The relative gene expression level of each target gene in amplified cDNA (gray column) was compared with that in unamplified cDNA (white column). Data represent the average of three experiments. Error bars indicate s.d. (B) Scatter plot showing the relationship between relative gene expression values obtained from amplified and unamplified cDNA. Data obtained from Fig. 3B were plotted. (C) Reproducibility of cDNA amplification. Expression values of the melanogenic genes were determined by Q-PCR using four replicates of amplified cDNA. The expression value of each gene was consistent among the four amplifications. (D) Detection of transcripts at known copy number in cDNA amplification. Control poly(A)-tailed RNAs of known concentrations were mixed in lysis buffer containing 20 pg total RNA prepared from Melb-a cells, reverse transcribed followed by cDNA amplification. Four different RNA spikes, corresponding roughly to 40, 400, 4000 and 40,000 copies/sample, were spiked into four different tubes containing an identical amount of total RNA. Relative expression values were determined by Q-PCR using a primer pair specific for each spike RNA.

View larger version (44K): [in a new window]   Fig. 5. Gene expression profiles of MSCs, TA cells/DCs and Mbs. The expression profiles of various genes in individual MSCs from the lower permanent portion of the hair follicle, and in hair matrix melanocytes (TA/DCs) and Mbs from the E16.5 epidermis are shown. The relative expression values for each gene were normalized against one of the mRNA spikes. To display gene expression profiles graphically, each relative expression value was transformed to a logarithmic value. Cells with a value of higher than 0 were classified as red, between 0 and -1 as orange, between -1 and -2 as light orange, between -2 and -3 as yellow, between -3 and -4 as pale yellow, and lower than -4 as white. Black shows that gene expression was not detected.

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