Thaned Kangsamaksin, Rebecca J. Morris 

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Bone Morphogenetic Protein 5 Regulates the Number of Keratinocyte Stem Cells from the Skin of Mice  Thaned Kangsamaksin, Rebecca J. Morris  Journal of Investigative Dermatology  Volume 131, Issue 3, Pages 580-585 (March 2011) DOI: 10.1038/jid.2010.378 Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Localization of bone morphogenetic protein 5 (Bmp5) in mouse hair follicles and interfollicular epidermis of wild-type but not mutant mouse skin. Adult mouse skin was collected for paraffin-embedded histological sections for (a) C57BL/6, (b) BALB/c, (c) wild-type, and (d) homozygous short-ear mutant mice at ∼7 to 8 weeks of age, and BMP5 immunohistochemistry was performed. B, hair follicle bulge; D, dermis; E, epidermis; HF, hair follicle; SG, sebaceous gland. Note the intense staining in the hair follicle and in an occasional interfollicular cell of the wild-type epithelium. In the homozygous short-ear mutant skin, Bmp5 staining was consistently absent from hair follicles and interfollicular epidermis. Scale bars=30μm. Journal of Investigative Dermatology 2011 131, 580-585DOI: (10.1038/jid.2010.378) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Quantification of mouse keratinocyte colonies. Keratinocytes were isolated from 7- to 8-week-old mice. (a) The photographs of the colony assays in 60-mm dishes are shown. The (b) number and (c) size of mouse keratinocyte colonies from all genotypes were quantified after 2 weeks of cultivation on irradiated 3T3 fibroblasts as a feeder layer. Note that the number of colonies is significantly less in the homozygous short-ear mutant mice, whereas colony number and size were relatively greater in the heterozygous and wild-type congenic littermates. Average number of colonies±SD. *P-value <0.003, N=3. Journal of Investigative Dermatology 2011 131, 580-585DOI: (10.1038/jid.2010.378) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Quantification of label-retaining cells (LRCs) in mouse epidermis. (a–d) Immunohistographs for LRCs are shown. Mice received twice-daily (1000 and 1700hours) subcutaneous injections of 50μg per g body weight of BrdU at postnatal days D3, D4, and D5. After 8–10 weeks, dorsal skins were collected and fixed with 10% formalin for at least 24hours. Cells retaining the label are identified as LRCs. (e) The bar graph shows the quantification of labeled cells in the interfollicular epidermis and hair follicles combined. Labeled cells were counted in the same region of the dorsal skin for every 500 cells. Mean±SD. *P-value <0.05. N=3. Scale bars=30μm. Journal of Investigative Dermatology 2011 131, 580-585DOI: (10.1038/jid.2010.378) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Exogenous recombinant human bone morphogenetic protein 5 (BMP5) increases keratinocyte colony size and number. (a) To determine the functional significance of Bmp5 on keratinocyte colony formation, we treated wild-type and short-ear keratinocytes cultured on 3T3 feeder layers with different concentrations of recombinant human BMP5 (0.01 and 0.1μgml–1). The recombinant peptide was added to the medium that was changed every other day up to 2 weeks of culture. Note that exogenous BMP5 added to culture increased the number and size of colonies in keratinocytes from all three genotypes. (b) The photographs of the colony assays each correspond to the bar graph directly above. Average number of colonies±SD. *P-value <0.05. N=3. Journal of Investigative Dermatology 2011 131, 580-585DOI: (10.1038/jid.2010.378) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Bone morphogenetic protein 5 (Bmp5) regulates the number of tumors in mice in the two-stage skin carcinogenesis model. (a) The bar graphs represent the average number of skin tumor across all genotypes. Mouse skin was initiated with 200nmol of 7,12-dimethylbenz[a]anthracene (DMBA) in 200μl of spectral grade acetone when the mice were 7 weeks of age. After 1 week, there was twice-weekly promotion with 17nmol of 12-O-tetradecanoylphorbol-13-acetate (TPA) in 200μl of acetone. Skin tumors were counted weekly for 20 weeks. Note that Bmp5 status was associated with the number of skin tumors, with short-ear mutant and BALB/c mice developing more skin tumors than heterozygous and wild-type littermates, or C57BL/6J wild-type mice. Wild-type C57BL/6 and BALB/c mice were used, respectively, as low- and high- tumor controls. (b) The marked-line chart shows the appearance of tumors across all genotypes. Mean±SD. *P-value <0.05. N=5. Journal of Investigative Dermatology 2011 131, 580-585DOI: (10.1038/jid.2010.378) Copyright © 2011 The Society for Investigative Dermatology, Inc Terms and Conditions