Gene Expression of Mouse S100A3, a Cysteine-Rich Calcium-Binding Protein, in Developing Hair Follicle Kenji Kizawa, Suguru Tsuchimoto, Keiko Hashimoto,

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Gene Expression of Mouse S100A3, a Cysteine-Rich Calcium-Binding Protein, in Developing Hair Follicle Kenji Kizawa, Suguru Tsuchimoto, Keiko Hashimoto, Hideyo Uchiwa Journal of Investigative Dermatology Volume 111, Issue 5, Pages 879-886 (November 1998) DOI: 10.1046/j.1523-1747.1998.00385.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Nucleotide and deduced amino acid sequence of mouse S100A3. Amino acid sequence is indicated below the corresponding nucleic acid sequence. Only the amino acids of human S100A3 that differ from the mouse S100A3 sequence are indicated on the third line. The two helix-turn-helix domains that define EF-hand motifs are boxed. Calcium-binding loops within them are italicized. The stop codon and a consensus polyadenylation signal are underlined. This sequence has been deposited in the Genbank data base and is available under accession number AF004941. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Clustering organization of mouse S100A3, S100A4, and S100A6. (a) Southern blot analysis of genomic DNA. Genomic DNA (5 μg) digested with restriction enzymes were separated by electrophoreses in 0.8% agarose and transferred to nylon membrane. The membrane was hybridized with digoxigenin-labeled full-length S100A3 riboprobe. Size markers are shown on the left. (b) Southern blot analysis of BAC clone DNA. Blots of BAC clone DNA (0.1 ng) digested with restriction enzymes were hybridized with digoxigenin-labeled S100A3, S100A4, or S100A6 riboprobe indicated below. Similar results were obtained with two BAC clones. (c) Restriction map of S100 gene cluster on mouse chromosome 3. B, BamHI; E, EcoRI; H, HindIII. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Tissue-specific and periodical expression of mouse S100A3. (a) Tissue distribution of the mouse S100A3 transcript. Northern hybridization was performed with total RNA from mouse various tissues. Total RNA (each lane contains 5 μg) was separated by electrophoresis in 1.2% agarose and then transferred to a nylon membrane. The membrane was hybridized with digoxigenin-labeled full-length S100A3 or G3PDH riboprobe. (b) Time course of the S100A3 transcript in mouse skin during cycles of hair growth. Northern hybridization performed as above with total RNA (each lane contains 5 μg) from whole dorsal skin of ICR mice at various ages. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Hair cycle dependent in situ localization of S100A3 mRNA in mouse skins.In situ hybridization with digoxigenin-labeled S100A3 anti-sense riboprobe was performed on paraffin sections of ICR mouse dorsal skin containing hair follicles at various developmental stages. (a) Longitudinal section of 3 d old mouse skin. Developing cuticle (arrow) within the early anagen hair follicle is stained. (b) Longitudinal section of 18 d old mouse skin. The bottom part of club hair (arrows) within the ascending catagen hair follicles are stained. (c) Longitudinal section of 19 d old mouse skin. No signal was observed with any telogen hair follicle. (d) Longitudinal section of 22 d old mouse skin. Cuticular layer equipped with hair shaft tip (arrow) within the regenerating hair follicle is stained. Scale bars: 100 μm. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Comparative gene expression of S100A3 and hacl-1 in the synchronized hair cycle. Total RNA prepared from whole dorsal skin of C3H mice at various days after artificial depilation was subjected to northern blot analysis. Total RNA (each lane contains 1 μg) was separated by electrophoresis in 1.2% agarose and then transferred to a nylon membrane. The membrane was hybridized with digoxigenin-labeled anti-sense riboprobe for S100A3, hacl-1, or G3PDH. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Complementary distribution of S100A3 and S100A6 within hair follicles.In situ hybridization was performed with digoxigenin-labeled anti-sense riboprobes for S100A3 and S100A6. S100A3 probe was hybridized to longitudinal sections of an anagen hair follicle (a), and to oblique skin sections (b). S100A3 mRNA signal is detected strongly in cuticle and weakly in cortex. S100A6 probe was hybridized to the longitudinal sections (c), and to the oblique sections (d). S100A6 mRNA signal is detected in medulla and inner root sheath. Complementary tissue expression pattern of the two S100 proteins is evident from the comparison of (a) vs (c) and (b) vs (d). Cu, cuticle; Co, cortex; M, medulla; I, inner root sheath. Scale bars: 50 μm. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 Localization of translation product of S100A3 gene. Immunohistochemical localization of S100A3 protein was examined on paraffin sections of anagen hair follicles. Transverse sections were reacted with the anti-serum against S100A3 (a), and with the control preimmune serum (b). The immunostainings are observed with cuticle and cortex in the follicle sections of the keratogenous zone and the isthmus region (arrows). (c) Longitudinal sections were reacted with the anti-serum against S100A3. The intense signal with the cuticular layer persists from the keratogenous zone upward. Nuclei are counter stained with light blue by hematoxylin. Cu, cuticle; Co, cortex; M, medulla; I, inner root sheath; O, outer root sheath. Scale bars: 50 μm. Journal of Investigative Dermatology 1998 111, 879-886DOI: (10.1046/j.1523-1747.1998.00385.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions