Expression of the Homeobox Gene, Barx2, in Wool Follicle Development Guy Sander, C. Simon Bawden, Philip I. Hynd, Antonietta Nesci, George Rogers, Barry C. Powell  Journal of Investigative Dermatology  Volume 115, Issue 4, Pages 753-756 (October 2000) DOI: 10.1046/j.1523-1747.2000.00122.x Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Comparison of vertebrate Barx proteins. The partial sequence of the ovine Barx2 homeodomain and C-terminal domain are compared with chicken Barx2b (Smith & Tabin, 1999), human Barx2 (accession number AF031924), mouse Barx1 (Tissier-Seta et al. 1995), and mouse Barx2 (Jones et al. 1997). The partial homeodomain is boxed. Dashes indicate sequence identity with ovine Barx2. Residues that are shared with each Barx2 protein are typed in bold. The carboxy-terminal end of each protein is indicated by an asterisk. To maximize alignment, ‘‘extra’' leucine and glycine residues in mouse Barx1 and an ‘‘extra’' alanine residue in chick Barx2b relative to ovine Barx2 are shown below the sequence comparisons. A + indicates that a space has been added between residues to maximize alignment. Note that the glutamic acid (E) residue at the N-terminal end of the ovine sequence may be a result of degenerate primer mismatch during RT-PCR amplification. Journal of Investigative Dermatology 2000 115, 753-756DOI: (10.1046/j.1523-1747.2000.00122.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Analysis of Barx2 expression in adult sheep wool follicles. (A) A transcript of approximately 2 kb was identified by northern blot. M = BRL RNA ladder. (B) RNA protection analysis of adult sheep esophagus, spleen, thymus, tongue, and wool follicle with an ovine Barx2 3′ coding probe. The position of protected Barx2 RNA is indicated by the arrow. Some full-length undigested probe of 293 bases is present in each lane (arrowhead). (C, D) Barx2 expression in vertical sections of adult sheep tongue. Note expression in the lower half of the tongue epithelium. Part (C) is photographed in bright field and (D) in dark field. Dashes mark the underlying basal layer. Scale bar: 150 μm. Journal of Investigative Dermatology 2000 115, 753-756DOI: (10.1046/j.1523-1747.2000.00122.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 In situ hybridization analysis of Barx2 expression during embryonic follicle development. (A-H) Barx2 expression in the developing embryonic epidermis and follicle. (I-L) Barx2 is expressed in the outer root sheath of a vibrissa follicle. (K, L) Higher magnification of the boxed section in part (I) showing expression of Barx2 in the outer root sheath. (M, N) Barx2 expression in the epidermis several days prior to birth. (O, P) Barx2 is weakly expressed in adult epidermis. Note the intense expression of Barx2 in a cell in the upper dermis (arrow) just below the epidermal basal layer. (Q, R) Barx2 expression is primarily associated with the basal layer of the sebaceous gland. (S, T) Barx2 is abundantly expressed in cells scattered throughout the upper adult dermis (arrowheads) and in the outer root sheath of pelage follicles. The artificial fluorescence in the cortex is due to its high refractivity. (U, V) Higher magnification of dermal cells showing Barx2 expression (black grains). In all panels, the basal layer is indicated by the dashed line. Arrows and arrowheads in all figures point to features described in the text. The following stages of follicle development followHardy (1992): (A, B), stage 0; (C, D), stage 1; (E, F), stage 2; (G, H), stage 3; (I-L), stage 8; (M, N), E135 skin; (O-V), adult skin of old sheep. Note that parts (S) and (T) show partial sections of stage 8 follicles. PDP, predermal papilla; B, bulge; O, outer root sheath; C, hair cortex and cuticle; I, inner root sheath. Scale bar: (A) 40 μm; (C) 55 μm; (E) 30 μm; (G) 40 μm; (I) 70 μm; (K, M, O) 35 μm; (Q) 25 μm; (S) 210 μm; (U) 15 μm. Journal of Investigative Dermatology 2000 115, 753-756DOI: (10.1046/j.1523-1747.2000.00122.x) Copyright © 2000 The Society for Investigative Dermatology, Inc Terms and Conditions