Identification of Differentially Expressed Genes During a Wool Follicle Growth Cycle Induced by Prolactin  Nicholas W. Rufaut, Allan J. Pearson, Allan J. Nixon, Thomas T. Wheeler, Richard J. Wilkins  Journal of Investigative Dermatology  Volume 113, Issue 6, Pages 865-872 (December 1999) DOI: 10.1046/j.1523-1747.1999.00775.x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 The induced growth cycles. Follicle activity scores (squares) and circulating prolactin concentrations (circles) are plotted for sheep killed at various times after cycle induction. (a) Shows the first experiment, cycle induction after bromocriptine treatment. For the day 0 animal, prolactin levels rose unexpectedly early. (b) Shows the second experiment, induction after exposure to a short photoperiod. In (b), the mean values for two animals are plotted at each time-point. Filled boxes represent activity scores for skin samples subsequently used for RNA extraction. Follicle activity scores were determined histologically, with follicles in the anagen phase or in early catagen or proanagen scored as active and colleagues as inactive. Prolactin concentrations were measured in blood plasma samples collected 0–2 d before being killed. Journal of Investigative Dermatology 1999 113, 865-872DOI: (10.1046/j.1523-1747.1999.00775.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Alignment of sequence tags with known genes. (a) WCG1 aligned with KRTAP3.2. (b) WCG2 aligned with bovine Dsg1. (c) WCG3 aligned with human stratifin and HME1, and with our ovine cDNA (GenBank accession no. AF071008). (d) WCG4 aligned with human, rat, and mouse Clk3 and with a human (GenBank accession no. AA489053) and mouse (GenBank accession no. AA174898) expressed sequence tag. See text for references for gene sequences. Vertical bars (¦) represent residues identical to aligning ones above, dots (.) indicate gaps introduced to maximize alignment. Consensus polyadenylation signals are shaded. Sequence representing incorporated PCR primers is shown in lower case. A few residues were lost from the extreme ends of the PCR products when their ends were blunted. The GenBank accession numbers for these tags are: WCG1, AF071004; WCG2 AF071005; WCG3, AF071006; WCG4, AF071007. Journal of Investigative Dermatology 1999 113, 865-872DOI: (10.1046/j.1523-1747.1999.00775.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Northern analyses confirming differential expression of tagged genes. RNA from the second cycle induction experiment was used (see (Figure 1b) for corresponding follicle activity scores). The RNA samples were extracted from duplicate animals killed at 0, 7, 14, 21, and 28 d after release from a short photoperiod (labeled d0–d28 above the lanes). Follicles in the skin of control animals not subjected to an artificial photoperiod were in anagen. Expression levels of β-actin were determined to ensure equal loadings of RNA. The sizes of hybridizing mRNA are shown on the left, their identities on the right. Journal of Investigative Dermatology 1999 113, 865-872DOI: (10.1046/j.1523-1747.1999.00775.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Localization of expression in follicles by in situ hybridization. Sense and anti-sense riboprobes for each sequence tag were hybridized to anagen and telogen skin sections. Longitudinal sections through lower follicles are shown. (a, e, i) Show KRTAP3.2 expression; (b, f, j) Dsg1 expression; (c, g, k) stratifin expression; (d, h, l) Clk3 expression. (a–d) Show anagen follicles with sense probe; (e–h), anagen follicles with anti-sense probe; (i–l), telogen follicles with anti-sense probe. The labeled structural features are: OS, outer root sheath; IS, inner root sheath; CO, cortex; CU, cuticle; BE, brush end; ES epithelial strand. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 113, 865-872DOI: (10.1046/j.1523-1747.1999.00775.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Localization of Dsg1, stratifin, and Clk3 expression in epidermis by in situ hybridization. Sense and anti-sense riboprobes for each sequence tag were hybridized to sections of sheep skin. (a) Dsg1 sense probe; (b) Dsg1 anti-sense probe; (c) stratifin anti-sense probe; (d) Clk3 anti-sense probe. The epidermis is indicated between the left pointing arrows, and is continuous with the outer root sheath of the upper follicle neck, which is indicated by the right pointing arrows. Scale bar: 100 μm. Journal of Investigative Dermatology 1999 113, 865-872DOI: (10.1046/j.1523-1747.1999.00775.x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions