Epididymal Cysteine-Rich Secretory Protein 1 Encoding Gene Is Expressed in Murine Hair Follicles and Downregulated in Mice Overexpressing Hoxc13  Ron.

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Epididymal Cysteine-Rich Secretory Protein 1 Encoding Gene Is Expressed in Murine Hair Follicles and Downregulated in Mice Overexpressing Hoxc13  Ron L. Peterson, Tatiana V. Tkatchenko, Nathanael D. Pruett, Christopher S. Potter, Donna F. Jacobs, Alexander Awgulewitsch  Journal of Investigative Dermatology Symposium Proceedings  Volume 10, Issue 3, Pages 238-242 (December 2005) DOI: 10.1111/j.1087-0024.2005.10114.x Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Detection of Cysteine-Rich Secretory Protein 1 (Crisp1) mRNA in skin of 5 d postnatal mice by RT-PCR. Standardized complementary DNA (cDNA) samples of 5 d normal (A) and GC13 (B) mice were used in PCR reactions containing primers specific for Crisp1 (lane 3), and for Crisp1 and GAPDH (lane 4); reactions were run on an agarose gel stained with ethidium bromide as shown. The reactions containing only Crisp1 primers generated a product within the predicted size range of 430 bp that was clearly visible with normal cDNA (white arrow in A), and only weakly detectable with GC13 cDNA (white arrow in B). The reactions containing both Crisp1 and GAPDH control primers resulted in a quenching of the Crisp1-specific band (black arrow in A) probably because of primer competition under these reaction conditions. Lane 1, MW standards in descending order from top are 1000, 800, 600, 400 bp; lane 2, control reaction containing Crisp1 primers without substrate. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 238-242DOI: (10.1111/j.1087-0024.2005.10114.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Localization of cysteine-rich secretory protein 1 (Crisp1) expression in murine hair and epididymis. Digoxygenin-labeled antisense RNA probes (probe 1 and probe 2 as indicated at the lower right of each panel) specific for Crisp1 were hybridized to 10 μm frozen sections of scapular skin derived from 5 d postnatal normal (A, B, and E) and GC13 mice (C, D); panel F shows hybridization to a longitudinal section of the epididymis where the proximal-caudal polarity is indicated. Hybridization signals were visualized by NBT/BCIP color reaction (purple blue). Expression in skin is restricted to the lower medulla of the hair shaft in normal mice (A, B), whereas in hair shafts of GC13 mutants (C, D), expression is difficult to discern. Hybridization to a cross-section (E) shows restriction in expression to the medulla at a higher resolution. The inset in panel F (arrow) shows a close-up of Crisp1 expression in ductal epithelium of the caudal epididymis (framed subregion). Ctx, cortex; Cu, cuticle; IRS, internal root sheath; M, medulla; ORS, outer root sheath. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 238-242DOI: (10.1111/j.1087-0024.2005.10114.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Medulla defects in hair of Hoxc13 overexpressing mice. Hair shafts (n≥10) plucked from the thoracic flank of GC13 mutant (A) and normal control mice (B) were viewed under a light microscope using a × 40 phase contrast objective. The highly regular septation seen in the medulla (arrows) of a normal hair shaft (B) is severely disrupted in mutant hair (A), which was overall difficult to image because of its greater light reflection compared with normal hair. Journal of Investigative Dermatology Symposium Proceedings 2005 10, 238-242DOI: (10.1111/j.1087-0024.2005.10114.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Interaction of Hoxc13 with putative cognate binding site in cysteine-rich secretory protein 1 (Crisp1) promoter region. (A) Map of putative Hoxc13 (light gray boxes) and TCF/LEF1 (dark gray boxes) binding sites are shown within 4.5 kb promoter region of Crisp1. Location of presumptive transcription start site is indicated by angled arrow. Sites examined by electrophoretic mobility shift assay (EMSA) are marked by asterisks. (B, C) Autoradiogram of EMSA examining interaction between Hoxc13 and double-stranded oligonucleotides (oligos) 2382 and 809 as indicated at the lower right of each panel. Lane 1, labeled oligo (oligo 2382 in B and oligo 809 in C); lane 2, labeled oligo plus luciferase; lane 3, labeled oligo plus Hoxc13; lane 4, labeled oligo plus Hoxc13 and anti-Hoxc13 antiserum; lane 5, labeled oligo plus Hoxc13 and anti-Trp1 antiserum; lane 6, labeled oligo plus Hoxc13 and cold competitor oligo 2382 (B) or 809 (C); lane 7, labeled oligo plus Hoxc13 and cold oligo 809 (B) or 2382 (C). Specific Hoxc13-DNA complexes are indicated by the strong bands seen in lanes 3 and 5 of panel B at the level of the black arrow shown at the left of that panel. This band is shifted after addition of anti-Hoxc13 antiserum (gray arrow, lane 4, panel B) but not with anti-Trp1 antiserum (lane 5). Excess cold oligo 2382 completely abolishes the formation of labeled Hox-DNA complex (lane 6, panel B), whereas excess cold oligo 809 achieves this only insufficiently (lane 7, panel B). Oligo 809 apparently does not form specific complexes with Hoxc13 protein under the same conditions (C). Journal of Investigative Dermatology Symposium Proceedings 2005 10, 238-242DOI: (10.1111/j.1087-0024.2005.10114.x) Copyright © 2005 The Society for Investigative Dermatology, Inc Terms and Conditions