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First published online 27 July 2004
doi: 10.1242/dev.01303

Development 131, 4189-4200 (2004)
Published by The Company of Biologists 2004
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The co-repressor hairless has a role in epithelial cell differentiation in the skin

Joanna M. Zarach1,2, Gerard M. J. Beaudoin, III1,2, Pierre A. Coulombe3,4 and Catherine C. Thompson1,2,*

1 Kennedy Krieger Research Institute, Baltimore, MD 21205, USA
2 Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
3 Department of Biological Chemistry, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA
4 Department of Dermatology, Johns Hopkins University School of Medicine, Baltimore, MD 21205, USA



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  		Fig. 1. Generation of Hr-/- mice. (A) Expression of Hr mRNA in Hr mutant mice. Northern analysis of mRNA from backskin of adult (10 week old) mice. hr, Hrhr allele (HRS/J); hrrh, Hrrh-j allele (RHJ/LeJ); +, wild type allele. Asterisks indicate high molecular weight RNA species detected only in Hrhr allele. (B) Expression of HR protein in Hr mutant mice. Western analysis of extracts from adult backskin shows protein detected by HR-specific antibody ({alpha}-Hr). hr, Hr allele (SKH2/J); hrrh, Hrrh allele (CBA-hrrh); +, wild type allele; Ponceau, Ponceau Red stained membrane. Molecular weight is shown in kDa. (C) Targeting vector used to create Hr null (Hr-) allele. Exons 8-10 were replaced with a neo cassette (PGKneo) using homologous recombination. Exons are not drawn to scale. RI, EcoRI sites. (D) Southern analysis of mice with targeted gene deletion. Southern blot of EcoRI-digested genomic DNA. Probe is region indicated by black bar in C. Band of 9 kb represents wild-type allele; 6 kb band represents recombinant allele; +, wild type allele; -, recombinant (Hr-) allele. (E) Hr mRNA is disrupted in mice carrying targeted gene deletion. Northern analysis of RNA prepared from skin of P15 mice. +/+, wild type; -/-, Hr-/-. (F) Mice carrying targeted gene deletion do not express HR protein. Western analysis of extracts from skin with HR-specific antibody ({alpha}-Hr). (G) Skin wrinkling in Hr-/- mouse (1 year old). (H) Comparison of 1-year-old Hr mouse mutants. Null, Hr-/- mouse; rhino, Hrrh/Hrrh mouse (CBA-Hrrh); Hr, Hrhr/Hrhr mouse (SKH2/J).

 


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  		Fig. 2. Utricle formation in Hr-/- mice. (A) Histology of backskin from Hr-/- mice. Hematoxylin-Eosin staining of sagittal sections from backskin at the indicated ages and genotypes. Arrowheads indicate morphological change in the infundibulum of Hr-/- skin. (B) Expression of keratin markers in utricles. Immunohistochemical detection of keratin markers in consecutive serial sections of backskin from P19 Hr-/- (top panels; -/-) and wild-type (+/+) mice (bottom panels). H&E, Hematoxylin-Eosin; K10, keratin 10; K14, keratin 14; K17, keratin 17. Arrowheads indicate positive signal in utricle; arrows indicate epithelial remnants; thick arrows indicate sebaceous glands. (C) Utricles (arrowheads) do not express hair markers. Immunohistochemical detection of Trichohyalin, a hair marker, in P15 Hr-/- backskin. (D) Immunofluorescent detection of K10 (red) and K17 (green) in sagittal skin sections from P19 Hr-/- and wild-type mice. Brackets indicate expansion of K10-positive region. Scale bars: 50 µm.

 


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  		Fig. 7. Developmental expression of caspase 14 and filaggrin in epidermis. In situ hybridization of sagittal sections from mouse backskin at the indicated ages using caspase 14-specific (left columns) or filaggrin-specific (right columns) cRNA probes. Shown is a comparison between wild type (+/+) and Hr-/- (-/-). Arrowheads, specific signal. epi, epidermis; inf, infundibulum; u, utricle. Scale bar: 50 µm.

 


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  		Fig. 3. Increase in cell proliferation accompanies utricle formation. (A) Immunohistochemical detection of PCNA (proliferating cell nuclear antigen) in sagittal sections from backskin of P16 mice. Arrowheads indicate PCNA-positive cells in interfollicular epidermis, infundibulum (in wild type, +/+) and utricle (in Hr-/-, -/-). (B) Proliferating cells in utricle express K14. Immunofluorescent detection of BrdU (red) and keratin 14 (green) in sagittal skin sections from P20 mice. White arrowheads indicate BrdU-positive cells. DAPI (blue), nuclear marker. (C) Utricle epithelium does not express keratin 16 (K16), a marker of abnormal epidermal differentiation. Immunohistochemical detection of K16 in sagittal sections from backskin at P20; K16 expression is detected in club hair sheath (chs). Arrowheads in C indicate infundibulum. -/-, Hr-/-; +/+, wild type. Scale bar: 50 µm.

 


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  		Fig. 4. Expression of hair bulb markers in Hr-/- skin. (A) Normal expression of hair bulb markers during initial hair growth. In situ hybridization of sagittal sections from backskin with cRNA probes specific for Lef1 (top panels, P6) and sonic hedgehog (Shh) (bottom panels, P9) in wild-type (+/+), Hr-/- (-/-). mice (B) Absence of hair markers during second anagen. In situ hybridization of sagittal sections from backskin of P28 mice with cRNA probes specific for Lef1 and Shh in wild-type (+/+), Hr-/- (-/-). Arrowheads, gene-specific signal; arrows, pigmented cells; m, matrix; dp, dermal papilla; epi, epidermis; u, utricle. Scale bar: 50 µm.

 


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  		Fig. 5. Development of dermal cysts in Hr-/- skin. (A) Sagittal sections of Hr-/- mouse skin at 3 months (3 mo), 6 months (6 mo) and 1 year (1 yr) stained with Hematoxylin-Eosin. Scale bar: 100 µm. (B) In situ hybridization with a sebaceous gland marker (SCD1). In situ hybridization of adjacent sagittal sections of backskin from adult (1 year) Hr-/- mouse with Scd1-specific antisense (AS, left panel) or sense (S) probes (right panel). Arrowheads indicate positive signal in subset of cells surrounding cyst. Scale bar: 50 µm. (C) Lipids detected in dermal cysts. Oil red O staining on sagittal sections from backskin of adult (1 year) Hr-/- mouse. Scale bar: 50 µm. (D) Immunohistochemical detection of keratinocyte differentiation markers. Positive staining in the cyst walls for K14 (green) and K17 (red) and both (K14/K17, yellow) in sagittal sections of backskin from adult (1 year) Hr-/- mouse. Scale bar: 20 µm. (E) Cysts do not express epidermal or hair markers. Immunohistochemical detection of loricrin, filaggrin and trichohyalin in adult Hr-/- backskin. Scale bar: 50 µm. (F) Proliferation in cyst wall colocalizes with K14. Immunofluorescent detection of BrdU (red) and K14 (green) in sagittal section of Hr-/- mouse backskin (P45). DAPI, nuclear stain (blue). Scale bar: 20 µm.

 


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  		Fig. 6. Specific changes in gene expression in skin of Hr-/-mice. (A) Northern analysis of total RNA extracted from backskin of P12 mice using the indicated probes. casp-14, caspase 14; calm-4, calmodulin 4/Scarf. ß-Tubulin shows equal RNA loading on a representative blot. (B) Developmental expression of K10, loricrin, filaggrin and caspase 14. Northern analysis of RNA prepared from the skin of mice at indicated ages with the designated probes. ß-Tubulin is shown as an RNA loading control. +/+, wild type; -/-, Hr-/-.

 


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  		Fig. 8. Gene expression is upregulated in keratinocytes cultured from Hr-/- mice. Northern analysis of RNA from primary keratinocytes cultured from Hr-/- (-/-) and heterozygous (+/-) mice with the indicated probes. ß-Tubulin shows equal RNA loading on a representative blot; signal was quantitated by phosphorimager and normalized to ß-tubulin value.

 

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© The Company of Biologists Ltd 2004