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First published online 14 November 2006
doi: 10.1242/jcs.03282

Journal of Cell Science 119, 4901-4912 (2006)
Published by The Company of Biologists 2006
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Targeted ablation of Arnt in mouse epidermis results in profound defects in desquamation and epidermal barrier function

Songmei Geng1,*,{ddagger}, Alexandre Mezentsev1,{ddagger}, Sergey Kalachikov2, Klaus Raith3, Dennis R. Roop4 and Andrey A. Panteleyev1,§

1 Departmnet of Dermatology, Columbia University, New York, NY, USA
2 Departmnet of Columbia Genome Center, Columbia University, New York, NY, USA
3 Institute of Pharmaceutics and Biopharmaceutics, Martin Luther University, Halle, Germany
4 Departments of Molecular and Cellular Biology and Dermatology, Baylor College of Medicine, Houston, TX, USA


Figure 1
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  		Fig. 1. Arnt immunohistochemistry in human (A) and mouse (B) skin. Deparaffinized skin sections were incubated with anti-Arnt antibodies and processed using the ABC procedure. (A-1) In normal human epidermis, Arnt-positive nuclei are seen in the basal and spinous layers whereas the granular layer (arrows) is mostly negative. (A-2) Intensive Arnt expression is seen in the nuclei of the sweat gland (SwG) epithelium and in the nuclei of cebocytes (arrows). (A-3) Follicular papilla (fp) fibroblasts are negative, whereas hair matrix cells are positive. (A-4) A close-up of the middle portion of a human hair follicle (HF) shows cells with positive nuclei in the outer root sheath (ORS), whereas the inner root sheath (IRS) and precortex cells (pk) are negative. (B-1) In embryonic mouse skin (E15.5), moderate Arnt expression is seen in the epidermis, concentrated above the hair placode. The mesenchymal compartment is negative. (B-2) During the initial stages of HF morphogenesis, the interfollicular epidermis shows low-to-moderate Arnt expression, whereas in growing HFs high expression is seen in the matrix and in the middle ORS. (B-3) With completion of HF morphogenesis, Arnt expression in both HF and epidermis significantly increases; dermal cells and follicular papilla fibroblasts are also positive. (B-4) In catagen-telogen, follicular papilla (FP) cells (arrows) are highly positive, whereas in the HF and the epidermis Arnt immunoreactivity declines and is seen mainly in the basal layer and HF ORS. (C) Western blot with protein samples isolated from dermis and epidermis at different stages of postnatal development of normal C57BL/6 mice (days 1-23 post partum). (D) Quantification of western blot data using a FluorChem 8800 digital image system showed gradual decline of Arnt expression in the mouse epidermis during the first days of life whereas in the dermis (including HFs) and in total skin (E) it increases between days 1 and 3 and then decreases. Bars, 40 µm (A-4, B-1); 60 µm (A-1, A-2, B-2, B-3, B-4); 120 µm (A-3).

 

Figure 2
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  		Fig. 2. Generation of Arnt-deficient mice. (A) Domain structure of Arnt. NLS, nuclear localization signal; bHLH, basic helix-loop-helix domain; PAS, Per-Arnt-Sim domain; TAD, transcription activation domain. (B) Generation of a K14-driven Arnt-deficient mouse model. LoxP sites flank exon 6 of the Arnt gene (see Tomita et al., 2000Go). Activity of Cre recombinase is introduced by crossbreeding of Arnt-floxed and K14-Cre animals, resulting in the excision of the exon 6 encoding the bHLH domain. (C) PCR analysis of the progeny obtained from crossbreeding of Arntflox/flox and K14-Cre mice. Lane 1, Arnt wild-type allele; lane 2, Arntflox/-:K14-Cre-; lane 3, Arntfloxflox:14-Cre-; lane 4, Arntflox/-:K14-Cre+, deleted ({Delta}) band is present; lane 5, Arntflox/flox:K14-Cre+, deleted ({Delta}) band is present. The presence of the undeleted (floxed) bands in lanes 4 and 5 is explained by contamination of the sample with genomic DNA from K14-negative tissues (e.g. cartilage or dermis). (D) Western blot performed with proteins isolated from epidermis of Arnt{Delta}/{Delta} pups and cultured Arnt{Delta}/{Delta} keratinocytes shows absence of Arnt protein, thus confirming the efficiency of gene targeting in our mouse model.

 

Figure 3
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  		Fig. 3. Arnt-null skin phenotype in mice. (A-1) The Arnt{Delta}/{Delta}:Cre+ newborn (on the left) is smaller and does not have milk in its stomach in contrast to control littermate (on the right; milk is seen through transparent skin). (A-2) Skin permeability assay (X-gal) in Arntflox/flox (Cre-) and Arnt{Delta}/{Delta} fetuses (E18.5). In Arnt{Delta}/{Delta} fetuses, the permeability barrier is impaired with most effect in the throat, chest and groin regions. Control fetus (on the left) has a fully functional barrier and does not show any X-gal penetration. (B) Skin histology (Hematoxylin and Eosin staining) in Arnt{Delta}/{Delta} (B-1) and control (B-2) newborn mice. Note the thinner epidermis, loss of the granular layer, compact corny layer and prominent parakeratosis in Arnt{Delta}/{Delta} epidermis. (C) Grafting of Arnt-null skin onto SCID mice. 30 days after grafting, Arnt{Delta}/{Delta} grafts (C-1) show lower rates of hair growth compared to control grafts (C-2). (C-3) Immunohistochemistry with anti-Arnt antibodies revealed the total absence of Arnt expression in epithelial (K14-positive) compartment of Arnt-null skin including sebaceous gland (arrows) and HF (C-5) whereas the mesenchymal component including dermal fibroblasts, perifollicular dermal sheath and dermal papilla cells of the HF (K14-negative structures) are positive for Arnt. Control skin had normal patterns of Arnt expression in both, epithelial and mesenchymal compartments (C-4,C-6). No difference in HF morphology between Arnt-null and wild-type grafts was observed. Bars, 40 µm.

 

Figure 4
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  		Fig. 4. (A-D) Scanning electron microscopy of the skin surface in Arnt{Delta}/{Delta} (A,C,D) and control (B) mouse newborns. The surface of Arnt{Delta}/{Delta} epidermis is flat and taut (A) whereas control epidermis is extensively folded, forming longitudinal ridges (B). At high magnification, corneocytes in the control epidermis have a perfectly smooth surface (not shown), whereas in Arnt{Delta}/{Delta} skin surface of outermost cells is rough, irregular, and often perforated (C,D). Surface cells in Arnt{Delta}/{Delta} epidermis often contain nuclei (C, arrow) suggestive of parakeratosis. (E-H) Transmission electron microscopy identified fragments of cytoplasmic organelles in the upper corny layer of Arnt{Delta}/{Delta} epidermis (F, white arrowheads). Corneocytes in Arnt{Delta}/{Delta} epidermis are much thicker and are tightly packed together (compare E with G). Whereas in normal epidermis (H) corneosomes are degraded in the lower part of corny layer (arrows), in Arnt{Delta}/{Delta} skin (F), corneosomes are intact even in the outermost portion of the corny layer (arrows) thus providing strong bonds between corneocytes. Black arrowheads in F and H indicate cornified envelope.

 

Figure 5
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  		Fig. 5. Expression of cornified envelope proteins (loricrin, filaggrin and involucrin) in paraffin sections of skin and tongue in control (A,C,E,G) and Arnt-null (B,D,F,H) mouse newborns. Note the abnormal corny layer and loss of anterior-posterior polarity in the tongue papillae in the Arnt{Delta}/{Delta} newborns. Bars, 20 µm.

 

Figure 6
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  		Fig. 6. Results from real-time PCR for epidermal differentiation complex genes and serine protease inhibitors performed with mRNA isolated from Arnt-null and control newborn mouse epidermis. (A) Genes with magnitude of expressional changes at or below 10. (B) Genes with magnitude of expressional changes significantly above 10.

 

Figure 7
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  		Fig. 7. The content (by weight) of the major ceramide species in the epidermis of Arnt{Delta}/{Delta} (black bars) and control (gray bars) mouse newborns (from HPTLC).

 

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© The Company of Biologists Ltd 2006