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First published online July 2, 2007
doi: 10.1242/10.1242/jcs.004481

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Conditional targeting of plectin in prenatal and adult mouse stratified epithelia causes keratinocyte fragility and lesional epidermal barrier defects

Reinhard Ackerl^1,*, Gernot Walko^1,*, Peter Fuchs¹, Irmgard Fischer¹, Matthias Schmuth² and Gerhard Wiche^1,

¹ Department of Molecular Cell Biology, Max F. Perutz Laboratories, University of Vienna, 1030 Vienna, Austria
² Department of Dermatology and Venereology, Medical University of Innsbruck, 6020 Innsbruck, Austria

View larger version (119K): [in this window] [in a new window]   Fig. 1. Phenotypic analysis of K5-Cre KO mice. (A-C) Two-day-old K5-Cre KO pups showing severe skin detachments and skin lesions on their upper extremities and in their armpits. (D) Hematoxylin and Eosin (H/E) staining of K5-Cre KO skin, showing epidermal detachment (asterisks) at the level of the basal keratinocyte layer. (E) Size comparison of 2-day-old Plecflox/flox (control) and K5-Cre KO pups. (F) Huge blister (asterisk) detected in the upper palate of a 2-day-old K5-Cre KO pup. Parts of the epithelial tissue (arrowheads) are still attached to the connective tissue, indicating disruption of basal oral keratinocytes. (G-J) H/E stainings of cross sections of proximal esophagus from K5-Cre KO (H,J) and control mice (G,I). I and J show magnifications of the boxed areas in G and H, respectively. No detachment of the epithelium was observed. Arrowheads in I,J denote basement membrane. (K,L) Comparison of stomachs and upper intestines of newborn control and K5-Cre KO pups. Note the translocation of the milk from the stomach to the gut, showing that the K5-Cre KO pup has not been affected by pyloric atresia. The inset in L shows the empty stomach of a 2-day-old KO pup. Arrows point to the pyloric regions. (M,N) Toluidine Blue dye penetration assay. Note localized breaches in skin barrier of K5-Cre KO pups allowing penetration of dye. (O,P) Representative H/E stainings of Toluidine-Blue-stained spots, revealing microlesions. In O, note the newly formed acanthotic epidermis under a crust (asterisk) that is covered by pieces of the old (dead) epidermis (arrowheads). The inset in O shows a photograph of a microlesion (arrow). The lesion in P is in the process of re-epithelialization (dashed line). (Q-T) Induction of microlesions by mild trauma. Flank skins of newborn mice were tape-stripped 10 times and subsequently stained with Toluidine Blue (Q,R). Note blue-stained microlesions (arrowheads) in stripped skin of K5-Cre KO (R). Tape stripping caused epidermal detachment (S, asterisks) and blister formation (T) in K5-Cre KO skin.

View larger version (113K): [in this window] [in a new window]   Fig. 2. High efficiency of Cre-mediated plectin deletion. (A) Immunoblotting of epidermal and dermal tissue homogenates from newborn Plecflox/flox (control) and K5-Cre KO mice. After separating epidermis and dermis using dispase, proteins were extracted and subjected to immunoblotting using anti-pan-plectin antiserum. Tubulin served as loading control. Note, very faint plectin signal in K5-Cre KO epidermis, testifying to nearly complete abolition of plectin in this tissue, whereas in dermis, plectin expression was hardly reduced. (B) Southern blot analysis of genomic DNAs from epidermis and dermis of newborn K5-Cre KO mice and their Plecflox/flox (control) littermates. DNAs were digested with NcoI (releasing 15 kb and 9 kb fragments from the Plecflox and Plec alleles, respectively) and hybridized with the Nco probe (see Fig. S1A in supplementary material; Plecflox and Plec alleles, respectively). Note complete conversion of the Plecflox to the Plec allele in the epidermis of K5-Cre KO mice and the presence of some Cre activity in the dermis, as indicated by the 9 kb Plec allele. (C) Immunofluorescence microscopy of frozen skin (a,b), tongue and palate (c,d), esophagus (e,f) and heart (g,h) tissue sections of newborn control and K5-Cre KO specimens using anti-pan-plectin antiserum. Note strong plectin signal in control skin along the basal membrane (arrowheads in a) and throughout the epidermis. In the oral cavity, the plectin signal is most prominent along the basal membranes of palate and tongue epithelia (arrowheads in c). Plectin was also expressed in the dermis, foremost at dermo-epidermal junctions of hair follicles (asterisks in a). No plectin-specific label was detectable in K5-Cre KO epidermis, palate, and tongue epithelia, except for a few single epidermal cells, most likely melanocytes and Langerhans cells (arrows in b). Also note strongly reduced plectin-specific label in K5-Cre KO esophagus (f), but in K5-Cre KO heart, plectin expression was not reduced (h). p, palate; t, tongue. Dashed lines (in b and d) indicate dermo-epidermal borders. Bars, 20 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 3. Immunolocalization of Int4 (A,B), BPAG1 (C,D), K5 (E,F) and K6 (G-I) in skin sections from 1-day-old Plecflox/flox (control) and K5-Cre KO mice. (A,B) In control epidermis, the Int4 signal was confined to the basal membrane (arrowheads) of basal keratinocytes and the outer root sheath of hair follicles, whereas in K5-Cre KO epidermis, it was slightly reduced and less polarized. In blistered areas (inset in B, asterisk), Int4 was located at blister floors. (C,D) Note more discontinuous BPAG1 staining along basal keratinocyte membranes (arrowheads) in K5-Cre KO compared to control epidermis. Inset in D shows BPAG1 localization at the floor (arrowheads) of blisters (asterisk). (E,F) The K5 signal was restricted to basal keratinocytes and rare transit amplifying cells (arrow), without noticable differences between control and K5-Cre KO epidermis. In blistered areas (inset in F, asterisks), K5 was located at blister roofs (arrowheads denote the blister floor). (G-I) In control skin, K6 was exclusively expressed in hair follicles (G), while in leg skins of K5-Cre KO mice, in addition to hair follicles (H), patches of suprabasal keratinocytes above microblisters were K6-positive (I). In I the dashed line indicates the dermo-epidermal border and the dotted line, a microblister separating the epidermis from the underlying dermis. Asterisks, blister. Bar, 20 µm.

View larger version (92K): [in this window] [in a new window]   Fig. 4. Inducible knockout of plectin in mice and examination of epidermal integrity. (A) Southern blot analysis (autoradiography) of DNA isolated from the epidermis of a PLECflox/–:K5-Cre-ERT mouse after treatment with 4-hydroxy-tamoxifen (OHT). DNA was digested with NcoI as described in Fig. 2, and hybridized with the Nco probe shown in Fig. S1A. The 2.8 kb signal stems from the plectin-null allele. Note partial Cre-mediated recombination after OHT treatment. (B-D) Immunofluorescence microscopy of frozen leg (B), ear (C) and tail (D) skin sections of OHT-treated PLECflox/–:K5-Cre-ERT mouse specimens using anti-pan-plectin antiserum. Note patches of plectin-deficient keratinocytes in intrafollicular epidermis (brackets). Arrows in B indicate non-keratinocyte plectin-positive cells, most likely melanocytes and Langerhans cells. HF, hair follicle. SG, sebaceous gland. (E-G) H/E stainings of tail skin (E,F) and shaved back skin (G) specimens from OHT-treated PLECflox/–:K5-Cre-ERT mice, showing normal skin morphology and absence of microblisters in unstressed skin (E) and formation of microblisters (asterisks) after repeated (10x) tape strippings with D-Squame disks (F,G). (H,I) Transepidermal water loss (TEWL; H) and quantification of stratum corneum protein removal (I) after serial tape strippings of skins from Plecflox/–:K5-Cre-ERT mice, that had been either untreated (–OHT) or treated (+OHT). Basal TEWL was measured before the first stripping. Values are mean ± s.e.m. from 5 OHT-treated and 5 untreated (control) Plecflox/–:K5-Cre-ERT littermates. Measurements were taken after two subsequent tape strippings. For statistical analyses the Student's t-test was used (*P<0.1; **P<0.05). Note that for quantification of stratum corneum protein removal, disks from two subsequent strippings were pooled. Bars, 20 µm.