Mice lacking desmocollin 1 show epidermal fragility accompanied by barrier defects and abnormal differentiation

Abstract

The desmosomal cadherin desmocollin (Dsc)1 is expressed in upper epidermis where strong adhesion is required. To investigate its role in vivo, we have genetically engineered mice with a targeted disruption in the Dsc1 gene. Soon after birth, null mice exhibit flaky skin and a striking punctate epidermal barrier defect. The epidermis is fragile, and acantholysis in the granular layer generates localized lesions, compromising skin barrier function. Neutrophils accumulate in the lesions and further degrade the tissue, causing sloughing (flaking) of lesional epidermis, but rapid wound healing prevents the formation of overt lesions. Null epidermis is hyperproliferative and overexpresses keratins 6 and 16, indicating abnormal differentiation. From 6 wk, null mice develop ulcerating lesions resembling chronic dermatitis. We speculate that ulceration occurs after acantholysis in the fragile epidermis because environmental insults are more stringent and wound healing is less rapid than in neonatal mice. This dermatitis is accompanied by localized hair loss associated with formation of utriculi and dermal cysts, denoting hair follicle degeneration. Possible resemblance of the lesions to human blistering diseases is discussed. These results show that Dsc1 is required for strong adhesion and barrier maintenance in epidermis and contributes to epidermal differentiation.

Figure 1.

Exon-intron organization of the murine Dsc1 gene. The analysis was conducted using a PAC clone (RPCI21 346c17) and two λ clones (λC1 and λC4). The location of each exon is shown relative to the Dsc1 gene and a schematic representation of the mouse (M) Dsc1a and Dsc1b proteins. Results from RNase protection assay (unpublished data) suggest that there are no additional 5′ exons. Alternative splicing of RNA can occur; the shorter “b” form of the protein is produced if RNA encoded by exon 16 (which contains an in-frame stop codon) is included in the final message. PRE, signal peptide; PRO, propeptide; EC1-EC5, extracellular homologous repeat domains 1–5; TM, transmembrane domain; CYT, cytoplasmic domain. The locations of introns in human (H) DSC2 (Greenwood et al., 1997) are shown for comparison and indicated by arrowheads.

Figure 2.

Targeted disruption of the mouse Dsc1 gene. (A) Targeting strategy. The targeting vector consisted of pUC DNA, a neomycin (neo) resistance cassette, and portions of the Dsc1 gene including 2.1 kb of homology (5′ arm) and 5.2 kb of homology (3′ arm). Filled vertical boxes represent exons. B, BamHI; P, PvuII; H, HindIII. (B) Southern blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA was digested with BamHI, subjected to agarose gel electrophoresis, and transferred to nitocellulose. Wild-type (20 kb) and mutant (6 kb) alleles were detected with probe 1, which hybridized to DNA just outside of the 5′ region of homology. Similarly, probe 2, which hybridized to DNA just outside the 3′ region of homology, detected wild-type (20 kb) and mutant (14 kb) bands (unpublished data).

Figure 3.

Expression of desmosomal constituents in Dsc1 mutant mice. (A) RT-PCR showing absence of Dsc1 message in Dsc1^−/− mice. Amplification of Dsc1 mRNA was performed using primer pairs P1 and P2 and P3 and P4 (Fig. 2 A) to give 476- and 358-bp products, respectively, in wild-type and heterozygous mice only. Control amplifications using glyceraldehyde-3-phosphate dehydrogenase primers (983-bp product) were performed. (B) Western blot analysis of epidermal extracts using antibodies specific for Dsc1, Dsc2, Dsc3, Dsg1+2, PG, and DP. Equal protein loadings were determined by prior staining with Coomassie brilliant blue. (C–F) Immunofluorescence of epidermis from 2-d-old normal and null mice. Dsc1 is expressed in the epidermis and hair follicles of normal mice (C) but is absent from the skin of null animals (D). DP distribution is unaffected apparently by the absence of Dsc1 and is similar in normal (E) and null mice (F). Vertical bars indicate position of epidermis. Skin samples were taken from the backs of mice. hf, hair follicle. Bars, 25 μm.

Figure 4.

Eye, skin, and growth abnormalities in Dsc1-null mice. (A) Eyelids from a 2-d-old normal mouse. f, fused eyelid epidermis; c, cornea. (B) Eyelids from a Dsc1 ^−/− littermate showing a failure of eyelid fusion, inflammation, and corneal damage. t, eyelid tips which have failed to fuse. (C) Flaky skin in two null mice at 2-d-old. At this age, null mice were indistinguishable in size from their normal littermates. (D) Flaky skin and characteristic runted appearance in an 8-d-old null mouse (bottom animal) when compared with a normal littermate (top animal). (E) Delayed weight gain in Dsc1-null mice. Results from a typical litter consisting of nine pups in which a null (Nu) mouse and its normal (No) littermates were born with equal weights, but at weaning (21 d) the null mouse weighed 30% less than the average of the others. Bars represent range of weights of normal littermates. 10 litters were examined with similar results. Bars, 150 μm.

Figure 9.

Phenotype of Dsc1-null mice increases in severity with age. (A) Scruffy and untidy coat appearance in an adult null mouse (bottom) compared with a normal littermate (top). Mice shown are 21-d-old (day of weaning). Note that the null mouse is smaller. (B) Alopecia in an adult null mouse showing the most typical pattern observed. Some ulceration on the ventral surface of the head and neck is visible also. (C) Snout ulceration in an adult null mouse showing a moderately severe example of the pattern found in all null animals and commencing after 1 mo of age. (D) Hyperplastic epidermis from the ventral surface of an adult null mouse showing acanthosis, hyperkeratosis with utriculi (ut), and cysts (cy). (E) Ulcerated skin from the abdomen of an adult Dsc1^−/− mouse showing complete loss of epidermis from the site of the ulcer and severe inflammation. The right hand margin of the ulcer is indicated by an arrow. At this low magnification, the presence of inflammatory cells is indicated by the dermal basophilia of this region. (F) Higher magnification of a utricule (ut) and a cyst (cy). (G) Whisker pad of an adult normal mouse. vf, vibrissa follicle. (H) Whisker pad of an adult null mouse. Severe hyperplasia is present in the null as indicated by the increased number of epidermal cell layers. Approximate thickness of epidermis indicated by bars (ep) in G and H. Bars: (D, E, and G) 150 μm; (F) 25 μm.

Figure 5.

Hyperplasia and increased cell proliferation in Dsc1-deficient epidermis. (A) Epidermis from a 2-d-old normal mouse. (B) Epidermis from a Dsc1 ^−/− littermate showing hyperplasia. Bars (e) indicate thickness of living layers of epidermis. (C) Epidermis from a Dsc1-null mouse showing localized hyperkeratosis and parakeratosis (retention of nuclei in the cornified layer) (arrow). (D) Expression of Ki67 in the epidermis in a normal mouse. (E) Ki67 staining in the epidermis of a Dsc1 ^−/− mouse. Only one proliferating cell located in the basal layer is present in D (arrow), whereas in E almost all of the cells in the basal layer and numerous suprabasal cells (arrows) are undergoing cell division. A–C show head skin; D and E show back skin. Bars: (A–C) 100 μm; (D and E) 50 μm.

Figure 6.

Immunofluorescence of differentiation markers in Dsc1-null mice. Distribution of P-cadherin in normal (A) and null (B) 2-d-old animals. In both cases, expression is confined to the basal cell layer and hair follicles. However, dramatic upregulation of P-cadherin is found in lesioned epidermis from adult (∼3-mo-old) null animals (C). Similarly, distribution of β₄-integrin expression is similar in normal (D) and null (E) 2-d epidermis but is increased dramatically in all cell layers of lesioned epidermis from older null mice (F). Keratin 6 is expressed in hair follicles of normal mice (G), whereas in unlesioned epidermis from 2-d-old null animals it is found both in hair follicles and interfollicular epidermis (H). Dramatic upregulation of keratin 6 is found in lesioned epidermis from adult null animals (I). In normal animals, superficial keratin 16 staining is detected in suprabasal layers (J), whereas in null mice it is expressed strongly throughout the epidermis including the basal cell layer in both nonlesioned (K) and lesioned epidermis (L). Arrowheads indicate the basement membrane. All micrographs show back skin. Bars: (A–F and J–L) 25 μm; (G–I) 150 μm.

Figure 7.

Absence of Dsc1 weakens adhesion in upper epidermis despite the presence of ultrastructurally normal desmosomes. (A) Epidermis from a 2-d-old null mouse showing hyperplasia and acantholysis. The granular layer appears to be separating from the upper spinous layer. (B) Epidermis isolated from a 2-d-old Dsc1 ^−/−-null mouse after treatment with EDTA showing that the tissue fragments allow only small pieces of epidermis to be separated from the dermis. (C) Epidermis isolated using the same technique from a normal littermate. (D) Ultrastructure of outer layers of epidermis from a normal mouse showing desmosomes (arrows). (E) Ultrastructure of outer layers of epidermis from a Dsc1-null mouse with desmosomes (arrows) of a normal appearance. (F) Ultrastructure of lesional epithelium in a Dsc1-null mouse showing splitting between cells and an absence of cell lysis. Arrows indicate possible remains of split desmosomes. All samples were from back skin. Bars: (A) 20 μm; (D and E) 1 μm; (F) 2 μm.

Figure 8.

Dsc1-null mice have skin barrier defects. (A) Localized breaches in the skin barrier allow penetration of dye into the epidermis of a null mouse but not its normal littermate, which was completely white, that is, unstained (unpublished data). (B and C) Early lesions in the skin of 2-d-old null mice showing infiltration of epidermis with polymorphonuclear cells (neutrophils). (D) More developed lesion in 2-d epidermis showing epidermal degradation and detachment from the underlying dermis. (E) Higher magnification showing epithelial cell migration from intact tissue into wound site. (F) Healed lesion in the epidermis of a 2-d-old null mouse. The damaged epidermis has completely detached from the underlying healed tissue. Arrowheads indicate polymorphonuclear cells. (G) Measurement of permeability coefficients (Kp) for water and mannitol under passive conditions show that skin from 2-d-old null mice is more permeable to both water and solutes (p < 0.05 in both cases) than that of a normal littermate. Nu, null; No, normal. Bars: (B, C, and E) 25 μm; (D and F) 100 μm.