Gene targeting of envoplakin, a cytoskeletal linker protein and precursor of the epidermal cornified envelope

Abstract

Envoplakin, a member of the plakin family of cytoskeletal linker proteins, is localized in desmosomes of stratified epithelial cells and is a component of the epidermal cornified envelope. Gene targeting in mouse embryonic stem cells was used to generate a null allele of envoplakin. No envoplakin transcripts from the targeted allele could be detected in the skin of newborn mice. Mice homozygous for the targeted allele were born in the normal Mendelian ratio and were fertile. They did not develop any discernible pathological phenotype up to the age of 1 year. The ultrastructural appearance of cornified envelopes from adult epidermis was indistinguishable between wild-type and knockout mice, and there was no evidence that the absence of envoplakin affected the subcellular distribution of periplakin or desmoplakin, two other plakins found in desmosomes. The proportion of immature cornified envelopes in the epidermis of newborn mice was greater in envoplakin-null animals than in heterozygous littermates or wild-type mice, and the envelopes had a larger surface area. This correlated with a slight delay in barrier acquisition during embryonic development. We conclude that although envoplakin is part of the scaffolding on which the cornified envelope is assembled, it is not essential for envelope formation or epidermal barrier function.

FIG. 1

Gene targeting of envoplakin locus. (a) Targeting strategy used to generate the envoplakin null allele. Top, 5′ end of the envoplakin gene. Black boxes represent the exons. Middle, the targeting vector inserts into the first exon of the envoplakin gene a cassette with stop codons in all reading frames, an internal ribosome entry site-dependent lacZ reporter gene, and a neomycin resistance gene. Bottom, diagnostic 5′ and 3′ end fragments to recognize correctly integrated targeting vector. (b) Genotyping the offspring of heterozygous crosses by Southern blotting. The −/− animals are homozygous for the targeted allele, as revealed by both the 5′ and 3′ probes. (c) RT-PCR analysis of mRNA from the skin of newborn mice. Both 5′-end primers (five first lanes from the left) that flank the deleted part of the gene and 3′-end primers (three right-hand lanes) from the last exon of the gene were used to verify the absence of mRNA in null animals. Primers for γ-actin were used as a control for the quality of the template RNA. Genotypes were determined by Southern blotting using the 5′-end probe.

FIG. 2

Epithelial histology of envoplakin null mice. (a and b) Whole-mount β-galactosidase staining was used to detect expression of the knockin lacZ reporter gene in tail (a) and tongue (b) epithelium of homozygous gene-targeted animals. (c to f) Hematoxylin and eosin staining of sections of intact (c and d) and wounded (e and f) back skin of heterozygous (c and e) and homozygous (d and f) mice. Arrows in e and f indicate the migrating front of wound edge keratinocytes 4 days after wounding. Bar, 60 μm (a to d) or 250 μm (e and f).

FIG. 3

Barrier acquisition and cornified envelope morphology. (a and b) Transmission electron microscopy of isolated CE from +/+ (a) and −/− (b) epidermis. Arrows indicate residual desmosomes. (c) Cornified envelopes isolated from 2-day-old wild-type or envoplakin knockout mice. f, fragile envelope; m, mature envelope. (d) E16.5 heterozygous (+/−) and envoplakin null homozygous (−/−) embryos stained with toluidine blue. Dark blue areas of the skin have not yet formed the epidermal barrier. Bar, 500 nm (a and b) or 25 μm (c).

FIG. 4

Subcellular distribution of desmoplakin and periplakin in cultured keratinocytes. Primary keratinocytes grown in high-calcium medium were stained with antibodies against desmoplakin (a and b) and periplakin (c and d). (a and c) Wild-type keratinocytes; (b and d) envoplakin-deficient keratinocytes. Bar, 10 μm (a to c) or 7 μm (d).