Focal activation of a mutant allele defines the role of stem cells in mosaic skin disorders

Abstract

Stem cells are crucial for the formation and maintenance of tissues and organs. The role of stem cells in the pathogenesis of mosaic skin disorders remains unclear. To study the molecular and cellular basis of mosaicism, we established a mouse model for the autosomal-dominant skin blistering disorder, epidermolytic hyperkeratosis (MIM 113800), which is caused by mutations in either keratin K1 or K10. This genetic model allows activation of a somatic K10 mutation in epidermal stem cells in a spatially and temporally controlled manner using an inducible Cre recombinase. Our results indicate that lack of selective pressure against certain mutations in epidermal stem cells leads to mosaic phenotypes. This finding has important implications for the development of new strategies for somatic gene therapy of dominant genodermatoses.

Figure 1

Targeting of the mouse keratin 10 gene. (A) Genomic organization of the mouse keratin 10 locus. Exon 1 contains codon 154 encoding arginine (R154). (B) The targeting vector contains a C to T mutation encoding cysteine (C154) which also destroys an AciI restriction site. (C) Recombinant mut^neo allele after homologous recombination in ES cells. (D) Recombinant mut^loxP allele after Cre-mediated excision of PGKneo. 5′ and 3′: probes used for Southern blot analysis. R, EcoRV; B, BglII; X, XhoI; S, ScaI; A, ApaI; N, NotI; K, KpnI.

Figure 3

Chimeric mice derived from +/mut^loxP ES cells. Thick hyperkeratoses on the paws at 5 wk (A) and 3 mo of age (B). (C) Normal paw.

Figure 2

Inducible mouse model for EHK. (A) Bigenic +/mut^neo. CrePR1 pup after four topical applications of RU486. Scaling around the arm folds after rupture of the blister. No blisters developed in untreated bigenic +/mut^neo.CrePR1 pups or +/mut^neo or CrePR1 pups treated with RU486 (data not shown). (B) Same mouse 3 mo after induction. Note the thick brownish hyperkeratoses on the paws. (C) Light micrograph of lesional skin from B. Note the thickened stratum corneum, vacuolization, and translucency in the suprabasal layer. Hematoxylin and eosin, bar = 50 μm. (D) PCR analysis of keratinocytes captured by LCM. PCR analysis to detect the loxP site (top panel) and neo cassette (bottom panel). 1, suprabasal cells from previously treated area; 2, untreated area; 3, wild-type control mouse; 4, +/mut^loxP mouse; and 5, +/mut^neo mouse. M, DNA size marker.

Figure 4

Gross appearance and skin morphology of mut^neo/mut^neo mice. (A) mut^neo/mut^neo pup shortly after birth with severe blistering and erosions. (B) Histological analysis of a skin biopsy from A shows a split in the suprabasal layer of the epidermis. Hematoxylin and eosin, bar = 50 μm. (C) Immunolabeling shows abundant mutant K10 (yellow) throughout the disintegrating suprabasal layer against the red K14 background stain. Bars = 50 μm.