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Human Molecular Genetics, 2003, Vol. 12, No. 14 1737-1744
DOI: 10.1093/hmg/ddg183
© 2003 Oxford University Press

Targeted epidermal expression of mutant Connexin 26(D66H) mimics true Vohwinkel syndrome and provides a model for the pathogenesis of dominant connexin disorders

George Bakirtzis1, Rukhsana Choudhry1, Trond Aasen1, Leonard Shore2, Ken Brown2, Sheila Bryson2, Stephen Forrow2, Laurence Tetley3, Malcolm Finbow4, David Greenhalgh1 and Malcolm Hodgins1,*

1Section of Squamous Cell Biology and Dermatology, Division of Cancer Sciences and Molecular Pathology, Robertson Building, University of Glasgow, Glasgow G12 8QQ, UK, 2Beatson Institute for Cancer Research, Bearsden, Glasgow G61 1BD, UK, 3Electron Microscopy Unit, Faculty of Biomedical and Life Sciences, University of Glasgow, Glasgow G12 8QQ, UK and 4Division of Biomedical Sciences, Glasgow Caledonian University, Glasgow G4 0BA, UK

Received February 24, 2003; Revised May 7, 2003; Accepted May 14, 2003


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
To investigate the role of connexins in dominantly inherited skin disease, transgenic mice were produced which expressed mutant connexin 26 [gjb2/connexin 26(D66H)], from a keratin 10 promoter, exclusively in the suprabasal epidermis (the cells in which Connexin 26 is up-regulated in epidermal hyperproliferative states). From soon after birth, the mice exhibited a keratoderma similar to that in humans carrying the Connexin 26(D66H) mutation (true Vohwinkel syndrome). Transgene expression was associated with loss of Connexin 26 and Connexin 30 from epidermal keratinocyte intercellular junctions and accumulation in cytoplasm. Light and electron microscopy showed marked thickening of the epidermal cornified layers and increased epidermal TUNEL staining, indicative of premature keratinocyte programmed cell death. The K10Connexin 26(D66H) mouse may provide a valuable model to study the role of gap-junctional intercellular communication in epidermal differentiation. Similarities in phenotype between individuals (man and mouse) carrying Connexin 26(D66H) and those carrying insertional mutants of Loricrin, a major cornified envelope protein of the epidermis, suggest a possible link between connexin function and cornified envelope formation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Connexins (Cx), a family of transmembrane proteins encoded by 20 known genes in man, form gap junctions, intercellular communication channels that permit direct exchange of small molecules (<1 kDa), and associate in heteromeric or heterotypic complexes to define the channel properties (1–3).

Mutations in members of the connexin gene family cause a spectrum of human inherited diseases, including the commonest type of non-syndromic recessive sensorineural deafness (DNFB1, OMIM 220290) (4–7), dominant hearing loss (OMIM 600101, 601544) (8–10), peripheral neuropathy (X-linked Charcot–Marie–Tooth disease, OMIM 302800) (11), cataracts (OMIM 601885 and 116200) (12,13), ectodermal dysplasia (Clouston syndrome, OMIM 129500) (14), occulodentodigitaldysplasia (OMIM 164200) (15), skin disease (erythrokeratodermia variabilis, OMIM 133200) (16), and syndromes in which keratoderma and deafness are associated (OMIM 148350; 148210, KID syndrome; 124500 true Vohwinkel syndrome) (17–19). Furthermore, the occurrence of connexin polymorphic variants with altered channel properties suggests that they could act as modifiers in an even wider range of complex disease (20).

It remains unclear how the connexin mutations cause disease, although recent studies have suggested differences in cellular processing of different disease-associated mutations in the same connexin (21). Mouse knockouts of individual connexin genes have had limited success as disease models owing to functional redundancy and the complex, overlapping expression domains of connexins, both within tissues and during development (2,3,22–24). Furthermore, in contrast to human, generalized knockout of Cx26 in mouse is embryonic-lethal (due to placental insufficiency), although recent studies have shown that conditional Cx26 knockout in the mouse inner ear causes deafness (25) as does generalized knockout of Cx30 (26). However, many of the mutant connexin phenotypes (especially many mutations of Cx26, Cx30 or Cx31 involving the skin) in man are associated with dominantly acting amino acid substitutions (2,3,8–20) for which appropriate mouse models are not available (e.g. knockout of Cx30 causes deafness but not skin disease in mouse (26). An understanding of the modes of action of dominant mutant connexins in causing disease requires the accurate recapitulation of the disease in model systems. To this end, transgenic mice were produced that express the true Vohwinkel syndrome-associated mutant, Cx26(D66H) (17) specifically in the suprabasal epidermal keratinocytes—the cells where Cx26 is up-regulated following minor skin trauma (27). Affected individuals in all true Vohwinkel syndrome families analysed to date are carriers of the Cx26D66H mutation (17). In addition to relatively mild sensorineural deafness, the clinical features include a characteristic hyperkeratosis of soles, palms and knuckles, with constriction rings on the digits sometimes leading to autoamputation (pseudo-ainhum) (17). In a preliminary study of a single patient with true Vohwinkel syndrome, we observed cytoplasmic accumulation of Cx26 immunostaining in upper palmar epidermis (28), while Di and co-workers (29) also reported cytoplasmic location of human Cx26(D66H). Thus strong expression of Cx26(D66H) in mouse suprabasal keratinocytes generally might produce skin abnormalities similar to but more widespread than those in true Vohwinkel syndrome patients. In fact the skin phenotype of the mice is indeed similar to but more extensive than that of humans carrying Cx26(D66H) and suggests a pathomechanism involving an effect on programmed cell death during epidermal differentiation.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
By employing the Keratin 10 (K10) promoter (Fig. 1A) to express the Cx26(D66H) mutant in suprabasal (differentiating) epidermal keratinocytes, three lines of transgenic founder animals were produced and propagated by crossing with outbred ICR mice. Transgenic mice were identified by PCR and Southern blot (Fig. 1B and C), while expression of the Cx26(D66H)–SV40 mRNA in skin was detected by RT–PCR (Fig. 1D). Animals expressing the transgene in skin accumulated immunoreactive Cx26 in keratinocyte cytoplasm of suprabasal keratinocytes instead of in the plasma membrane at intercellular junctions (Fig. 2A–D). This was consistent with perinuclear localization of mouse Cx26(D66H) when overexpressed in the human cervical keratinocyte cell line C33a, which expresses endogenous Cx43 but not Cx26, thereby permitting unambiguous identification of transfected mouse Cx26. Note how transfected wild-type mouse Cx26 localizes with endogenous Cx43 in large junction plaques, while Cx26(D66H) accumulates around the nucleus (Fig. 3A and B). Moreover, the aberrant connexin distribution in transgenic epidermis was not restricted to Cx26, as areas of high transgene expression (identified by strong cytoplasmic Cx26 stain) also showed focal loss of the closely related Cx30 from plasma membranes and its accumulation in cell cytoplasm with Cx26 (Fig. 4A and B). In contrast, as in the transfected C33a cells (Fig. 3), the distribution of endogenous Cx43 in epidermis was not obviously changed (data not shown).



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  		Figure 1. Genotyping and Cx26(D66H)–SV40 mRNA expression. (A) Drawing of the K10D66H transgene showing specific PCR primers. (B) Photograph of the PCR products used to identify the transgenic and wild-type animals. Lanes 1, 3 and 6, wild-type animals; lanes 2, 4 and 5 transgenic animals; lane 7, negative control; lane 8, known positive control; lane 9, water control; PCR product 250 bp. (C) Southern blot analysis. Genomic DNA was digested with SalI and hybridized to the probe shown in (A). The 6 kb band indicates the transgene. Lanes 1, 2 and 4, transgenic; lane 3, non-transgenic. (D) RT–PCR photograph showing expression of RNA from transgenic newborn skin. Lanes 1–8, all samples without reverse transcriptase; lanes 9, 13 and 15, negative samples; lanes 10, 11, 12, 14 and 16, positive samples; lane 17, negative control; lane 18, positive control; lanes 19 and 20, water control with and without reverse transcriptase. Size of the product, 250 bp.

 


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  		Figure 2. Cx26 accumulates in the cytoplasm of suprabasal keratinocytes in K10Cx26D66H transgenic epidermis. (A) Staining of newborn wild type mouse epidermis with Cx26 (day 4). Note the staining in plasma membranes of keratinocytes in the granular layer. (B) Staining of newborn mouse epidermis (day 4) with Cx26. Note strong patchy staining of Cx26 across the suprabasal epidermis, indicating mosaicism. Scale bars for (A) and (B)=100 µm. (C) Confocal image showing the distribution of Cx26 in the newborn wild-type mouse footpad epidermis (day 4). (D) Confocal image showing mainly cytoplasmic distribution of Cx26 in the newborn transgenic mouse footpad epidermis (day 4). Scale bars for (C) and (D)=50 µm.

 


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  		Figure 3. Cx26(D66H) accumulates in the cytoplasm and fails to translocate to gap junctions. Triple immunofluorescence of C33a tumour cells transfected with plasmids directing expression of Cx26(D66H) and Cx26 (WT) under control of the CMV promoter. (A) Staining of cells transfected with wild-type Cx26 shows accumulation of Cx26 in the points of cell to cell contact colocalized with endogenous Cx43 (yellow plaques, arrow). (B) Staining of cells transfected with Cx26(D66H) shows accumulation of Cx26(D66H) (green, arrow) around the nucleus (blue), while endogenous Cx43 is located in a large intercellular junction plaque (red, arrowhead).

 


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  		Figure 4. Cx26(D66H) causes reduction and cytoplasmic location of Cx30 in epidermis. Fluorescence micrographs indicating the distribution of Cx26 (red) and Cx30 (green) in the epidermis in newborn mice. (A) Wild-type mouse epidermis: note the plasma membrane location of Cx30 in the suprabasal layers (arrow). The arrowhead indicates non-specific staining in the stratum corneum. (B) Transgenic mouse epidermis: Cx26 (red, arrowhead) is strongly stained in cytoplasm; Cx30 (green) is lost from plasma membranes and is seen colocalized with Cx26(D66H) in the cytoplasm of some cells (yellow, arrow). Scale bar=50 µm.

 
By postnatal day 4, mice expressing the transgene showed transient epidermal scaling with zonal hyperkeratosis of the ears, tail and feet, leading to constriction bands, particularly on the tail (Fig. 5A–C). Generalized erythema was not seen to accompany the transient scaling, although erythema was evident around tail constrictions. The tail constrictions eventually caused autoamputation (not shown), in a similar fashion to constriction bands on digits of some patients carrying the Cx26(D66H) mutation. In the mice, the less frequently observed toe constrictions persisted into adulthood without resulting in amputation (Fig. 5D). The above phenotypes were never seen in non-transgenic mice. The severity of the phenotype varied and was not correlated with transgene copy number. All phenotypic mice analysed showed strong cytoplasmic Cx26 staining in the epidermis, as in Figure 2. Histology of transgenic epidermis showed zones of orthohyperkeratosis with a prominent granular cell layer and abnormally thick, densely packed stratum corneum (Fig. 6A and B). This was confirmed by scanning and transmission electron microscopy (Fig. 6C–F). Limited areas of parakeratotic scaling were also noted on the tail (not shown). No coat abnormalities have been noted, but eruption of hairs on the tail was inhibited at sites of heavy scaling (Fig. 6E and F) and some erupting hair shafts close to constriction rings were distorted (not shown). Abundant TUNEL positive nuclei in the tail epidermis at 4 days indicated either excess apoptosis or premature terminal differentiation (Fig. 7A and B), whereas a high level of PCNA staining in the basal epidermis (where the transgene is not expressed) indicated active cell proliferation (Fig. 7C and D).



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  		Figure 5. Epidermal scaling and constriction bands in K10Cx26(D66H) transgenic mice. (A) Transgenic mouse at day 8, note significant scaling on the back of the mouse. (B) Transgenic mouse at day 8, note scaling on the forelimb and digits of the front leg. (C) Transgenic mouse at day 8, note the appearance of constriction rings along the tail. (D) Transgenic mouse at 3 months. Appearance of a constriction ring on the toe of the hind leg. The constriction ring (indicated by the arrow) appeared on the mouse 4 weeks after birth and persisted on the same point without leading to autoamputation, throughout his life.

 


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  		Figure 6. Failure of stratum corneum shedding in transgenic mice. (A) Haematoxylin and eosin stained wild-type tail mouse epidermis (day 4). Note the open basket weave structure of the stratum corneum. (B) Haematoxylin and eosin stained transgenic mouse tail epidermis (day 4). Note the thick, compacted stratum corneum. Scale bar for (A) and (B)=50 µm. (C) Transmission electron microscopy of the wild type mouse tail epidermis (day 4). Note separation of corneocytes. (D) Transmission electron microscopy of the transgenic mouse tail epidermis (day 4). Note the lack of separation of corneocytes. Scale bar for (C) and (D)=10 µm. (E) Scanning electron microscopy of the wild type tail (day 4), showing normal scale pattern with hairs erupting. (F) Scanning electron microscopy of the transgenic tail (day 4). Note the dense, even scale and absence of hairs. Scale bar for (E) and (F)=1000 µm.

 


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  		Figure 7. Increased apoptosis and cell proliferation in the transgenic epidermis. (A) Staining of wild-type mouse epidermis (day 4) using TUNEL (brown nuclei). (B) Staining of transgenic mouse epidermis (day 4) using TUNEL (brown nuclei). Note the increased number of apoptotic cells present in the transgenic tissue as in contrast with the wild-type tissue. (C) Staining of wild-type mouse epidermis (day 4) using PCNA (purple nuclei). (D) Staining of transgenic mouse epidermis (day 4) using PCNA (purple nuclei). Note the increased number of proliferating cells present in the transgenic tissue as in contrast with the wild-type tissue. Scale bar=50 µm.

 
Repeated attempts were made to express mouse wild-type Cx26 using the K10 transgene vector as above but no transgenic animals were recovered after day E12. The reason for this is unknown at present.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
Dominantly acting mutant connexins might interact with other connexins to disrupt connexon assembly, as appears to happen in the lens of Lop10 mice carrying mutant Connexin 50 (30) or could inhibit channel functions as with the keratoderma-associated D66H, R75W and del42E mutants of Cx26 when assayed in Xenopus oocytes (31). Alternatively, mutant connexin proteins could have entirely novel properties that perturb cell function.

A plausible hypothesis to explain the skin phenotypes associated with Cx26(D66H) in mouse and man would be that expression of the mutant protein in the suprabasal keratinocytes disrupts the epidermal gap junction network, leading to premature terminal differentiation, with retention of cohesion between corneocytes in the stratum corneum. Premature keratinocyte death might produce compensatory basal cell proliferation, leading to the massive thickening of the stratum corneum. Previous studies on Cx26(D66H) expressed in Xenopus oocytes suggested that it could inhibit the channel function of wild-type Cx26 and Cx43 (31). The failure of Cx26(D66H) to form gap junction plaques in keratinocytes suggests that such inhibition would most likely involve interference with trafficking of wild-type connexins, as appears to happen with Cx30 in the K10Cx26(D66H) mouse epidermis. However, Cx26(D66H) did not appear to disrupt membrane localization of Cx43 in C33a cells (Fig. 3) or in the epidermis (not shown), even though it inhibited Cx43 channel activity when expressed in Xenopus oocytes (31). Further work will be directed towards determining the effects of Cx26(D66H) on gap-junctional communication between keratinocytes in the mouse epidermis. It is noteworthy that a targeted deletion of Cx26 in mouse cochlea or a general knockout of the close homologue Cx30 resulted in hearing loss associated with apoptosis in the cochlear sensory epithelium (25,26). Interestingly, homozygous loss of Cx26 or Cx30 in man (or of Cx30 in mouse), causes hearing loss without clinically discernible epidermal abnormality (4–7), although recent data suggest there may be subtle changes in epidermal thickness (32), compared with the more extensive hyperkeratosis associated with Cx26(D66H). A likely explanation for these findings is that one or more of the several other connexins expressed in epidermis (33) can compensate for homozygous loss of either Cx26 or Cx30. However, while other connexins may compensate for loss of Cx26 or Cx30 from epidermis (but not in the inner ear), they cannot overcome the effects of a dominant mutant such as Cx26(D66H) when this is expressed at high level as in hyperproliferative human epidermis (27) or as in the epidermis of the K10-Cx26(D66H) mouse. This hypothesis receives further support from recent reports that Cx31 mutants, that cause erythrokeratodermia variabilis, also cause cell death when overexpressed in cell culture (21,34). Marziano and co-workers (35) have recently confirmed the cytoplasmic location of Cx26(D66H) when expressed alone in HeLa cells. Interestingly, co-expression of wild-type Cx30 caused Cx26(D66H) to target the membrane, supporting an interaction between the proteins. Furthermore, when co-expressed at similar level with wild-type connexin, Cx26(D66H) inhibited channel activity of wild-type Cx26 but not wild-type Cx30. Taken together, these results and our observations suggest that the precise phenotypes (deafness versus skin disease) resulting from the different dominant Cx26 mutants may depend upon both the nature of the physical interactions between different mutant and different wild-type connexins and upon their relative levels of expression in particular tissues.

It is possible that the cell death associated with dominant connexin mutants results from causes other than direct disruption of tissue gap junction networks. Mutant connexins may exhibit entirely novel interactions with other key cellular proteins, reflected by their mislocation within the cell. This could lead to premature cell death independently of gap-junction networks. A variant of Vohwinkel syndrome (OMIM 604117), distinguished by more widespread ichthyosis—like hyperkeratosis, without deafness, also features palmoplantar keratoderma with digital constrictions, but is associated with heterozygous mutation (insG230/231) of the gene encoding Loricrin, a component of the keratinocyte cornified envelope and expressed in the granular layer (36–38). The frameshift generates a protein with an arginine and leucine rich c-terminal tail, 22 amino acids longer than the normal protein, lacking the glycine loop motifs and lysine residues, that locates abnormally in the cell nucleus (39). Expression of the mutant Loricrin in mouse suprabasal epidermis caused hyperkeratosis and tail constrictions (40), whereas expression of the normal human Loricrin gene produced no abnormalities (41). Thus, mutations in a gene encoding a gap junction protein and a gene encoding a cornified envelope precursor cause a similar, distinctive phenotype in human and in mouse epidermis. Taken together with the studies of the human diseases, our findings may have uncovered a functional relationship between the epidermal gap junction network (Cx26) and cornified envelope formation. This would be consistent with the evidence of subtle thickening of epidermis and stratum corneum in humans homozygous for loss of Cx26 (32), a possibility which can now be investigated using the mouse models. Alternatively, Cx26(D66H) and Loricrin (insG230/231) may act independently to disrupt the final stages of keratinocyte terminal differentiation by virtue of their mislocation within the cell. Preliminary immunofluorescence staining has demonstrated the presence of Loricrin in the upper layers of Cx26(D66H) tail epidermis (data not shown). Both explanations would be consistent with the more restricted phenotype of true Vohwinkel syndrome (hands and feet) compared with that of the variant, as Cx26 is expressed at a significant level in human epidermis only around the openings of eccrine sweat glands (acroinfundibulum), or at pressure sites (e.g. palms and soles), or after injury or in hyperproliferative disease (27,31), whereas Loricrin is generally expressed. Thus, current findings suggest that, while the pathomechanisms underlying connexin diseases may involve direct disruption of the tissue gap junction networks, they could also involve novel interactions of mutant proteins leading to cell death. Further mouse models, expressing mutant connexins from tissue specific promoters, should help elucidate the mechanisms underlying connexin disorders.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Generation of K10Cx26 (D66H) mice
The targeting vector consists of the backbone PSP72 (Promega) carrying a 4.8 kb fragment of the bovine keratin 10 (K10) promoter, including the TATA box, as previously described (42), immediately 5' of the complete coding exon 2 fragment of the mouse Cx26 (680 bp) (containing the D66H mutation—confirmed by sequencing the entire Cx26 insert) ligated to an SV40 3' untranslated sequence containing a polyadenylation site (520 bp; Fig. 1A). The transgene was excised from the vector with Sal I, purified then injected into the pronucleus of single cell embyos of B6CBAF1/j mice. Surviving embryos were cultured overnight at 37°C/5% CO2, then two cell embryos were transferred to the oviduct of 0.5 day post coitum pseudopregnant MF1 foster mothers. Transgenic offspring were identified at weaning by genotyping of tail DNA. Founder animals were crossed with C57BL/6 mice to generate stable lines, which were then propagated by further crosses with ICR mice.

Genotyping
Genotyping was performed using PCR and Southern blot analysis. Genomic DNA was isolated following complete digestion of the tail tissue using phenol/chloroform extraction. A 200 ng aliquot of genomic DNA was used as a template in a PCR reaction using sense and antisense primers spanning the exon 2 region of Cx26 and the 3' untranslated region of SV40 (Fig. 1A). The forward primer is the 588f (sense) (5'CTTC ATGCAACGTCTGG 3'). The reverse primer is the polyA360 (antisense) (5'TCTAGTTGTGGTTTGTCC). For Southern blot analysis, 5 µg of DNA from each sample were digested with SalI overnight at 37°C, separated by electrophoresis in a 0.8% agarose gel, blotted and hybridized to a [32]P-labelled random primed DNA probe fragment (Prime-ItII kit, Stratagene) corresponding to the 1.2 kb transgene (Cx26D66H+3'UTR of SV40).

RT–PCR
For RT–PCR analysis, RNA was extracted from newborn mouse skin using TRI-Reagent (Sigma) according to the manufacturers protocol and treated with RNAase-free DNAase-1. The superscriptII (GIBCO-BRL) kit was used for the first-strand cDNA synthesis, followed by PCR with the 588f (sense) and polyA360 primer pair. For negative controls the reverse transcriptase step was omitted; for a positive control the targeting vector was used.

Transfection of Connexin 26 into C33a cells
Wild-type Cx26 and Cx26(D66H) coding exons were inserted into the mammalian expression vector pcDNA3.1+ (Invitrogen) under control of the CMV promoter for high-level expression. C33a cervical epithelial tumour cells (which express endogenous Cx43 but not Cx26) were grown in Eagles MEM with 10% fetal bovine serum on glass coverslips until about 60–70% confluent, then transiently transfected for 23 h with 1 µg of plasmid DNA using Lipofectamine (Invitrogen) according to the manufacturer's instructions. Following a culture medium change, cells were incubated for a further 24 h, then fixed in methanol for 10 min at -20°C for immunofluorescent staining of Cx26 and Cx43.

Immunofluoresence analysis of Connexins
Mouse skin tissue was snap frozen in liquid nitrogen and stored at -70°C. Cryosections (7 µm) were cut and stained by single and dual immunofluorescence for connexins 26 and 30 as previously described (25,27). Primary antibodies were mouse monoclonal anti Cx26 (Catalogue no. 33-5800) and rabbit anti Cx30 (Catalogue no. 71-2200, Zymed, San Francisco, CA, USA). Secondary antibodies were FITC-labelled horse anti-mouse IgG and Texas Red-labelled goat anti-rabbit IgG (Vector laboratories, Peterborough, UK). Methanol-fixed C33a cells were similarly stained for Cx26 and Cx43 with primary antibodies described previously (27). Stained sections were viewed and images collected with a BioRad MRC500 confocal laser scanning microscope as described previously (27). Stained cells were viewed and images collected with a Zeiss Axiovision system.

Electron microscopy
Tissue, specimens were fixed in 2.5% glutaraldehyde in phosphate buffer (pH 7.4) for 24 h. For transmission electron microscopy tissues were dehydrated through graded alcohols and propylene oxide, and embedded in Araldite resin. Thin sections were stained with uranyl acetate/lead citrate and viewed in a Zeiss 902 transmission electron microscope. For scanning electron microscopy, fixed tissues were dehydrated in graded acetone solutions, critical point dried, gold sputter-coated and viewed in a Philips 500 scanning electron microscope.

Light microscopy
Tissues were fixed in 4% formaldehyde in phosphate buffered saline, dehydrated and embedded in paraffin wax. Sections (3 µm) were cut, rehydrated and stained with haematoxylin and eosin for histology. For PCNA (cell proliferation marker) staining, sections were rehydrated, subjected to antigen retrieval by microwaving in citrate buffer, then stained by standard indirect immunoperoxidase technique using a goat polyclonal antibody to PCNA (sc-9857, Santa Cruz, CA, USA). For detection of apoptosis by TUNEL assay, the FrageL kit (Oncogene Research products) was used according to the manufacturer's instructions.


   ACKNOWLEDGEMENTS
 
This work was supported by the BBSRC, The British Skin Foundation and Cancer Research UK. The study was performed following Local Ethical Committee review and under licences 60/2495 and 60/2929 of the Animals [Scientific Procedures] Act, 1986.


   FOOTNOTES
 
* To whom correspondence should be addressed. Email: m.b.hodgins{at}clinmed.gla.ac.uk Back


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 

  1. Lo, C.W. (1999) Genes, gene knockouts, and mutations in the analysis of gap junctions. Dev. Genet., 24, 1–4.[CrossRef][Web of Science][Medline]

  2. Kelsell, D.P., Dunlop, J. and Hodgins, M.B. (2001) Human diseases: clues to cracking the connexin code? Trends Cell Biol., 11, 2–6.[CrossRef][Web of Science][Medline]

  3. Evans, W.H. and Martin, P.E.M. (2002) Gap junctions: structure and function. (Review.) Mol. Membr. Biol., 19, 121–136.[CrossRef][Web of Science][Medline]

  4. Kelsell, D.P., Dunlop, J., Stevens, H.P., Lench, N.J., Liang, J.N., Parry, G., Mueller, R.F. and Leigh, I.M. (1997) Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature, 387, 80–83.[CrossRef][Medline]

  5. Zelante, L., Gasparini, P., Estivill, X., Melchionda, S., D'Agruma, L., Govea, N., Mila, M., Monica, M.D., Lutfi, J., Shohat, M. et al. (1997) Connexin26 mutations associated with the most common form of non-syndromic neurosensory autosomal recessive deafness (DFNB1) in Mediterraneans. Hum. Mol. Genet., 6, 1605–1609.[Abstract/Free Full Text]

  6. Estivill, X., Fortina, P., Surrey, S., Rabionet, R., Melchionda, S., D'Agruma, L., Mansfield, E., Rappaport, E., Govea, N., Mila, M., Zelante, L. and Gasparini, P. (1998) Connexin-26 mutations in sporadic and inherited sensorineural deafness. Lancet, 351, 394–398.[CrossRef][Web of Science][Medline]

  7. del Castillo, I., Villamar, M., Moreno-Pelayo, M.A., del Castillo, F.J., Alvarez, A., Telleria, D., Menendez, I. and Moreno, F. (2002) A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. New Engl. J. Med., 346, 243–249.[Abstract/Free Full Text]

  8. Denoyelle, F., Lina-Granade, G., Plauchu, H., Bruzzone, R., Chaib, H., Levi-Acobas, F., Weil, D. and Petit, C. (1998) Connexin 26 gene linked to a dominant deafness. Nature, 393, 319–320.[CrossRef][Medline]

  9. Grifa, A., Wagner, C.A., D'Ambrosio, L., Melchionda, S., Bernardi, F., Lopez-Bigas, N., Rabionet, R., Arbones, M., Monica, M.D., Estivill, X. et al. (1999) Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nat. Genet., 23, 16–18.[Web of Science][Medline]

  10. Xia, J., Liu, C., Tang, B., Pan, Q., Huang, L., Dai, H., Zhang, B., Xie, W., Hu, D., Zheng, D. et al. (1999) Mutations in the gene encoding gap junction protein beta-3 associated with autosomal dominant hearing impairment. Nat. Genet., 20, 370–373. [Erratum, Nat. Genet., 21, 241 (1999).]

  11. Bergoffen, J., Scherer, S.S., Wang, S., Oronzi Scott, M., Bone, L.J., Paul, D.L., Chen, K., Lensch, M.W., Chance, P.F. and Fischbeck, K.H. (1993) Connexin mutations in X-linked Charcot–Marie–Tooth disease. Science, 262, 2039–2042.[Abstract/Free Full Text]

  12. Mackay, D., Ionides, A., Kibar, Z., Rouleau, G., Berry, V., Moore, A., Shiels, A. and Bhattacharya, S. (1999) Connexin46 mutations in autosomal dominant congenital cataract. Am. J. Hum. Genet., 64, 1357–1364.[CrossRef][Web of Science][Medline]

  13. Shiels, A., Mackay, D., Ionides, A., Berry, V., Moore, A. and Bhattacharya, S. (1998) A missense mutation in the human connexin50 gene (GJA8) underlies autosomal dominant ‘zonular pulverulent’ cataract, on chromosome 1q. Am. J. Hum. Genet., 62, 526–532.[CrossRef][Web of Science][Medline]

  14. Lamartine, J., Essenfelder, G.M., Kibar, Z., Lanneluc, I., Callouet, E., Laoudj, D., Lemaitre, G., Hand, C., Haylick, S.J., Zonana, J. et al. (2000) Mutations in GJB6 cause hidrotic ectodermal dysplasia. Nat. Genet., 26, 142–144.[CrossRef][Web of Science][Medline]

  15. Paznekas, W.A., Boyadjiev, S.A., Shapiro, R.E., Daniels, O., Wollnik, B., Keegan, C.E., Innis, J.W., Dinulos, M.B., Christian, C., Hannibal, M.C. and Jabs, E.W. (2003) Connexin 43 (GJA1) mutations cause the pleiotropic phenotype of oculodentodigital dysplasia. Am. J. Hum. Genet., 72, 408–418.[CrossRef][Web of Science][Medline]

  16. Richard, G., Smith, L.E., Bailey, R.A., Itin, P., Hohl, D., Epstein, E.H., DiGiovanna, J.J., Compton, J.G. and Bale, S.J. (1998) Mutations in the human connexin gene GJB3 cause erythrokeratodermia variabilis. Nat. Genet., 20, 366–369.[CrossRef][Web of Science][Medline]

  17. Maestrini, E., Korge, B.P., Ocana-Sierra, J., Calzolari, E., Cambiaghi, S., Scudder, P.M., Hovnanian, A., Monaco, A.P. and Munro, C.S. (1999) A missense mutation in connexin26, D66H, causes mutilating keratoderma with sensorineural deafness (Vohwinkel's syndrome) in three unrelated families. Hum. Mol. Genet., 8, 1237–1243.[Abstract/Free Full Text]

  18. Richard, G., Rouan, F., Willoughby, C.E., Brown, N., Chung, P., Ryynanen, M., Jabs, E.W., Bale, S.J., DiGiovanna, J.J., Uitto, J. and Russell, L. (2002) Missense mutations in GJB2 encoding connexin-26 cause the ectodermal dysplasia keratitis–ichthyosis–deafness syndrome. Am. J. Hum. Genet., 70, 1341–1348.[CrossRef][Web of Science][Medline]

  19. van Steensel, M.A.M., van Geel, M., Nahuys, M., Smitt, J.H.S. and Steijlen, P.M. (2002) A novel connexin 26 mutation in a patient diagnosed with keratitis–ichthyosis–deafness syndrome. J. Invest. Dermatol., 118, 724–727.[CrossRef][Web of Science][Medline]

  20. Kelsell, D.P., Di, W.L. and Houseman, M.J. (2001) Connexin mutations in skin disease and hearing loss. Am. J. Hum. Genet., 68, 559–568.[CrossRef][Web of Science][Medline]

  21. Di, W.L., Monypenny, J., Common, J.E., Kennedy, C.T., Holland, K.A., Leigh, I.M., Rugg, E.L., Zicha, D. and Kelsell, D.P. (2002) Defective trafficking and cell death is characteristic of skin disease associated connexin 31 mutations. Hum. Mol. Genet., 11, 2005–2014.[Abstract/Free Full Text]

  22. White, T.W. and Paul, D.L. (1999) Genetic diseases and gene knockouts reveal diverse connexin functions. A. Rev. Physiol., 61, 283–310.[CrossRef][Web of Science][Medline]

  23. Plum, A., Hallas, G., Magin, T., Dombrowski, F., Hagendorff, A., Schumacher, B., Wolpert, C., Kim, J., Lamers, W.H., Evert, M. et al. (2000) Unique and shared functions of different connexins in mice. Curr. Biol., 10, 1083–1091.[CrossRef][Web of Science][Medline]

  24. White, T.W. (2002) Unique and redundant connexin contributions to lens development. Science, 295, 319–320.[Abstract/Free Full Text]

  25. Cohen-Salmon, M., Ott, T., Michel, V., Hardelin, J.-P., Perfettini, I., Eybalin, M., Wu, T., Marcus, D.C., Wangemann, P., Willecke, K. and Petit, C. (2002) Targeted ablation of connexin26 in the inner ear epithelial gap junction network causes hearing impairment and cell death. Curr. Biol., 12, 1106–1111.[CrossRef][Web of Science][Medline]

  26. Teubner, B., Michel, V., Pesch, J., Lautermann, J., Cohen-Salmon, M., Söhl, G., Jahnke, K., Winterhager, E., Herberhold, C., Hardelin et al. (2003) Connexin30 (Gjb6)-deficiency causes severe hearing impairment and lack of endocochlear potential. Hum. Mol. Genet., 12, 13–21.[Abstract/Free Full Text]

  27. Lucke, T., Choudhry, R., Thom, R., Selmer, I.S., Burden, A.D. and Hodgins, M.B. (1999) Up-regulation of connexin 26 is a feature of keratinocyte differentiation in hyperproliferative epidermis, vaginal epithelium and buccal epithelium. J. Invest. Dermatol., 112, 354–361.[CrossRef][Web of Science][Medline]

  28. Hodgins, M.B., Munro, C.S., Shore, L.J., Finbow M.E., Taylor, A.E.M. and Richard, G. (1999) The skin phenotypes of dominant Connexin 26 mutations. J. Invest Dermatol., 113, 444.

  29. Di, W.L., Common, J.E. and Kelsell, D.P. (2001) Connexin 26 expression and mutation analysis in epidermal disease. Cell Commun. Adhes., 8, 415–418.[Web of Science][Medline]

  30. Chang, B., Wang, X., Hawes, N.L., Ojakian, R., Davisson, M.T., Lo, W.-K. and Gong, X. (2002) A Gja8 (Cx50) point mutation causes an alteration of alpha-3 connexin (Cx46) in semi-dominant cataracts of Lop10 mice. Hum. Mol. Genet., 11, 507–513.[Abstract/Free Full Text]

  31. Rouan, F., White, T.W., Brown, N., Taylor, A.M., Lucke, T.W., Paul, D.L., Munro, C.S., Uitto, J., Hodgins, M.B. and Richard, G. (2001) Trans-dominant inhibition of connexin 43 by mutant connexin 26: implications for dominant connexin disorders affecting epidermal differentiation. J. Cell Sci., 114, 2105–2113.[Abstract/Free Full Text]

  32. Meyer, C.G., Amedofu, G.K., Brandner, J.M., Pohland, D., Timmann, C. and Horstmann R.D. (2002) Selection for deafness? Nat. Med., 8, 1332–1333.[CrossRef][Web of Science][Medline]

  33. Di, W.L., Rugg, E.L., Leigh, I.M. and Kelsell, D.P. (2001) Multiple epidermal connexins are expressed in different keratinocyte subpopulations including connexin 31. J. Invest. Dermatol., 117, 958–964.[CrossRef][Web of Science][Medline]

  34. Diestel, S., Richard, G., Doring, B. and Traub, O. (2002) Expression of a connexin31 mutation causing erythrokeratodermia variabilis is lethal for HeLa cells. Biochem. Biophys. Res. Commun., 296, 721–728.[CrossRef][Web of Science][Medline]

  35. Marziano, N.K., Casalotti, S.O., Portelli, A.E., Becker D.L. and Forge, A. (2003) Mutations in the gene for connexin 26 (GJB2) that cause hearing loss have a dominant effect on connexin 30. Hum. Mol. Genet., 12, 805–812.[Abstract/Free Full Text]

  36. Maestrini, E., Monaco, A.P., McGrath, J.A., Ishida-Yamamoto, A., Camisa, C., Hovnanian, A., Weeks, D.E., Lathrop, M., Uitto, J. and Christiano, A.M. (1996) A molecular defect in loricrin, the major component of the cornified cell envelope, underlies Vohwinkel's syndrome. Nat. Genet., 13, 70–77.[CrossRef][Web of Science][Medline]

  37. Ishida-Yamamoto, A., McGrath, J.A., Lam, H., Iizuka, H., Friedman, R.A. and Christiano, A.M. (1997) The molecular pathology of progressive symmetric erythrokeratoderma: a frameshift mutation in the loricrin gene and perturbations in the cornified cell envelope. Am. J. Hum. Genet., 61, 581–589.[Web of Science][Medline]

  38. Korge, B.P., Ishida-Yamamoto, A., Punter, C., Dopping-Hepenstal, P.J.C., Iizuka, H., Stephenson, A., Eady, R.A.J. and Munro, C.S. (1997) Loricrin mutation in Vohwinkel's keratoderma is unique to the variant with ichthyosis. J. Invest. Dermatol., 109, 604–610.[CrossRef][Web of Science][Medline]

  39. Ishida-Yamamoto, A., Kato, H., Kiyama, H., Armstrong, D.K.B., Munro, C.S., Eady, R.A.J., Nakamura, S., Kinouchi, M., Takahashi, H. and Iizuka, H. (2000) Mutant loricrin is not crosslinked into the cornified cell envelope but is translocated into the nucleus in loricrin keratoderma. J. Invest. Dermatol., 115, 1088–1094.[CrossRef][Web of Science][Medline]

  40. Suga, Y., Jarnik, M., Attar, P.S., Longley, M.A., Bundman, D., Steven, A.C., Koch, P.J. and Roop, D.R. (2000) Transgenic mice expressing a mutant form of Loricrin reveal the molecular basis of the skin diseases, Vohwinkel syndrome and progressive symmetric erythrokeratoderma. J. Cell Biol., 151, 401–412.[Abstract/Free Full Text]

  41. Yoneda, K. and Steinert, P.M. (1993) Overexpression of human loricrin in transgenic mice produces a normal phenotype. Proc. Natl Acad. Sci. USA, 90, 10754–10758.[Abstract/Free Full Text]

  42. Bailleul, B., Surani, M.A., White, S., Barton, S.C., Brown, K., Blessing M., Jorcano, J. and Balmain, A. (1990) Skin hyperkeratosis and papilloma formation in transgenic mice expressing a ras oncogene from a suprabasal keratin promoter. Cell, 62, 697–708.[CrossRef][Web of Science][Medline]


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