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First published online 2 July 2003
doi: 10.1242/jcs.00638

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Altered connexin expression and wound healing in the epidermis of connexin-deficient mice

Markus Kretz, Carsten Euwens, Sonja Hombach, Dominik Eckardt, Barbara Teubner^*, Otto Traub, Klaus Willecke and Thomas Ott

Institut für Genetik, Abteilung Molekulargenetik; Römerstraße 164, 53117 Bonn, Germany
^* Present address: DLR-Projektträger, Gesundheitsforschung, Südstr. 125, 53175 Bonn, Germany

View larger version (110K): [in a new window]   Fig. 1. Immunofluorescence analysis of connexins in cryosections of wild-type epidermis. Cx31 immunoreactivity was mainly detected in the granulous layer and the upper part of the spinous layer (A). In 20% of all specimen analyzed, Cx30 (B) and Cx26 (D) were weakly expressed in the granulous layer. Cx43 expression occurred in the basal as well as in the spinous layer (C). Nuclei were stained with Hoechst 33258 fluorescent dye. The basal lamina is marked by white dots. Bar, 25 µm. Epidermal layers: bs, basal; co, cornified; gr, granulous; sp, spinous.

View larger version (62K): [in a new window]   Fig. 2. ß-Galactosidase staining of Cx31–/– (A) and Cx30–/– (B) tail epidermis. In Cx31–/– mice (no nuclear localisation signal in the lacZ gene) granulous and upper spinous layers were strongly stained. The innermost spinous layers were labelled more weakly and the basal layer showed punctuate staining. Cx30-deficient mice (B) showed ß-galactosidase staining in all epidermal layers. The basal lamina is marked by white dots. Bars, 25 µm. Epidermal layers: bs, basal; co, cornified; gr, granulous; sp, spinous.

View larger version (39K): [in a new window]   Fig. 3. Immunofluorescence signals of Cx43 protein in cryosections of the Cx31–/– epidermis (A) are significantly weaker than in the epidermis of wild-type mice (cf. Fig. 1C). (B) Immunoblot analysis of Cx43 protein in Cx31+/+ and Cx31–/– tail skin. Cx31+/+ skin lysate showed about threefold more Cx43 protein than Cx31–/– lysate. Ponceau-stained nitrocellulose membranes (C) indicated equal protein loading of the lanes shown in (B). Nuclei were stained with Hoechst 33258 fluorescent dye. The basal lamina in A is marked by white dots. Bar, 25 µm. Epidermal layers: bs, basal; co, cornified; gr, granulous; sp, spinous.

View larger version (51K): [in a new window]   Fig. 4. Immunofluorescence labelling of Cx30 in cryosections of 4-hydroxytamoxifen-induced Cx43Cre-ER(T)/fl mouse tail epidermis (A) showed prominent immunoreactivity of Cx30 in the granulous and spinous layers, in contrast to barely visible Cx30 immuno signals in wild-type epidermis (B). Nuclei were stained with Hoechst 33258 fluorescent dye. The basal lamina is marked by white dots. Bars, 25 µm. Epidermal layers: bs, basal; co, cornified; gr, granulous; sp, spinous.

View larger version (151K): [in a new window]   Fig. 5. Paraffin sections (5 µm) of wounded epidermis of mouse tail, stained with haematoxylin and eosin. During the first 12 to 24 hours after wounding (A), the incision wound could be recognized as a cut through the epidermis and dermis. On the second day of the healing process (B), the epidermis at the wound edges was thickened and the rounded keratinocytes (see enlargement of boxed area in B) began their migration under the coagulum. Three days after wounding (C), the migrating keratinocytes met under the coagulum, while closing the epidermal wound and beginning with their process of terminal differentiation (see enlargement C). After 4 to 5 days of wound healing (D), the epidermis was again completely stratified (enlargement D). Bars in main images, 250 µm; bars in inserted micrographs, 25 µm. Bs, basal; Co, cornified; Coa, coagulum; E, epidermis; Gr, granulous; Sp, spinous.

View larger version (39K): [in a new window]   Fig. 6. Altered expression levels of Cx26, Cx30, Cx31 and Cx43 relative to the corresponding level in uninjured epidermis. (A,C) Cx26 and Cx30 immunosignals increased in all epidermal layers to maximal expression on day 3. (D) The Cx31 protein level decreased in the suprabasal layers accompanied by an increase in the basal layer until day 4. (B) Cx43 protein decreased within the first 4 days of wound healing. On day 5, all of the connexins analyzed were readjusted to the expression level of uninjured epidermis. Cx30 (E) and Cx31 protein (F) in mice with decreased amount of Cx43 protein showed an altered expression pattern which resembled that in wounded wild-type epidermis one day later. Labelling of the columns: grey, basal layer; horizontal lines, spinous layer; vertical lines, granulous layer. Arrows indicate the shifted maximal expression of Cx30 and Cx31. Asterisks above the lanes indicate significant changes of signal abundance (P<0.05) (Student's t-test) compared with corresponding uninjured epidermis.

View larger version (67K): [in a new window]   Fig. 7. Left panel: Immunostaining of Cx30 expression in wild-type epidermis on days 1-5 after wound healing. Cx30 protein increased after wounding to maximal expression on day 3. On days 4 and 5, Cx30 protein level decreased again. Right panel: Cx30 expression in tail epidermis of mice with decreased amounts of Cx43 protein. The maximal expression of Cx30 after wounding was shifted to one day earlier than in wild-type epidermis. On day 2 after wounding the epidermis was already closed under the coagulum, in contrast to wild-type epidermis, which fused on day 3 after wounding. Nuclei were stained with Hoechst 33258 fluorescent dye. The basal lamina is marked by white dots. Bars, 25 µm. Coa, coagulum.

View larger version (67K): [in a new window]   Fig. 8. Left panel: Immunostaining of Cx31 expression in tail epidermis on days 1-5 after wound healing. Cx31 protein amount decreased during the first 2 days postwounding and Cx31 protein was mainly localized in the basal layer. On days 4 and 5, the expression level increased again to the level of uninjured epidermis. Right panel: Cx31 expression in tail epidermis of mice with decreased amount of Cx43 protein (Cx43CreER(T)/fl, induced). Maximal Cx31 expression after wounding occurred one day earlier than in wild-type epidermis. Nuclei were stained with Hoechst 33258 fluorescent dye. Basal lamina is marked by white dots. Bars, 25 µm. Coa, coagulum.

View larger version (25K): [in a new window]   Fig. 9. Immunoblot analyses of connexin protein in uninjured and wounded tail epidermis from wild-type mice with antibodies to Cx26 (A) and Cx30 (B). Numbers indicate days after wounding. Ponceau stained nitrocellulose membranes (C, D) show equal protein loading of the lanes depicted in A and B. Cx26 and Cx30 expression was increased from day 1 and reached its peak on day 3 after wounding. On day 4, the level of Cx26 and Cx30 protein had clearly decreased and rose again on day 5 before it dropped to the level observed in uninjured epidermis.

View larger version (40K): [in a new window]   Fig. 10. Microinjection of Alexa Fluor 488 fluorescent dye into single cells in thick sections (250 µm) of ear epidermis. (A) Skin section derived from Cx43Cre-ER(T)/fl mouse after induction with 4-hydroxytamoxifen, photographed under UV light, indicating strongly decreased dye transfer compared with section of wild-type (C57BL/6) mouse (B). Arrows mark the injected cell. Bars, 20 µm. ba, basal layer; de, dermis; la, basal lamina; sb, suprabasal cell layers. (C) Dye transfer in Cx43Cre-ER(T)/fl epidermis was reduced by 40% compared with several controls (P<0.001). The numbers of mice and injections for each experiment are given in Materials and Methods.