Figure 8: Impaired skin wound healing in Daple^−/− mice.

(a) Western blot analysis of brain lysates from wild-type and Daple^−/− P7 mice, using anti-Daple, revealed the absence of Daple in the homozygous mutant mice. (b, c) Wounds were made on the backs of wild-type and Daple^−/− P56 mice, and the gross appearance of the wounded areas (dotted lines) was observed for 12 days (b). Wound areas were quantified and expressed as a percentage of the initial wound size (100%) (c). The results represent the mean values±s.e. of six animals. (d) The wound tissue sections prepared from wild-type (left panels) and Daple^−/− (right panels) mice 2 days after wounding. The regions in boxes indicate the location of fibroblasts (1, 3) and keratinocytes (2, 4), which are shown at higher magnification in lower panels. Immunostaining with anti-Daple antibody revealed the expression of Daple in fibroblasts (1; arrow heads) and keratinocytes (2; arrows) in the wild-type, but not Daple^−/− wound tissues. HE, haematoxylin and eosin; IHC, immunohistochemistry. (e, f) Dermal fibroblasts (e) or epithelial cells (keratinocytes) (f) isolated from skin samples of wild-type and Daple^−/− mice were subjected to GST-PAK-PBD pull-down assays (upper panels) and wound-healing assays in vitro (lower panels). In wound-healing assays, the dotted lines indicate the wound edge at 0 h. The closure rate was expressed as a ratio of the migration distance in each group divided by the migration distance in Daple^+/+. Bars represent the mean values ±s.d. of three experiments. (g, h) Fibroblasts harvested from wild-type and Daple^−/− mice were incubated in a medium supplemented with 2% FBS. Each monolayer was scratched to induce cell migration in the presence (+) or the absence (−) of Wnt5a, and incubated for 16 h (g). The wound closure rate was expressed as a ratio of the migration distance in each group divided by the migration distance in control (Wnt5a (−)) cells (h). Bars represent the mean values±s.d. of three experiments. Significant difference was determined by Student's t-test. *P<0.05; n.s., not significant. Scale bars represent 200 (d) and 300 μm (e–g).