liver was then removed by hepatectomy. In such conditional c-Met mutant mice, liver regeneration was impaired and, in partic- ular, cell cycle progression of hepatocytes was altered (Borowiak et al., 2004). We show that c-Met signaling is also crucial for wound healing, which is a regeneration process of the skin. No other paracrine or autocrine system can compensate for a loss of c-Met function in skin regeneration because c-Met mutant cells were completely unable to contribute to the HE, which is the source of the keratinocytes that reepithelialize the wounds. Few residual keratinocytes, which escaped recombination, close the wound in the skin of conditional c-Met mutant mice The question arises how the few remaining nonrecombined cells in the conditional c-Met mutant skin ( 5%) could close the wound in vivo. Wound closure in vivo was delayed, but occurred and required only twice as much time, despite the fact that the vast majority, i.e., the 95% mutant cells, did not participate in the process. Moreover, the overall kinetics of wound closure was similar in both control and mutant wounds, but was shifted to the right in the mutants, which may indicate that the identity of cells contributing to the HE was the same. Thus, reepithelialization started after a delay in the skin of the conditional mutant mice (3 d after injury), but recovered fast because the proliferation of the keratinocytes in the HE was increased during early wound healing. Moreover, in the control skin, the size of the HE comes to a halt 5 d after the injury, but was still increasing at that time in the conditional c-Met mutant mice. A compensatory overpro- liferation of the control cells occurred because of this, resulting in a selection of cells that escaped recombination over mutant cells within a few days in vivo, and results in a wound that heals. Regeneration of the epidermis after wounding involves activa- tion, migration, and proliferation of keratinocytes from the sur- rounding epidermis, but keratinocytes derived from hair follicles and sweat glands may also participate in the healing process. In response to injury, bulge stem cells leave their niche and con- tribute to the HE (Miller et al., 1998; Ito et al., 2005; Levy et al., 2005). Thus, bulge cells of the skin are another potential source of cells that reconstitute the injured epidermis. However, K14-cre–mediated recombination also occurs in the hair bulge, and we provide evidence that cre-mediated deletion is efficient in the bulge and in the epidermis. Moreover, we did not detect clusters of dividing cells that could correspond to cells that had escaped recombination close to hair follicle remnants. We also observe that keratin 6–positive cells form continuous layers in the wound epithelium in c-Met mutant skin, and do locate to areas close to hair follicles. Repopulating cells that escaped recombi- nation could thus originate from both cells of the epidermis and of the hair bulge. It is possible that the underlying dermis also contributes in this compensatory process, e.g., by producing an increased amount of other growth factors or cytokines. Wound healing in vitro and the importance of signal transduction and cytoskeletal changes We also analyzed the effects of c-Met signals on wound closure of primary keratinocytes in a culture system, and analyzed if 160 JCB • VOLUME 177 • NUMBER 1 • 2007 c-Met–positive keratinocytes preferentially contribute to wound closure in a culture that contains few nonrecombined (c-Met+) and the majority of mutant (c-Met−) cells. In such cultures, we observed that, in the presence of HGF/SF, nonrecombined (c-Met+) cells migrate preferentially into the scratch wounds. We also observed that control keratinocytes at the edges of scratch wounds orient themselves, i.e., focal adhesions and stress fibers pointed toward the wound edges (Ridley et al., 1995; Nobes and Hall, 1999). In contrast, such a pronounced orientation of cells was not observed in the scratch wounds of the keratinocytes de- rived from conditional c-Met mutant mice. However, mutation of c-Met did not disturb orientation of the cells toward the wound in the presence of TGFα. Our genetic data thus indicate that HGF/ SF and c-Met are the major signal that is available to reorient cells at the wound edges in vitro, and to join the HE in vivo. In vitro, the loss of c-Met signaling can be compensated for by the admin- istration of other growth factors like TGFα, but in vivo, the avail- ability of other growth factors that can elicit similar responses to HGF/SF appears to be limited. We found that c-Met signaling in primary keratinocytes activates Erk1/2, Akt, Gab1, and PAK1/2. The phosphorylation of PAK1/2 might be of particular impor- tance in the motility response; PAK1/2 is a target of Rho signaling that regulates actin polymerization and protrusion formation (Frost et al., 1998; Royal et al., 2000). In the last decade, several growth factors have been impli- cated in wound healing, e.g., FGFs, factors that signal via the EGF receptor, and members of the TGFβ superfamily (Werner and Grose, 2003). Ablation of these factors or their receptors in mice affected the kinetics of wound healing, but mutant cells appear to have contributed to the newly formed epithelium. As yet, c-Met is the only example of a receptor that is essential in wound healing. The application of HGF/SF and/or HGF/SF variants in the therapy of wounds (Bevan et al., 2004) therefore appears to be an attractive possibility. Materials and methods Wound healing experiments, histology, immunohistochemistry, and Western blotting To mutate c-Met in the skin, the K14-creneo mouse line was used, which expresses cre after E15 of embryogenesis (Huelsken et al., 2001). 2–3-mo- old mice were anesthetized, and two full-thickness 4-mm-diam excisional wounds were made on both sides of the dorsal midline, as previously described (Werner et al., 1994). Mice were kept separately in cages to prevent ﬁghting, and no self-induced trauma was observed in control or mutant mice. Littermates of the same sex were used for the analysis of wound closure. Wound closure was determined as the percentage of the distance covered by the epidermis between the wound edges. For histolog- ical analysis, dissected wounds were ﬁxed overnight in 4% formaldehyde in PBS, followed by dehydratation through a graded ethanol series and embedding in parafﬁn. Sections from the middle of the wound were stained with hematoxylin/eosin, and immunoﬂuorescence was performed as previ- ously described (Huelsken et al., 2001). Histological sections were used to determine the area of HE by using the measure function of the Adobe Photoshop program. BrdU incorporation was assessed by immunohisto- chemistry 2 h after injection of 75 μg BrdU/g of body weight. The prolifer- ation index represents the percentage of BrdU-positive cells within the HE. We used antibodies directed against keratin 6 (Covance), phospho-histone H3 (Millipore), PCNA (Oncogene Science), BrdU (Sigma-Aldrich), CD34 (Biozo), phospho-Met (Sigma-Aldrich), and K14 (Covance). Sections were incubated overnight with primary antibodies at 4°C, followed by ﬂuorescent-conjugated secondary antibodies (Jackson ImmunoResearch Laboratories). TUNEL staining was performed using an in situ cell-death