depicted; Fuchs et al., 1992; Byrne et al., 1994). Similarly, we did not observe changes in hair cycle progression when control and conditional mutant mice were compared. For instance, the first and second anagen phases occurred at P5 and P30, respec- tively (Fig. 2 J). Catagen and telogen occurred at P18 and P20 (Paus and Cotsarelis, 1999). We have now kept conditional c-Met mutant mice for nearly 2 yr, and we have not observed unusual hair loss or other changes in the appearance of the skin. We introduced full-thickness wounds into the dorsal skin (Werner et al., 1994) of control and conditional c-Met mutant mice at 12 wk of age, and we analyzed wound closure by histol- ogy 3–15 d after the injury. Wound healing did occur in condi- tional c-Met mutant mice, but was delayed and required about twice as much time as in the control mice (Fig. 3, A–F; see quantification in Fig. 4 A). For instance, 5 d after the injury, 50% wound closure was observed in control mice; in conditional c-Met mutant mice, this required 9 d (Fig. 4 A). Histological examinations showed that the HE was thinner, and its formation was delayed in the conditional c-Met mutant mice, as assessed by hematoxylin/ eosin (Fig. 3, A and D) and by Masson tri- chrome staining; the latter stains the epithelium in red (Fig. 3, B and E). Immunohistological analysis using keratin 6 antibodies also demonstrated a reduction in the thickness of the HE in the conditional c-Met mutant mice (Fig. 3, C and F). Keratin 6 is expressed in activated keratinocytes of the HE and in hair folli- cles (Fuchs, 1990; Takahashi et al., 1998; Wankell et al., 2001). Quantification showed that the formation of the HE was delayed in the conditional c-Met mutant mice; compared with control mice, an 80% reduction of the area of the HE was observed 3 d after injury, a 65% reduction 5 d after injury, and only a 25% re- duction 7 d after injury (Fig. 4 B). In contrast, the proportion of proliferating keratinocytes in the HE was increased after injury in the conditional c-Met mutants, as assessed by phospho- histone 3 and BrdU staining (Fig. 4, C and D). We did not observe accumulations of proliferate-positive cells at particular sites in the HE, and, in particular, we did not observe an accumulation of proliferating cells close to the remnants of the hair follicle. We also did not observe any difference in the number of apop- totic cells in the skin of control and conditional c-Met mutant mice, as assessed by TUNEL staining (unpublished data). Thus, wound healing occurred in the skin of c-Met conditional mutant mice, but reepithelialization of wounds was delayed and re- quired about twice as much time as in control mice. Residual keratinocytes that escaped recombination reepithelialize the wounds in conditional c-Met mutant mice To assess if the c-Met mutant cells (95% of the keratinocytes in the epidermis) could contribute to the newly formed epithelium of the wounds, we collected hyperproliferative epithelia of many control and mutant wounds by laser capture microdissection (see Materials and methods) and performed Southern blotting. An example of a section of a wound before and after micro- dissection is shown in Fig. 5, A and B. Remarkably, Southern blot analysis demonstrated the absence of c-Met mutant cells (i.e., of the c-Met∆ allele) in the microdissected hyperproliferative epithelia of the mutant mice at day 5 (Fig. 5 C). Instead, all cells in the mutant HE contained the nonrecombined c-Metflox allele, despite the fact that this cell population constituted only 5% in the skin before injury. At day 3, a 1:1 mixture of c-Metflox and c-Met∆ cells was seen. Immunofluorescence and immunohisto- chemistry staining with anti–phospho-c-Met antibodies revealed that the majority of cells in the HE at day 5 contained the acti- vated and phosphorylated c-Met receptor, both in control and mutant skin (Fig. 5; compare D and F with E and G). We have observed that staining of activated c-Met is more pronounced in the upper layers of the HE, but staining is also visible in lower layers (Fig. 5, D–G, arrows). We also generated wounds in the skin of K14-cre; Z/AP reporter mice, and could show that K14-cre recombination occurred in the wound epithelium (Fig. 2 H). Immunohistological analyses confirmed that K14 was strongly expressed in the HE (Fig. 2 I). We conclude from these data that Figure 4. Quantiﬁcation of wound healing in control and conditional c-Met mutant mice. (A) Wound closure kinetics in control and mutant mice (see Materials and methods). (B) Quantiﬁcation of the area of HE 3, 5, and 7 d after wounding in control and mutant mice; only sections of the middle of the wounds were used for quantiﬁcation. (C) Proliferation of keratinocytes in the HE from control and mutant mice 3, 5, and 7 d after wounding, as as- sessed by the proportion of phospho-Histone 3–positive nuclei in the epithelium (see Materials and methods). A t test was performed, and signiﬁcant differences be- tween control and mutant were observed 3 d after injury. P = 0.01. (D) Proliferation of keratinocytes in the HE from control and mutant mice 3, 5, and 7 d after wounding, as assessed by the proportion of BrdU-positive nuclei in the epithelium. Signiﬁcant statistical differences between con- trol and mutant were observed 5 d after injury. P = 0.01. Error bars represent the SD. c-MET IN WOUND HEALING • CHMIELOWIEC ET AL. 155