newly formed epithelium. Remarkably, we found that c-Met mutant keratinocytes were completely unable to reepithelialize the wounds. Instead, residual keratinocytes that escaped recom- bination (5%, c-Met–positive cells) closed the wounds, but the wound healing process was delayed. These results demonstrate that the c-Met signaling system is essential for skin wound heal- ing. Apparently, no other signaling system is able to compen- sate for a lack of c-Met in this process. Results HGF/SF and c-Met are expressed during wound healing and may act in an autocrine fashion We performed full-thickness dorsal skin wounding in mice in such a manner that the epidermis and the underlying dermis were damaged (Werner et al., 1994). We then analyzed HGF/SF and c-Met expression during the wound healing process by in situ hybridization of these sections, i.e., 1–10 d after the injury. Before wounding, HGF/SF was expressed in hair follicles, but not in the epidermis (Lindner et al., 2000). After wounding, HGF/SF was initially expressed in the dermis adjacent to the clot (Fig. 1 B, only the left halves of the wounds are shown; Fig. 1 A shows the scheme of entire wound; Cowin et al., 2001). 3 d after the injury, HGF/SF was strongly up-regulated in the HE at the edges of wounds (Fig. 1 C; control is shown in D). At this time point, the new epithelium is already formed and visible. The receptor tyrosine kinase c-Met is expressed in the epider- mis and hair follicles of normal skin (Lindner et al., 2000), but it is also strongly expressed in the HE during wound repair (Fig. 1, E and F; control is shown in G). We also performed immuno- fluorescence staining of phosphorylated c-Met on normal skin Figure 2. Generation and analysis of skin-speciﬁc c-Met mutant mice. (A) Schematic representation of nonrecombined allele and recombined allele of c-Met. Exon 15 of the c-Met gene that encodes the ATP-binding site (red box) was ﬂanked by loxP sites (triangles) and is excised after K14-cre–induced recombination. Blue boxes indicate exons 14 and 16. The sizes of the restriction fragments generated by BamHI digest before and after recombination are indicated. B, BamHI; P, Pst. (B) Southern blot analysis of epidermis from control and conditional c-Met mutant mice of different ages (12 wk old, E17.5, and P8). (C) Southern blot analysis of different organs of conditional c-Met mutant mice. (D–G) Double staining of skin section from Z/AP; K14-cre mice (Lobe et al., 1999; Huelsken et al., 2001) for lacZ (blue, nonrecombined) and alkaline phosphatase activity (yellow, recombined). (E) A higher magniﬁcation shows an area of nonrecom- bined epidermal keratinocytes (blue patch). Higher magniﬁcations show also two independent hair follicles (F and G); note that the bulge region contains re- combined (yellow) cells. Arrector pili muscle cells, which surround the follicle, are nonrecombined (blue). (H) Double staining of alkaline phosphatase and β-galactosidase activity of wound section from Z/AP; K14-cre mice. Note that K14-cre–induced recombination is observed in the unwounded epidermis and in the HE. (I) Immunohistological analysis of a wound section from control mice using antibodies directed against K14 (red) and ﬁbronectin (green). (J) Hair follicle cycle in control and conditional c-Met mutant. Sagittal sections of control and conditional c-Met mutant skin stained with hematoxylin/eosin at P1 (ﬁrst anagen), P5 (ﬁrst anagen), P8 (ﬁrst anagen), P18 (ﬁrst catagen), P20 (ﬁrst telogen), and P30 (second anagen). Bars: (D) 50 μm; (E–F) 20 μm; (G–I) 100 μm. c-MET IN WOUND HEALING • CHMIELOWIEC ET AL. 153