Figure 5. Only residual c-Met–positive keratino- cytes contribute to the HE of wounds in condi- tional c-Met mutant mice. (A and B) Isolation of HE by laser capture microdissection. A wound section before (A) and after laser capture microdissection (B) is shown. (C) Southern blot analyses of back epidermis and hyper- proliferative epithelia from conditional c-Met mutant mice. Microdissected, nonwounded epidermis from the back (left) and micro- dissected hyperproliferative epithelia of wounds 3 (middle) and 5 d (right) after injury were col- lected. Southern blotting of two independent preparations from different pools of micro- dissected tissues is shown. Note that the HE 5 d after injury in conditional c-Met mutant mice is formed exclusively by cells that contain the nonrecombined c-Metﬂox allele, but not the re- combined c-Met∆ allele. At day 3, a 1:1 mixture of recombined and nonrecombined cells is seen (middle). The c-Metnull allele is also present be- cause heterozygous c-Metﬂox/null mice were used for conditional mutagenesis. (D–G) Immuno- histological analysis of wound sections from control and conditional mutant mice 5 d after injury using anti–phospho-c-Met antibodies (red immunoﬂuorescence in D and E, and brown immunohistochemistry in F and G). Note that in the skin of conditional c-Met mutant mice, cells in the HE (outlined) are phospho-c-Met positive. Arrows mark phospho-c-Met–positive cells in the lower hyperproliferative epithelia layers. Bar, 100 μm. only nonrecombined c-Met keratinocytes, i.e., cells that express a functional c-Met, participate in the formation of the HE. In the skin of conditional c-Met mutant mice, the few remaining cells that escaped recombination can apparently compensate and gen- erate the entire hyperproliferative epithelia. These data demon- strate that c-Met plays crucial functions during wound closure. Closure of scratch wounds is impaired in cultured keratinocytes derived from conditional c-Met mutant mice Primary keratinocytes were isolated from the skin of newborn control and conditional c-Met mutant mice (Caldelari et al., 2000), and monolayers in culture were scratch wounded (Fig. 6, A–C; Sano et al., 1999). Analysis of K14 by immunohistology indicated that the isolated cells, indeed, correspond to epithelial cells (Fig. S2, available at http://www.jcb.org/cgi/content/full/ jcb.200701086/DC1). We found that in the presence of HGF/ SF, primary keratinocytes from control mice closed the wounds within 48 h, as did cells from conditional c-Met mutant mice in the presence of the EGF receptor ligand TGFα (Fig. 6, A and C). However, in the presence of HGF/SF, keratinocytes from the skin of conditional c-Met mutant mice did not close the scratch wounds within 48 h, but required 96 h (Fig. 6 B). Strong prolif- erate response toward HGF/SF was observed in control cells close to the wound edges at 24 h, but such a response was not observed in the mutant keratinocytes (Fig. 6 D, quantification is shown in E). We then examined if, while in culture, the few remain- ing nonrecombined keratinocytes from the skin of conditional c-Met mutant mice preferentially contribute to wound closure. 156 JCB • VOLUME 177 • NUMBER 1 • 2007