all hair types [13]. Mice lacking fgfr2b only in the epidermis developed similarly thin and silky pelage hair [19]. FGFs 7, 10 and 22 show distinct temporal expression patterns through the murine hair cycle, with both FGF7 and FGF10 expressed highly at anagen V (day 8), when hair grows vigorously, and FGF22 expression strong at anagen VI (day 18), when hair follicle reaches its maximum length [20]. This pattern of expression recapitulates that seen during the wound healing process [12]. FGF7 is expressed weakly in normal murine and human skin, but, upon injury, its expression is up-regulated dramatically [21]. FGF10 levels also increase rapidly following wounding [22] and levels of both growth factors decline once re-epithelialisation is complete [20]. In contrast, FGF22 expression declines during the first days after wounding and remains low until day 5 after injury. Subsequently, the expression increases above basal levels at day 7 after wounding and remains elevated until day 13, being localised to the hyperthickened epidermis of fully healed wounds [12]. Fgf7 knockout mice showed no defect in their ability to repair incisional wounds and the proliferation rate of keratinocytes at the wound edge was not impaired [9]. This was unexpected, since transgenic animals expressing a kinase-deficient, dominant-negative, FGFR2b displayed a severe delay in wound re-epithelialisation, with an 80–90% reduction in the number of proliferating keratinocytes in the hyperproliferative epithelium of five day old excisional wounds, compared with control mice [23]. Truncated FGFR2b abrogates the effects of FGF7, FGF10, FGF1 and FGF3, thus blocking the potential ligand redundancy seen in fgf7 knockout mice, where FGF10 may be sufficient to drive normal repair. Supporting this hypothesis, a significant delay in wound re- epithelialisation was seen in mice lacking dendritic epidermal T cells (DETC), an important source of FGF7 and FGF10 in the healing wound [24]. Finally, mice lacking FGFR1b, a receptor for FGF10 but not FGF7, did not display abnormalities in skin development or repair [10]. Epidermal specific deletion of fgfr2b resulted in a loss of sebaceous glands and abnormal hair development, with mice developing thickened epidermis over time and showing exquisite sensitivity to chemical-induced skin carcinogenesis [19]. Deletion of both fgfr1 and fgfr2 led to an even more extreme phenotype, with dramatic hair loss and epidermal hyperthickening, demonstrating the importance of FGFR signaling in skin homeostasis [25]. What emerges from the above picture is that there is a clear role for FGF signalling in the wound healing process and that this role is protected by redundancy at the level of both ligand and receptor. Our current study builds on these data as the first to investigate the role of FGF22 in the skin, using a germline knockout approach. Results Generation of fgf22 Knockout Mice Mice lacking fgf22 were generated by replacing the fgf22 genomic sequence with a Neomycin selection cassette, such that homologous recombination eliminated the entire coding region of the fgf22 gene (exons 1, 2 and 3) (Fig. 1A). Two G418-resistant clones were identified as homologous recombinants by Southern blot analysis, after EcoRI digestion (Fig. 1B), using a probe external to the homologous sequence to confirm correct targeting. The two mutant clones were injected separately into C57Bl6/J blastocysts. Male chimaeric animals of at least 70% 129ola contribution (as judged by coat colour) were mated with C57Bl6/J females and the offspring of these mice were screened for germline transmission. Heterozygous offspring were mated to produce FGF22 in Skin Development and Repair homozygous mutants, which were born at the expected Mendelian frequency of 25% for both clones (1C12 and 1E1). PCR genotyping was performed to identify fgf22 knockout mice, with primers for the wild type (wt) allele generating a 286 bp product and (ko) allele a 130 bp product (Fig. 1C). Knockout mice from both clones were indistinguishable in terms of cutaneous histology (data not shown), so just one clone (1E1) was used for the functional investigations presented. To verify that targeted homologous recombination resulted in abrogation of FGF22 mRNA expression, total RNA was isolated from mouse brain, known to express FGF22 at a detectable level [11]. Following cDNA synthesis, PCR was performed using primers recognising exon1 and exon3 of the fgf22 coding sequence. As expected, relative to control wild type mouse brain, FGF22 mRNA expression was reduced in heterozygous brains and completely absent in knockout mice (Fig. 1D). Unfortunately, we were unable to detect endogenous levels of FGF22 protein in cells or tissue from control mice, using a polyclonal antiserum known to detect FGF22 expression in cells transfected with a constitutive FGF22 expression construct [12]. Characterisation of fgf22 Knockout Mice Fgf22 knockout animals were viable, fertile and did not display any obvious abnormalities, demonstrating that FGF22 is not essential for embryonic development. As FGF22 is both a ligand for FGFR2b and is expressed in the skin, we first performed a detailed analysis of those epidermal structures affected by lack of FGFR2b in the epidermis. H&E stained sections of back and tail skin from wild type and knockout mice also showed identical histologically, in terms of epidermal, dermal and adipose thickness as well as hair follicle frequency (Fig. 2A1–A4). There were no evident differences in pelage growth or sebaceous gland morphol- ogy between knockout and wild type mice. All the major hair types (zigzag, guard and awl/auchene) were present in knockout mouse back skin, at the expected frequencies (70% zigzag, 28% awl/ auchene and 2% guard hairs) and these hairs did not demonstrate any morphological differences when compared with those of wild type littermates (Fig. 2B) or compared to published mouse hair phenotypes [26]. Furthermore, average length of zigzag hairs (8.7 mm) was identical between genotypes. Whole-mount staining of the sebaceous glands in adult tail skin confirmed no differences between knockout and wild type mice (Fig. 2C). Since there were no defects evident in the unchallenged skin of fgf22 knockout mice, we investigated their capacity to respond to skin injury and carcinogenic insult, both of which have been shown to depend at least in part on FGF signalling. Wound healing studies, where mice were subject to 3 mm diameter full thickness punch biopsy wounds, showed that mice lacking FGF22 were able to heal acute wounds just as efficiently as wild type mice, although 5 days after wounding knockout male wounds appeared to have healed faster (Fig. 3A), with more efficient re-epithelialisa- tion (Fig. 3F) and a high rate of proliferation of cells involved in the process at this time-point (Fig. 3B1 and B2, quantified in 3E). However, at all other time-points during wound healing, morphometric analysis showed very similar values for both male and female fgf22 wild type and knockout animals (Fig. 3E, F and G). Moreover, Sirius red staining of 14 day old wounds from male fgf22 knockout and wild type controls showed no difference in collagen fibre reorganization during healing (Fig. 3C and D). To complement the in vivo wound healing study, we isolated keratinocytes from back skin of wild type and knockout mice and compared their behaviour in a 2D scratch wound assay. Keratinocytes from fgf22 knockout mice were able to close the PLoS ONE | www.plosone.org 2 June 2012 | Volume 7 | Issue 6 | e39436