In vivo Role of TGase 3 Figure 1. Ablation of the TGM3 gene. The locus of exons 4 to 7 of the TGM3 gene (A). The targeting construct was produced by insertion of the neomycin resistance cassette into exon 6. Southern blot analysis of cells after restriction digestion using NheI for the 59 probe and PstI with the 39 probe was used to identify correctly targeted ES cells. The disruption of exon 6 resulted in the wild type 28.8 kb NheI fragment being reduced to 3.4 kb, and the 5.3 kb PstI fragment being reduced to 4.9 kb. Southern blot analysis of ES cell DNA after NheI digestion and hybridization with the 59 probe (B, left panel), and after PstI digestion and hybridization with the 39 probe (B, right panel). doi:10.1371/journal.pone.0034252.g001 or keratin related proteins such as involucrin (Fig. 5A). Neverthe- less, there was a slight increase in the extractability of trichohyalin, a minor component of the epidermis (Fig. 5A). Given that barrier formation was delayed, we tested the developing epidermis at E16.5. Again we found no difference in the extractability of either keratins or involucrin from the forming skin (results not shown). To test for alterations in transglutaminase enzyme activity in epithelia, we extracted soluble transglutaminase from the epider- mis and tongue epithelium. There was very little activity without activation by dispase in wild type mice (Fig. 5B) and no difference between dispase treated and untreated extracts from TGM32/2 mice. This shows that almost all soluble transglutaminase activity in these epithelia is contributed by TGase 3. TGase 3 does not alter the wound healing rate in epithelial wounds Transglutaminases are upregulated after wounding and are implicated in healing events [27–30]. To discover if TGase 3 is important in this process, full thickness wounds were excised with a 4 mm diameter biopsy punch. While TGase 3 staining is increased in the healing wild type epidermis (Fig. 6B), the rate of wound closure was unaltered between mutant and control (Fig. 6A). We then studied if the expression of keratinocyte markers in the healing wound was changed. While loricrin (Fig. 6G–I) and keratin10 (Fig. 6J–L) show the expected widened expression pattern seen in healing epidermis, keratin14 appears more restricted in the wounds of mutant mice (Fig. 6N and O) than in control animals (Fig. 6M), with an expression pattern closer to mature epidermis. Further, the healing epidermis was thinner in the mutant animals and appeared more differentiated (Fig. 6C–F). TGase 3 is required for proper hair development TGM32/2 mice could be identified by the second day after birth due to markedly irregular whiskers (Fig. 2E–F). Even though the whiskers were often later lost, the number, positioning and layering of the whisker follicles was normal. Microscopy showed that the vibrissae were twisted and thinner than in controls. Pelage and tail hair also showed a wavy pattern. Interestingly, this was most obvious in the first four weeks (Fig. 2B–D). While adult mice had grossly normal looking coat hair, their whiskers, when retained, continued to be irregular. Nevertheless, the mutant hair in adult mice appeared irregular with an increased number of uneven, thinner hairs, displaying irregular torsions when looked at PLoS ONE | www.plosone.org 3 April 2012 | Volume 7 | Issue 4 | e34252