In vivo Role of TGase 3 Figure 3. TGase 3 expression in epithelia. Upper panel, protein extracts separated by SDS-PAGE were incubated with a mouse monoclonal antibody (A) and a rabbit polyclonal antiserum (B) against TGase 3. The expected TGase 3 band of 77 kDa was observed in extracts from wild type animals and was especially strong in oesophagus and tongue epithelium. In protein extracts from homozygous animals the signal was completely absent. The 50 kDa band seen in (A) corresponds to the heavy chain of IgG. Lower panel, immunofluorescence analysis of tongue (C, D) and back skin (E–L) of wild type (C, E, G, I, K) and TGM32/2 animals (D, F, H, J, L). The sections were incubated with rabbit polyclonal antibodies against TGase 3 (C–F), TGase 6 (K and L) and monoclonal antibodies against TGase 1 (G, H) and TGase 2 (I and J). (scale bars represent 100 mm, the dotted line marks the dermal- epidermal junction). doi:10.1371/journal.pone.0034252.g003 Figure 2. Gross phenotype of TGM3 mice. Southern blot analysis of tail biopsies from mice born of TGM3+/2 interbreeding showed that homozygous mutants were born at the expected Mendelian ratios (A). The pelage hair of homozygous animals at 4 weeks of age showed a distinctive wavy pattern (B and C). This became less obvious as the mice matured (D). While gross hair abnormalities disappear with time, irregularities in the vibrissae, which are evident perinatally (E, mutant; F, control at P5) persist throughout life. Expression of TGase 3 is observed in pelage hair in the medulla and in the inner root sheath (G). doi:10.1371/journal.pone.0034252.g002 under the microscope (Fig. 7A). To verify presence and numbers of the four hair types (auchene, zig-zag, guard and awl), hairs were plucked and typed. While all hair types were present (Fig. 7M), the numbers of zig-zag type hairs were decreased (p = 0.028) when compared to control litter mates at 4 months age (78% in control, 63% in TGM32/2 mice). Further, they appeared brittle with damaged hairs present and the thinner hairs often broke during manipulation in the counting process. Despite this, baldness was not generally observed. Hairs were then examined by scanning electron microscopy. This showed an irregular twisting of many hairs in the mutant with the even groove, found in normal hairs, often being absent or highly deformed (Fig. 7G–J). The regular and overlapping shingle-like structure of the cuticular scales, while maintained in some TGM32/2 hairs, was often distorted. In some cases small cracks occurred in the cuticle, while in others scales could be seen lifting from the underlying cortex (Fig. 7G, I). In agreement with the finding that the mutant mice did not show alopecia, tape stripping suggested the hair bulb was no less well attached to the follicle and scanning electron microscopy of the anagen hair bulb indicated no differences between TGM32/2 and control mice (Fig. 7K, L). To determine if the integrity of the hair was compromised, plucked hairs were treated overnight with detergent and a reducing agent to break any disulfide bonds. In wild type hair the overall structure was maintained, with the cuticle being retained and the structure of the medulla remaining intact (Fig. 7D, F). In contrast, mutant hair showed long stretches where the cuticle was lost and in places the medulla distorted (Fig. 7C, E). We then examined the pelage hair and whiskers by transmission electron microscopy. The usual layers in the hair shaft and follicle were evident in the mutant hairs (Fig. 8A, B). Both the outer and inner root sheath were present, while the amorphous Henley’s layer appeared normal with an evident PLoS ONE | www.plosone.org 4 April 2012 | Volume 7 | Issue 4 | e34252