In vivo Role of TGase 3 Figure 8. Transmission electron microscopy of sections through the hair follicle of TGM32/2 (A, C) and wild type hair (B, D) (scale bar 10 mm). Arrows show the keratin filaments. The cuticle layer is distorted in the mutant (magnified region), and disruption of the Huxley’s layer is evident. Trichohyalin droplets (*) are seen as non-membrane-bound inclusions, in Huxley’s layer of the IRS in both TGM32/2 (E) and control mice (F) and cornification occurs on the Henle’s layer (arrow heads) (scale bar 2 mm). ORS-outer root sheath, Cp-companion layer, He-Henle’s layer, Hu- Huxley’s layer, Cl-cuticle of inner root sheath, Ch-hair cuticle, Co-hair cortex, Me-hair medulla. doi:10.1371/journal.pone.0034252.g008 wounding TGM32/2 skin more rapidly developed a keratin expression pattern closer to that of unwounded skin when compared to controls. Further, there was an obviously thinner epidermal layer at day 9 after wounding in the mutants, despite no statistical change in cell number when compared to control wounds. This suggests the ‘‘activated’’ keratinocytes seen in wound Figure 9. Protein extractability in TGM32/2 hair. Proteins were extracted from wild type and TGM32/2 hair with 2% SDS and 5% b- mercaptoethanol over night prior to SDS-PAGE separation and transferred to nitrocellulose. Membranes were probed with polyclonal antibodies against involucrin, trichohyalin, keratin10 and keratin14. Two prominent bands observed in the Coomassie stained SDS-PAGE of TGM32/2 protein lysates were identified by tryptic peptide mass fingerprinting as keratin6hf (A) and keratin17 (B). doi:10.1371/journal.pone.0034252.g009 healing changed in the absence of TGase 3. These cells produce increased levels of transglutaminases and growth factors [51]. TGase 3, like factor XIII and TGase 2, is released from cells [16,39] and may play a role in TGFb activation [52], fibronectin signaling [53], and cross-linking of the extracellular matrix [29,54]. Transglutaminases are up-regulated in migrating kerati- nocytes at the wound edges [27]. Interestingly, keratin6hf and keratin17, which are substrates for TGase 3, are also rapidly up- regulated here, with a concomitant reduction in keratin1 and 10 [42]. In conclusion, despite a widespread expression and much circumstantial evidence as to its importance in the formation of the cornified envelope, it appears that loss of TGase 3 is largely compensated in stratified epithelia. TGase 3 is highly significant in hair development and in particular the cuticle, where it appears to have a unique role in stabilizing the trichohyalin network. Materials and Methods Ethics statement Animal studies were performed in accordance with institutional guidelines. The protocol was approved by the District Council of Cologne, North Rhine-Westphalia, Germany (Permit Number: 52.203.2-K46, 22/02). Generation of TGM32/2 mice The 59 flanking arm of the targeting construct, a 3.1 kb fragment containing exon 5, intron 5 and a portion of exon 6 of the TGM3 gene was amplified by PCR from genomic mouse DNA with SacII and NheI/SalI restriction sites integrated into the 59 and 39 primers, respectively. The 3.2 kb 39 region directly flanking exon 6 and containing exon 7 was also amplified by PCR with SalI restriction sites included in the primers. These fragments were cloned into pBluescript and a neomycin-resistance gene expression cassette was then inserted into the NheI site. The targeting vector was linearized with ClaI and electroporated into E14 (subclone PLoS ONE | www.plosone.org 9 April 2012 | Volume 7 | Issue 4 | e34252