In vivo Role of TGase 3 Figure 4. Barrier function of the skin in TGM32/2 mice. Transmission electron microscopy of skin from TGM32/2 (A, C and E) and wild type (B, D and F) 4 week old animals. Cornified cell envelopes (closed arrowheads in A and B) and keratohyalin granules (*) were visible in mice of both genotypes and in some sections. The latter could be seen coalescing with the forming cornified envelope of granular layer keratinocytes (open arrowheads in C and D). The cells of the stratum corneum in both wild type and TGM32/2 animals consisted of a defined cell envelope surrounding compact, electron-dense cytoplasm containing condensed tonofibrils. Lucifer yellow failed to penetrate through the cornified envelope in either the newborn TGM32/2 (G) or wild type skin (H). (scale bars B 0.5 mm, D 7 mm, F and H 50 mm). At birth there was no retention of toluidine blue dye in the skin of either TGM32/2 or wild type neonates (I). Dye was retained in the basal region of the whiskers in TGM32/2 (L) neonates, a finding not seen in wild type mice (K). While formation of the epithelial barrier had occurred in control mice at E17.5, toluidine blue penetrated the skin of TGM32/2 litter mates (J). Sonication of corneocytes isolated from skin biopsy punches for various times revealed that TGM32/2 corneocytes were more susceptible to lysis (M). The number shown is that of intact corneocytes remaining as a percentage of those initially isolated (n = 4). doi:10.1371/journal.pone.0034252.g004 cornified cell membrane (Fig. 8E, F). In contrast, the Huxley’s layer, containing vacuoles filled with trichohyalin, was often torn or distorted (Fig. 8A, C). Further, the cuticle of the hair was abnormal, the outer exocuticular layer being irregular and having a reduced number and electron density of granules underlying the cell membrane. In the cortex and medulla, keratin filaments were obvious in the mid-shaft region but in mutant hair the keratin filaments in cortex cells were often shorter and less regular. Biochemical defects in cross-linking of the hair We then questioned whether there were definable defects in cross-linking in the hair due to the absence of TGase 3. Proteins were extracted under reducing conditions from equal amounts of plucked hair of 14 days and 4 months old mice. At both ages, far more protein was isolated from mutant hair (Fig. 9 and results not shown). The extracts were then submitted to SDS-PAGE and immunoblotting and probed for known substrates of TGase 3. In all cases there was a marked increase in extractability, but this was less pronounced for keratin10 and 14, which are present in the inner and outer root sheath of the hair follicle, respectively. A dramatic change was seen in the two keratin associated proteins, involucrin and trichohyalin, the former of which was only weakly extractable and the latter was not extractable from control hair. For both a strong signal was observed with the main protein bands being accompanied by an immunoreactive smear, suggesting partial cross-linking of these proteins into a matrix. The SDS- PAGE separation of TGM32/2 hair extracts also revealed two prominent protein bands upon Coomassie blue staining which were absent from control extracts (Fig. 9). These bands were cut from the gel, in-gel digested with trypsin and the peptides obtained analyzed by MALDI-TOF mass spectrometry. This allowed the identification of keratin6hf and keratin17, two keratins present in both the medulla of the hair and the companion layer of the outer root sheath of the hair follicle. PLoS ONE | www.plosone.org 5 April 2012 | Volume 7 | Issue 4 | e34252