ing keratinocytes, including loricrin and involucrin [8]. These are cross-linked to different degrees by the various transglutaminases [9]. For instance, while TGase 1 induces loricrin multimerization at the inner surface of the plasma membrane, TGase 3 causes intramolecular linkage or dimerization of loricrin in the cytoplasm [8]. Assembly of the cornified envelope is a highly ordered event, though surprisingly, loss of certain major structural proteins including involucrin and loricrin results in remarkably little change in its formation [21,22]. The differential localization of the epidermal transglutaminases, both with respect to the stages of keratinocyte differentiation and to their intracellular localization, combined with their different substrate usage, strongly suggests highly specific roles for these enzymes in keratinization. Tricho- hyalin is a glutamine-rich protein highly expressed in specialized epithelial structures with high mechanical strength [23], such as the inner root sheath which forms a collar about the hair. It is a substrate for TGase 3 and the in vivo expression of TGase 3 coincides with the modification of trichohyalin [24]. This finding is in agreement with in vitro results showing that TGase 1 or TGase 2, which are also present in the inner root sheath, cross-link trichohyalin to a far lesser degree [24,25]. The transglutaminase- modified trichohyalin is believed to serve as a matrix onto which keratin intermediate filaments are deposited. To understand the importance of TGase 3 in development and maintenance of tissues, we produced and analyzed the phenotype of a novel mouse line lacking this enzyme. Results Generation of mice lacking TGase 3 Embryonic stem cells were targeted at the TGM3 allele by disrupting the sixth exon and introducing a neomycin resistance gene (Fig. 1A). This destroys the active center of the enzyme and splicing of exon 5 to any of the following exons (7 to 11) will result in an out of frame product and/or an unstable mRNA transcript. Three clones were correctly targeted when analyzed by Southern blotting and probing 59, 39 and internal to the targeting site (Fig. 1B). Two of these were used to derive chimeras by injection of cells into C57/Bl6 blastocysts, and germ-line transmission was obtained. Breeding mice heterozygous for the TGM3 mutation produced offspring with the three possible genotypes at the expected Mendelian ratios (Fig. 2A), showing that mutation of the TGM3 allele is compatible with life. Western blotting and probing either with a mouse monoclonal antibody or with an affinity purified antiserum against recombinant TGase 3 verified the absence of the protein, with the expected 77 kDa band being absent in mutant tissues (Fig. 3A and B). Further, RT-PCR with TGM3 specific primers annealing 59 and 39 to exon 6 failed to amplify any product (Fig. S1), suggesting destabilization of the TGase 3 mRNA and a mutation in the TGM3 gene. TGM32/ 2 males and females display defects in hair development but are fertile and have a normal lifespan. (Fig. 2). TGM32/2 mice do not display changes in epidermal differentiation and develop a normal cornified envelope Since TGase 3 has been described as a major contributor of transglutaminase activity in the epidermis [23], which is a crucial factor in the development and stabilization of the skin, we initially carried out an extensive analysis of the epidermis. Grossly, the skin of mice appeared normal but there was an obvious alteration in hair development (see below). Histology showed no major changes (Fig. S2) nor was there evidence for increased scaling. The apparent lack of changes led us to suppose that there could be a compensation for the loss of TGase 3. However, immunostaining In vivo Role of TGase 3 of skin with antibodies raised against TGase 1, TGase 2 or TGase 6, the closest relative to TGase 3, showed no alteration in the expression pattern of these main skin transglutaminases (Fig. 3C– L). Interestingly, TGase 2 which is present in human basal keratinocytes, was not detected in mouse epidermis, although it was seen staining the epimysium (Fig. 3I and J). Expression of TGase 5 at the mRNA level was also unaltered (Fig. S3). TGase 3 is present in keratinizing compound squamous epithelia, yet there were no gross abnormalities of the oesophagus, filiform papillae of the tongue or the keratinized stomach and histology also failed to reveal any changes (Fig. S2). We then studied expression of markers for keratinocyte differentiation. These included keratin isoforms, filaggrin, loricrin and involucrin, all known substrates for TGase 3 and the latter two being highly expressed in the stratum granulosum. None showed an altered expression pattern (Fig. S2). Absence of TGase 3 does not affect epidermal barrier function in newborn or adult mice despite changes in corneocyte stability Mutations leading to loss of TGase 1 activity cause severe changes in the cornified layer and the lipid envelope. To test whether TGase 3 could have a physiological function in the cornified layer, we assayed the structure and function of the epithelial barrier. We investigated transepidermal water loss in newborn mice with a Tewameter and also the passive inward diffusion of Lucifer yellow dye through the stratum corneum (Fig. 4G and H). Both of these measurements showed no change in the absence of TGase 3 either in adult or neonatal mice. During gestation TGase 3 is first expressed by the periderm at E12.5 and then is seen in the developing epidermis before E16.5, a time point when the epidermal barrier function first becomes evident [26]. Toluidine blue staining was used to analyze the barrier function. Neither wild type nor TGM32/2 perinatal mice showed toluidine blue retention in the epidermis (Fig. 4I) but dye was maintained by a portion of the whisker directly where it emerged from the skin (Fig. 4L). The formation of a water impermeable barrier occurs from day E16.5 and skin of E17.5 control embryos retained no toluidine blue. In contrast, TGM3 mutants retained the pigment, particularly over the abdominal regions, suggesting a delay in formation of the skin barrier (Fig. 4J). These findings indicate that, while there may be retardation in its development, there is little change in epidermal barrier function at birth or later. To verify this, skin from the ear, back and belly of female TGM32/2 and wild type litter mates was analyzed by electron microscopy. A normal cornified envelope was present upon corneocytes (Fig. 4A and B) and keratohyalin granules were detected in both mutant and control skin in the upper granular layers, in some cases coalescing with the developing cornified envelope (Fig. 4C to F). Corneocytes isolated from the pinna appeared morphologically comparable in mutant and wild type mice and were obtained in similar yields. To assay for stability, corneocytes were kept under reducing conditions and subjected to mild sonication. Those from wild type mice showed a greater resistance to this treatment, with over 25% of corneocytes remaining intact after 5 minutes sonication, a time when all mutant cells had been disrupted (Fig. 4M). This suggested that despite a normal postnatal skin barrier there where changes in corneocyte stability. Covalent cross-links between keratins, i.e. disulfide bridges and Ne-(c- glutamyl)lysine isopeptide bonds, are crucial for epidermal integrity. To see if there was an obvious alteration in stability of the skin protein network in TGM3 mutants, skin from four week old mice was extracted under reducing conditions and subjected to immunoblotting for known TGase 3 substrates. No difference was seen in the extractability of epidermal keratins (results not shown) PLoS ONE | www.plosone.org 2 April 2012 | Volume 7 | Issue 4 | e34252