Wound healing in <i>TGM3^−/−</i> skin.

<p>The rate of wound closure in <i>TGM3</i>^−/− mice was not different compared to control animals (A) (n = 9), despite an increase in TGase 3 expression in healing epidermis (B). At day 9 post wounding the thickness of the epidermal layer in the wounded region was greater in wild type skin (C, E) when compared to mutant skin (D, F). Staining of healing skin with keratinocyte differentiation markers in wild type (G, J, M) and <i>TGM3^−/−</i> (H, I, K, L, N, O) animals demonstrated that while there was no change in the expression pattern of the keratinocyte markers loricrin (G–I) or keratin10 (J–L) between the mouse lines, keratin14 (M–O) already showed a more restricted staining pattern reminiscent of unwounded skin. (scale bars C, D 400 µm, E–G 100 µm)</p

Ablation of the <i>TGM3</i> gene.

<p>The locus of exons 4 to 7 of the <i>TGM3</i> 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 5′ probe and PstI with the 3′ 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 5′ probe (B, left panel), and after PstI digestion and hybridization with the 3′ probe (B, right panel).</p

TGase 3 expression in epithelia.

<p>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 <i>TGM3</i>^−/− 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 µm, the dotted line marks the dermal-epidermal junction).</p

Morphological changes in pelage hair lacking TGase 3.

<p>Whole-mount light microscopy of <i>TGM3</i>^−/− (A) and wild type (B) hair (scale bar 10 µm). Inserts show higher magnification of lower regions of the hairs. Hair incubated overnight at 65°C with agitation in 2% SDS and 20 mM dithiothreitol (C–F) <i>TGM3</i>^−/− (C, E) and control hairs (D, F). Arrowheads indicate regions lacking or retaining the cuticle cells in the mutant and control hairs, respectively. Scanning electron microscopy of mutant (G, I) and control (H, J) hair shafts shows severe distortion of the hair, in particular the cuticle, in the absence of TGase 3. In contrast, the roots of the mutant hairs (K) appear similar to the wild type ones (L) (scale bar 20 µm). Each of the four main pelage hair types was present in the mutant animals (M).</p

Barrier function of the skin in <i>TGM3^−/−</i> mice.

<p>Transmission electron microscopy of skin from <i>TGM3</i>^−/− (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 <i>stratum corneum</i> in both wild type and <i>TGM3^−/−</i> 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 <i>TGM3</i>^−/− (G) or wild type skin (H). (scale bars B 0.5 µm, D 7 µm, F and H 50 µm). At birth there was no retention of toluidine blue dye in the skin of either <i>TGM3^−/−</i> or wild type neonates (I). Dye was retained in the basal region of the whiskers in <i>TGM3</i>^−/− (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 <i>TGM3</i>^−/− litter mates (J). Sonication of corneocytes isolated from skin biopsy punches for various times revealed that <i>TGM3^−/−</i> 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).</p

Gross phenotype of <i>TGM3</i> null mice.

<p>Southern blot analysis of tail biopsies from mice born of <i>TGM3^+/−</i> 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).</p

Protein extractability in <i>TGM3^−/−</i> hair.

<p>Proteins were extracted from wild type and <i>TGM3^−/−</i> hair with 2% SDS and 5% β-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 <i>TGM3^−/−</i> protein lysates were identified by tryptic peptide mass fingerprinting as keratin6hf (A) and keratin17 (B).</p

Transmission electron microscopy of sections through the hair follicle of <i>TGM3^−/−</i> (A, C) and wild type hair (B, D) (scale bar 10 µm).

<p>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 <i>TGM3^−/−</i> (E) and control mice (F) and cornification occurs on the Henle's layer (arrow heads) (scale bar 2 µm). 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.</p

Analysis of collagen fibrils in wounds of wild type and <i>Col6a1</i> null mice and of basement membranes and tensile strength in skin of <i>Col6a1</i> null mice.

<p>(a) Picrosirius red staining of day 7 wounds. d =  dermis, e =  epidermis, g =  granulation tissue, st =  scar tissue. Bar, 200 µm. (b–g) Transmission electron microscopy of day 7 wounds (b–d) and of blood vessels (e), nerves (f) and muscle (g) in unwounded <i>Col6a1</i> null skin. Arrows indicate duplicated basement membranes. (h, i) Quantification of the distance between fibrils (wt = 903; <i>Col6a1</i> null  = 1776) (h) and of fibril diameter (wt = 890; <i>Col6a1</i> null  = 1091) (i) in areas from (b) and (c), (two electron micrographs from two animals per genotype were used for quantification). Load (j) and stress (k) are significantly reduced in unwounded <i>Col6a1</i> null skin. *<0.05, **<0.005, ***<0.0005. Bar, 250 nm.</p

Collagen VI in extracts of unwounded skin and wounds derived from wild type and <i>Col6a1</i> null mice.

<p>Extracts from unwounded skin and wounds were subjected to SDS-PAGE on 4–12% polyacrylamide gradient gels under reducing conditions, proteins transferred to a membrane and detected with affinity purified antibodies against the collagen VI α1, α2, α3, α5 and α6 chains. Boxed areas a and b on the right show a longer exposure. Arrows indicate the position of the full length proteins, asterisks indicate artefact bands.</p