The Ultrastructure of Collagen in the Dermis of Tight-skin (Tsk) Mutant Mice

David N. Menton, Rex A. Hess
1980 Journal of Investigative Dermatology  
A recently discovered dominant mutation in C57/BIO mice called tight-skin (Tsk) results in hypertrophy of certain collagenous tissues including the dermis and hypodermis. The skin of heterozygotes (Tsk/ +) is indurated and substantially stiffer than that of the normal animals (+ / +). In this study, an electron microscopical comparison of the skin of these animals revealed that the fibrous architecture of the hypertrophic reticular dermis of Tsk/ + mice is more disorganized than that of the + /
more » ... + mice and in many areas, the collagen fibrils are IDore densely packed. The abundance of fibroblasts with distended endoplasmic reticulum in both the dermis and hypodermis of Tsk/ + mice is consistent with increased collagen synthesis. Several of the changes in the dermis and hypodermis of the Tsk/ + mice are similar to changes reported in sclerodermatous skin of man. Surprisingly, an apparent abnormality in the morphology of some of collagen fibrils in the skin of Tsk/ + mice was found to be at least as prevalent in the"'normal" +/+ mice. The reticular dermis of both animals contain scattered fibrils which are pmch larger in diameter than normal and often have a twisted appearance resulting from e ither helical grooves in the surface of the fibril or discrete branches which twist about one another. A dominant mutation in th e mouse called " tight-skin" (Tsk) was discovered in 1967 by Hel en Bunker in th e BlO.D2/ Sn strain at the Jackson Laboratory. The integument of animals heterozygous (Tsk/ +) for this mutant gene lacks t he pliability, resilience and deformability typical of normal skin. Green, Sweet, and Bunker [1] have shown t hat the Tsk mutation primarily affects th e connective tissues and results in hypertrophy of small tendons, tendon sheaths, cartilage, bone and loose connective tissue. The subcutaneous connective tissue in particular was reported to be both thicker land more cellulaj' than normal. Electron microscopical study of th e hypodermal areas revealed scattered collagen fibrils in t he midst of masses of fin e "microfibrils" measuring about 10 nm in diameter and lacking a banded perodicity [1] . No abnormalities were reported in the dermis itself. In a recent light microscopical and rheological study of the skin of Tsk/ + mice, the dermis was found to be abnormal in both structural and tensile properties compared to that of normal siblings (+ / +) [2] . The dermis from the ear, back, and abdomen, for example, proved to be nearly twice as t hick a normal and appeared to lack the regular weave pattern typical of normal dermal collagen. Instead the fibrous archi tecture of the Tsk/+ dermis is generally disorganized and in some areas appears amorphous or hyaline in the superficial h alf of the Manuscrip t . Louis, Misso uri 63110. Abbreviations: PTA: phos photungstic acid RER: rough e ndoplasmic reticulum 139 dermis. In addition, t he Tsk dermis is usually more cellular t han normal. Rheological studies have shown t hat the Tsk/ + dermis is indurated and substantially stiffer than that of normal siblings [2] . Of particular interest was t he observation t hat t he elastic stiffness of tight skin is similar when stretched either in t he longitudinal axis of the body or transversely whereas the skin of the + / + mice shows a nisotropy and is substantially stiffer when str etched in t he transverse axis than in the longitudinal. These abnormal directional and tensile properties of the skin of Tsk/ + mice were in terpr eted to be at least in part a result of alterations in the synthesis a nd fibrous archi tecture of dermal collagen. The purpose of this study was to investigate t he affects of the T sk mutation on the fine structure and organization of the fibrillar and cellular elem ents of the dermis in the hope that this might reveal some morphological basis for the altered rheological properties of Tsk/ + skin. Particular attention was given to the hyalinized areas of the superificial dennis in an effort to determine what structw-al alterations might be responsible for t his homogenous a ppearance. MATERIALS AND METHODS Full thickness dorsal ski n was dissected from 10 t igh t s kin (Tsk/ +) mice and 10 mice homozygous for t he normal all ele (+/ +) which were raised by inbreeding (Tsk/ +) x (+ / +) from st.ock originally obtain ed from th e Jackson Labo ratory (courtesy of Dr. Margaret Green). All mice used for microsco pical study a nd co mpaJ"ison we re males between 2 and 4 mo old. For light mi croscopical stud y, large pieces of s kin were fix ed in Bouin's fluid, de hyd.rated, cleared and embedded in paraffin . Following the re moval of th e paraffin, the sections were stained in Harris' a lum hematoxy lin or Verhoeffs elast ic stain a nd cou nterstained wi t h Van G ieso n's stain [3] . Photomicrographs were made using a Wratten 58 filter to e mphasize t he coll agen pattern. E lectron microscopica l study was perfo rm ed on pieces of s kin from the mid-dors um which were dissected fir st in long narrow strips (l mm wide) para llel to t he long axis of the body, a nd t hen cut by mea ns of a Cambridge Vibratome in to 0.5 mm pieces perpendicular to th e long ax is of the strip. In t his way, t horough in filtra tion a nd embedment of t he tissue as well as co nsistent orientation of t he specimen was assured . The a mpl es were t hen fixed for 4 hr at 22°C with 4 % paraformaldehyde a nd 3% glu taraldehyde in 0.1 m cacodylate buffer with 0.0 1 M CaCI~ and postfix ed for 1 hr at 22°C with 2% osmium tetrox id e in the same buffer. Foll ow in g a distilled water rinse ~h e tissue was stained en bloc wi t h aqueous (1 %) ura nyl acetate for 1 hr at 22°C, de hydrated with 2,2dimethoxypropane [4] and embedded in Spurr's resin [51-Selected ar ea of the dermis were ul trathin section ed (800-900 A) with a diamond knife after ligh t micl"Oscopical evaluation of 1 Jl t.hick sections. To show general ult rastru ctural features, t hese sections were stain ed with 1 % ura nyl acetate in 50% ethanol a nd poststain ed in Reynold's lead citrate [6). Some sections were stained onl y in 1 % phosp hotun gstic acid (PTA) in 95 % etha nol to provide more electronde nse staining of co llage n. Electron mi crograp hs we re t.aken usin g a Philips EM 300. RESULTS Generai Light Microscopical Comparison of the Tsh! + and ± / + Dermis The reticular dermis of skin from the back of Tsk/ + mice is consistently thicker and often more cellula r tha n t hat of + / + siblings (compare Fig 1 and 2) . The wickerwork arrangement of collagen that characteri zes normal dermis is frequently more
doi:10.1111/1523-1747.ep12535041 pmid:7359004 fatcat:xhcmnef3nrgaldqfk2223qdz34