Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae

Iwasaki, S., Yoshizawa, H. and Aoyagi, H. 2012. Immunohistochemical analysis of the distribution of type VI collagen in the lingual mucosa of rats during the morphogenesis of filiform papillae. —Acta Zoologica (Stockholm) 93 : 80–87. We examined the distribution after immunostaining of immunofluorescence of type VI collagen, differential interference contrast (DIC) images, and images obtained using confocal laser‐scanning microscopy in transmission mode, after toluidine blue staining, during morphogenesis of the filiform papillae, keratinization of the lingual epithelium and myogenesis in the rat tongue on semi‐ultrathin sections of epoxy resin‐embedded samples. Immunoreactivity specific for type VI collagen was dispersed over a relatively wide range of connective tissue in the mesenchyme of fetuses on day 15 after conception (E15), at which time the lingual epithelium was composed of one or two layers of cuboidal cells and the lingual muscle was barely recognizable. Slight immunoreactivity specific for type VI collagen was scattered within the lamina propria in fetuses on E17 and on E19, and immunoreactivity was relatively distinct on the connective tissue around the lingual muscle during myogenesis. In fetuses on E19, the epithelium was already stratified squamous. At postnatal stages from P0 to P14, keratinization of the lingual epithelium advanced gradually as morphogenesis of the filiform papillae proceeded during postnatal development. In newborns on P0, myogenesis of the tongue was almost completed. The intensity of immunoreactivity specific for type VI collagen at postnatal stages was mainly restricted on the endomysium and perimysium around the lingual muscle, while scant immunoreactivity was evident in the connective tissue in the lamina propria. Immunoreactivity around the fully mature lingual muscle on P7 and P14 was weaker than that on E19 and P0. Thus, type VI collagen appeared in the connective tissue that surrounded the lingual muscles such as the endomysium and perimysium, in parallel with changes in extracellular components during myogenesis of the tongue.     

Developmental expression of extracellular matrix components in intramuscular connective tissue of bovine semitendinosus muscle

 We have investigated the expression patterns of extracellular matrix components in intramuscular connective tissue during the development of bovine semitendinosus muscle by means of indirect immunofluorescence techniques. Types I, III, V, and VI collagen and fibronectin were located in the endomysium and the perimysium. Type IV collagen, laminin, and heparan sulfate proteoglycans (PGs) were exclusively located in the endomysium, and dermatan sulfate PGs existed only in the perimysium. The localization of these components in the intramuscular connective tissue of semitendinosus muscle remained unchanged throughout prenatal and postnatal growth of cattle, suggesting that they are essential for forming and maintaining structures of the endomysium and perimysium in bovine semitendinosus muscle. On the other hand, decorin was undetectable in the endomysium of neonates, although other matrix components were already expressed. It was expressed slightly in the endomysium of 2-month-old calves, and clearly detectable in the endomysium of cattle more than 6 months old. Chondroitin sulfate PGs were barely detectable in the perimysium of fetuses and neonatal calves, and progressively appeared during postnatal development of the muscle. It seems likely that these PGs are closely related to the postnatal development of the endomysium and perimysium. Accepted: 30 October 1996     

Histological features of endomysium,perimysium and epimysium in rat lateral pterygoid muscle

The distribution of the endomysium, perimysium, and epimysium and their constituent connective tissue fiber types in the mature rat lateral pterygoid muscle was examined with the light microscope. The endomysium and perimysium were relatively thin and consisted mainly of reticular fibers. The epimysium was thicker than the intramuscular sheaths and consisted of both collagen and reticular fibers; however, the thickness and constituent connective tissue fiber types of these sheaths varied regionally. Near the articular capsule and disc, the endomysium, perimysium, and epimysium were all thicker than in other regions of the muscle and consisted of collagen, reticular, and elastic fibers. The perimysium bound the bundles of muscle fibers together and frequently included blood vessels and nerves. As the superior head of the pterygoid muscle approached its insertion, sheaths of perimysium divided this head into smaller and smaller bundles of muscle fibers. In the inferior head, some of the perimysial sheaths and part of the epimysium were aponeurotic, and many muscle fibers attached to them. There were few such aponeurotic regions in the superior head. © 1996 Wiley-Liss, Inc.     

Morphology of perimysial and endomysial connective tissue in skeletal muscle

The fibrous components of the endomysium and perimysium of various muscles from four animal species were examined by scanning electron microscopy. All muscles were qualitatively similar. Endomysium consisted of a dense feltwork of collagen fibrils completely covering individual muscle fibre surfaces. Perimysium consisted of three fibrous components, (1) coarse, crimped fibres laid down in a well-ordered criss-cross pattern, (2) a loose feltwork of non-crimped fibrils, (3) fine noncrimped bundles of fibrils with no directional organization. The perimysium showed gradation from dense sheets of collagen down to the most delicate of sheets found on every muscle fibre surface overlying the endomysium.     

TGF-β mediated FGF10 signaling in cranial neural crest cells controls development of myogenic progenitor cells through tissue-tissue interactions during tongue morphogenesis

Skeletal muscles are formed from two cell lineages, myogenic and fibroblastic. Mesoderm-derived myogenic progenitors form muscle cells whereas fibroblastic cells give rise to the supportive connective tissue of skeletal muscles, such as the tendons and perimysium. It remains unknown how myogenic and fibroblastic cell-cell interactions affect cell fate determination and the organization of skeletal muscle. In the present study, we investigated the functional significance of cell-cell interactions in regulating skeletal muscle development. Our study shows that cranial neural crest (CNC) cells give rise to the fibroblastic cells of the tongue skeletal muscle in mice. Loss of Tgfbr2 in CNC cells (Wnt1-Cre;Tgfbr2^flox/flox) results in microglossia with reduced Scleraxis and Fgf10 expression as well as decreased myogenic cell proliferation, reduced cell number and disorganized tongue muscles. Furthermore, TGF-β2 beads induced the expression of Scleraxis in tongue explant cultures. The addition of FGF10 rescued the muscle cell number in Wnt1-Cre;Tgfbr2^flox/flox mice. Thus, TGF-β induced FGF10 signaling has a critical function in regulating tissue-tissue interaction during tongue skeletal muscle development.     

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)     

Resident macrophages (ED2- and ED3-positive) do not phagocytose degenerating rat skeletal muscle fibres

Two soleus muscles with degenerating muscle fibres were serially sectioned and adjacent sections from various levels of the skeletal muscles were stained with antibodies that react with either monocytes and inflammatory macrophages (ED1) or with the major subpopulations of resident macrophages (ED2 and ED3). ED2⁺ and ED3⁺ resident macrophages were abundant throughout the muscles but were not present within the degenerating fibres. ED1⁺ cells, in contrast, were rarely observed within the undamaged regions of the muscles but were abundant within the degenerating fibres and in the perimysium between arterioles and degenerating fibres. It is concluded that the phagocytosis of damaged muscle fibres is not carried out by the major subpopulations of resident macrophages.     

Characterization of muscle epimysium, perimysium and endomysium collagens

In the past it has been proven difficult to separate and characterize collagen from muscle because of its relative paucity in this tissue. The present report presents a comprehensive methodology, combining methods previously described by McCollester [(1962) Biochim. Biophys. Acta 57, 427-437] and Laurent, Cockerill, McAnulty & Hastings [(1981) Anal. Biochem. 113, 301-312], in which the three major tracts of muscle connective tissue, the epimysium, perimysium and endomysium, may be prepared and separated from the bulk of muscle protein. Connective tissue thus prepared may be washed with salt and treated with pepsin to liberate soluble native collagen, or can be washed with sodium dodecyl sulphate to produce a very clean insoluble collagenous product. This latter type of preparation may be used for quantification of the ratio of the major genetic forms of collagen or for measurement of reducible cross-link content to give reproducible results. It was shown that both the epimysium and perimysium contain type I collagen as the major component and type III collagen as a minor component; perimysium also contained traces of type V collagen. The endomysium, the sheaths of individual muscle fibres, was shown to contain both type I and type III collagen as major components. Type V collagen was also present in small amounts, and type IV collagen, the collagenous component of basement membranes, was purified from endomysial preparations. This is the first biochemical demonstration of the presence of type IV collagen in muscle endomysium. The preparation was shown to be very similar to other type IV collagens from other basement membranes on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and was indistinguishable from EHS sarcoma collagen and placenta type IV collagen in the electron microscope after rotary shadowing.     

Localization of hyaluronan in various muscular tissues

Summary The histochemical distribution of hyaluronan (hyaluronic acid, HYA) was analysed in various types of muscles in the rat by use of a hyaluronan-binding protein (HABP) and the avidin-biotin/peroxidase complex staining procedure. Microwave-aided fixation was used to retain the extracellular location of the glycosaminoglycan. In skeletal muscles, HYA was detected in the connective tissue sheath surrounding the muscles (epimysium), in the septa subdividing the muscle fibre bundles (perimysium) and in the connective tissue surrounding each muscle fibre (endomysium). HYA was heterogeneously distributed in all striated muscles. In skeletal muscles with small fibre dimensions (e.g., the lateral rectus muscle of the eye and the middle ear muscles), HYA was predominantly accumulated around the individual muscle fibres. Perivascular and perineural connective tissue formations were distinctly HYA-positive. In cardiac muscles, HYA was randomly distributed around the branching and interconnecting muscle fibres. In comparison, smooth muscle tissue was devoid of HYA.     

Morphology of connective tissue in skeletal muscle

The arrangement and distribution of connective tissue in six different skeletal muscles and smooth muscle was examined by scanning electron microscopy. The endomysial arrangement of collagen was similar in all types of muscle and consisted of three components: (1) myocyte-myocyte connectives; (2) myocyte-capillary connectives; and (3) a weave network of collagen intimately associated with the basal laminae of the myocytes. The perimysium of the different muscles was qualitatively similar but quantitatively dissimilar. The perimysium consisted of large tendon-like bundles of interwoven collagen which connected with the dense weave collagen that surrounded groups of muscles. The arrangement of the collagen in the perimysium and endomysium would explain differences in the mechanical properties of the different muscle. The contribution of the connective tissue to mechanical properties of muscle is discussed.     

Analysis of fibronectin expression during human muscle differentiation

Fibronectin expression during human muscle differentiation was investigated by determining its distribution in foetal, normal adult and dystrophic muscle and in foetal, normal adult and dystrophic muscle cultures during myogenesis. Muscle sections and muscle cultures were studied by indirect immunofluorescence staining using polyclonal and monoclonal anti-human antibodies. Mass and clonal muscle cultures were prepared from foetal, adult and dystrophic muscle tissue. Immunofluorescence staining detected fibronectin on the epimysium, perimysium and endomysium of transverse sections of normal adult muscle, while sarcoplasm was devoid of this glycoprotein. In foetal muscle, some fibers showed a prominent ring of fibronectin. In mass and clonal cultures, myoblasts were found to synthesize and accumulate fibronectin while myotubes did not. No difference in fibronectin distribution was observed between Duchenne Muscular Dystrophy (DMD) and control myotubes. An enzyme-linked immunoassay (ELISA), performed on homogenated muscle, sonicated fibroblasts and muscle cells, showed a high fibronectin level in fibroblasts when compared with the other samples tested.     

Location of the integrin complex and extracellular matrix molecules at the chicken myotendinous junction

Summary The distribution of several extracellular matrix macromolecules was investigated at the myotendinous junction of adult chicken gastrocnemius muscle. Localization using monoclonal antibodies specific for 3 basal lamina components (type IV collagen, laminin, and a basement membrane form of heparan sulfate proteoglycan) showed strong fluorescent staining of the myotendinous junction for heparan sulfate proteoglycan and laminin, but not for type IV collagen. In addition, a strong fluorescent stain was observed at the myotendinous junction using a monoclonal antibody against the subunit of the chicken integrin complex (antibody JG 22). Neither fibronectin nor tenascin were concentrated at the myotendinous junction, but instead were present in a fibrillar staining pattern throughout the connective tissue which was closely associated with the myotendinous junction. Tenascin also gave bright fluorescent staining of tendon, but no detectable staining of the perimysium or endomysium. Type I collagen was observed throughout the tendon and in the perimysium, but only faintly in the endomysium. In contrast, type III collagen was present brightly in the endomysium and in the perimysium, but could not be detected in the tendon except when associated with blood vessels and in the epitendineum, which stained intensely. Type VI collagen was found throughout the tendon and in all connective tissue partitions of skeletal muscle. The results indicate that one or more molecules of the integrin family may play an important role in the attachment of muscle to the tendon. This interaction does not appear to involve extensive binding to fibronectin or tenascin, but may involve laminin and heparan sulfate proteoglycan.     

Immunohistochemical localization of genetically distinct types of collagen in muscle of kuruma prawn Penaeus japonicus

• 1.1. The location of genetically distinct types of collagen in muscular tissue of the kuruma prawn was examined using immunohistochemical techniques.
• 2.2. Collagen was distributed not only in muscle connective tissues, which were classified into three forms, epimysium, perimysium and endomysium, but also in subcuticular membrane, which was mainly composed of two layers, hypodermis and subcuticular connective tissue.
• 3.3. The α1(Pr) component existed in all connective tissues in the kuruma prawn muscle. Type AR-II collagen was distributed in all the connective tissues except for the hypodermis, while the α2(Pr) component existed in the thin connective tissues, the perimysium and endomysium, and in the hypodermis.

     

A Semiquantitative Scoring Tool to Evaluate Eccentric Exercise-Induced Muscle Damage in Trained Rats

Unaccustomed eccentric exercise is a well-documented cause of exercise-induced muscle damage. However, in trained subjects muscle injury involves only light or moderate tissue damage. Since trained rats are widely used as a model for skeletal muscle injury, here we propose a semiquantitative scoring tool to evaluate muscle damage in trained rats. Twenty male Sprague-Dawley rats were trained for two weeks following a two-week preconditioning period, and randomly divided into two groups: control rats (CTL; n=5) and rats with eccentric exercise-induced muscle damage (INJ; n=15). Injured rats were sacrificed at three time points: 1, 3 and 7 days post injury (n=5 each). Transverse sections from the right soleus were cut (10 µm) and stained with haematoxylin-eosin. Samples were evaluated by two groups of observers (four researchers experienced in skeletal muscle histopathology and four inexperienced) using the proposed tool, which consisted of six items organised in three domains: abnormal fibre morphology, necrotic/(re) degenerating fibres (muscle fibre domain), endomysial and perimysial infiltration (inflammatory state domain) and endomysium and perimysium distension (interstitial compartment domain). We observed the expected time course in the six evaluated items. Furthermore, agreement among observers was evaluated by measuring the Intraclass Correlation Coefficient (ICC). Within the experienced group, items from the muscle fibre and interstitial compartment domains showed good agreement and the two items from the infiltration compartment domain showed excellent agreement. In conclusion, the proposed tool allowed quick and correct evaluation of light to moderate muscle damage in trained rats with good agreement between observers.Key words: Histopathology, haematoxylin-eosin, histochemical method     

Biological resorption of fibers from chitosan in endomysium and perimysium of muscular tissue

The resorption of fibers from chitosan implanted into emdomysium and perimysium of the rat’s broadest muscle of the back is comparatively studied in vivo by the scanning electron microscopy and histologic analysis methods. It is shown that the mechanism and rate of resorption of the fibers from chitosan depend on the fiber localization in the muscular tissue. Implantation of chitosan fibers into endomysium, where they have been in direct contact with muscle fibers, results in 14 days in the formation of transverse cracks, fiber fragmentation, and their partial resorption. Complete resorption of fibers in endomysium is observed in 30 days. Fibers implanted into perimysium maintain integrity in 7 days of the experiment, and a fibrous tissue is formed around the fibers. There is no destruction of chitosan fibers in 45 days of the exposition. The biocompatibility of the chitosan fibers is confirmed by the effective adhesion and proliferation mesenchyme stem cells on their surface.     

Histochemical demonstration of monoaminergic and cholinesterase-positive nerve fibres in regenerating rat submandibular gland autografts

Summary A cholinesterase localization method and a monoamine histofluorescence technique were used to locate nerve fibres in regenerating rat submandibular gland autografts. Experimental rats had a portion of one submandibular gland excised and cut into small fragments which were autografted immediately into the middle one-thrid of the tongue. Control rats had a portion of one submandibular gland removed and discarded, and their tongues were sham-operated. Seven to ten weeks later, the rats were killed and the tongues were removed, frozen and sectioned in a cryostat. A light microscopical study of the tongue sections subjected to the cholinesterase technique showed that the submandibular gland autografts contained many nerve fibres that exhibited cholinesterase activity. These cholinesterase-positive nerve fibres were distributed throughout the autografts. The fibres were associated with the numerous duct-like structures and the less numerous acini. In addition, ultraviolet illumination of tongue sections after treatment with a glyoxylic acid mixture revealed histofluorescent monoaminergic nerves within the autografts. These fibres were less prominent than the cholinesterase-positive fibres and appeared to run primarily along blood vessels within the autografts. The results suggest that autonomic nerves are present within regenerating submandibular gland autografts.     

Fibre type regionalization of forelimb muscles in two mammalian species, Galea musteloides (Rodentia, Caviidae) and Tupaia belangeri (Scandentia, Tupaiidae), with comments on postnatal myogenesis

To investigate structural differences between propulsory and antigravity muscles, the spatial distribution of slow (type I) and fast (type II) muscle fibres in forelimb muscles of two species of small mammals was studied, Galea musteloides and Tupaia belangeri. Serial sections through complete forelimbs were prepared. Following histochemical fibre typing, the forelimbs were reconstructed three-dimensionally using product design software. Most forelimb muscles of both species showed a homogenous distribution of type I fibres. In the supraspinatus and triceps brachii (capita longum et laterale) muscles, however, a segregation of fibre types into ”fast” superficial areas and ”slow” deep regions was observed. Slow regions contained at least 60% type I fibres and were positioned along intramuscular extensions of the tendons of insertion. The functional implications of fibre type regionalization are discussed. An analysis of intramuscular fibre type distribution during postnatal myogenesis revealed no significant differences in muscle fibre differentiation between altricial and precocial juveniles. Differences in locomotor ability probably arise from heterochronic development of connective tissue components (endo- and perimysium). Accepted: 10 June 1999     

A TGFβ-Smad4-Fgf6 signaling cascade controls myogenic differentiation and myoblast fusion during tongue development

The tongue is a muscular organ and plays a crucial role in speech, deglutition and taste. Despite the important physiological functions of the tongue, little is known about the regulatory mechanisms of tongue muscle development. TGFβ family members play important roles in regulating myogenesis, but the functional significance of Smad-dependent TGFβ signaling in regulating tongue skeletal muscle development remains unclear. In this study, we have investigated Smad4-mediated TGFβ signaling in the development of occipital somite-derived myogenic progenitors during tongue morphogenesis through tissue-specific inactivation of Smad4 (using Myf5-Cre;Smad4(flox/flox) mice). During the initiation of tongue development, cranial neural crest (CNC) cells occupy the tongue buds before myogenic progenitors migrate into the tongue primordium, suggesting that CNC cells play an instructive role in guiding tongue muscle development. Moreover, ablation of Smad4 results in defects in myogenic terminal differentiation and myoblast fusion. Despite compromised muscle differentiation, tendon formation appears unaffected in the tongue of Myf5-Cre;Smad4(flox/flox) mice, suggesting that the differentiation and maintenance of CNC-derived tendon cells are independent of Smad4-mediated signaling in myogenic cells in the tongue. Furthermore, loss of Smad4 results in a significant reduction in expression of several members of the FGF family, including Fgf6 and Fgfr4. Exogenous Fgf6 partially rescues the tongue myoblast fusion defect of Myf5-Cre;Smad4(flox/flox) mice. Taken together, our study demonstrates that a TGFβ-Smad4-Fgf6 signaling cascade plays a crucial role in myogenic cell fate determination and lineage progression during tongue myogenesis.     

Chick myotendinous antigen. I. A monoclonal antibody as a marker for tendon and muscle morphogenesis

Extracellular matrix components are likely to be involved in the interaction of muscle with nonmuscle cells during morphogenesis and in adult skeletal muscle. With the aim of identifying relevant molecules, we generated monoclonal antibodies that react with the endomysium, i.e., the extracellular matrix on the surface of single muscle fibers. Antibody M1, which is described here, specifically labeled the endomysium of chick anterior latissimus dorsi muscle (but neither the perimysium nor, with the exception of blood vessels and perineurium, the epimysium ). Endomysium labeling was restricted to proximal and distal portions of muscle fibers near their insertion points to tendon, but absent from medial regions of the muscle. Myotendinous junctions and tendon fascicles were intensely labeled by M1 antibody. In chick embryos, " myotendinous antigen" (as we tentatively call the epitope recognized by M1 antibody) appeared first in the perichondrium of vertebrae and limb cartilage elements, from where it gradually extended to the premuscle masses. Around day 6, tendon primordia were clearly labeled. The other structures labeled by M1 antibody in chick embryos were developing smooth muscle tissues, especially aorta, gizzard, and lung buds. In general, tissues labeled with M1 antibody appeared to be a subset of the ones accumulating fibronectin. In cell cultures, M1 antibody binds to fuzzy, fibrillar material on the substrate and cell surfaces of living fibroblast and myogenic cells, which confirms an extracellular location of the antigenic site. The appearance of myotendinous antigen during limb morphogenesis and its distribution in adult muscle and tendon are compatible with the idea that it might be involved in attaching muscle fibers to tendon fascicles. Its biochemical characterization is described in the accompanying paper ( Chiquet , M., and D. Fambrough , 1984, J. Cell Biol. 98:1937-1946).