Sturctural analysis of cell:matrix association during the morphogenesis of atrioventricular cushion tissue.

Translocation of an endocardially seeded cushion cell progeny across a broad acellular expanse of extracellular matrix (ECM) constitutes a fundamental morphogenetic event in the development of atrioventricular (AV) cushion pads, the primordia of membraneous septa and cardiac valves. Transmission, scanning, and high-voltage electron microscopy together with light microscopic examination of living or fixed tissues were utilized to determine if (1) one component of the ECM more than any other interacted with the motility-like appendages of cushion cells in such a manner as to suggest a physical substratum; (2) any ECM components were organized into polarized “tracks” which could serve to guide cells centrifugally; and (3) cell:ECM associations varied among the cells comprising the migratory wave. Results indicated that two morphologically identifiable matrix components, microfibrils and a continuum of solid pleomorphic strands of heterogeneous composition called cetylpyridinium chloride (CPCL)-dependent matrix, comprised the bulk of the premigratory ECM. Contact of the premigratory matrix by cushion cells at the leading edge (pioneer cells) of the migratory wave coincided with modification in composition of the CPCL matrix and alignment of microfibrils into polarized tracks (an event seemingly dependent on motility appendage formation, since cells lacking processes after cytochalasin B treatment had altered track associations). Trailing cushion cells uniformly populated the ECM, never piled up against the myocardium, had no track associations, formed numerous cell to cell associations, and were coated with a granular remnant of disrupted CPCL-dependent matrix. The foregoing data suggest that active in vivo translocation and subsequent stabilization of cushion cells involve alignment and compositional changes in the premigratory ECM, events linked temporally with the passage of pioneer cells.     

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and "self-assembly" of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collage gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (less than 1 mg/ml) and CS at high concentrations (greater than 20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degrees, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG.(ABSTRACT TRUNCATED AT 400 WORDS)     

Cellular migration through the cardiac jelly matrix: A stereoanalysis by high-voltage electron microscopy

Formation and migration of cushion tissue in the developing chick heart was analyzed by scanning and high-voltage electron microscopic stereoanalysis. Two methods of fixation which enhance the preservation of water-soluble components of the extracellular matrix (cardiac jelly) were employed: 1% tannic acid in 3% glutaraldehyde (TAG) and 1% cetylpyridinium chloride (CPC) in 3% glutaraldehyde. Our results indicated that the preservation of the cell: matrix interaction exhibited by endocardial cells and migrating cushion tissue is dependent upon the method of fixation. In TAG-fixed embryos, filopodial extensions from the endocardium as well as filopodia of pioneering cells are most often associated with microfibrillar components of the matrix, whereas in CPC-fixed material these same cellular extensions are found in association with pleomorphic anastomosing strands rich in hyaluronate. Following these initial cell:matrix interactions by both the endocardium and pioneering cells, trailing cells invade the extracellular matrical region and clearly encounter in both types of fixation a different microenvironment in which to engage in cell:matrical associations. These observations support the hypothesis that filopodial probing by endocardial cells and pioneering cells results in macromolecular reorderings of the matrix and thus suggest an additional function for filopodia beyond translocation of the cells.     

Morphological effects of streptomyces hyaluronidase treatment on the outgrowth of the nasal processes in mouse embryos

The role of hyaluronic acid (HA) in embryonic mouse nasal process outgrowth was assessed following administration of Streptomyces hyaluronidase, an enzyme that degrades HA. Enzyme-treated and control embryos were compared morphologically 4 and 24 hr after treatment on day 11 of gestation. After 4 hr the nasal processes of treated embryos were reduced in volume compared to controls. This size reduction was associated with a decrease in the amount of extracellular space in the nasal processes and a change in mesenchymal cell shape. Extracellular matrix material observed in controls included collagenlike fibers, 25-30-nm granules, and a delicate meshwork of 3-4-nm filaments. Basal laminae exhibited filamentous and granular material that extended to the surface of underlying cells. Similar matrix constituents were observed in treated embryos with the exception of the 3-4-nm filaments, which probably represent HA. By 24 hr after treatment, embryonic circulation had ceased and heart beat was slow. The nasal processes of these embryos were very small, but their configuration was such that fusion had often begun. Thus the presence of HA appears to be important in maintenance of the normal volume of the nasal processes and in maintenance of normal mesenchymal cell morphology, but other factors appear to contribute to the change in process shape requisite for fusion.     

Canada goose posterior lobe pituicytes: seasonal ultrastructural changes in relation to axonal release of neurosecretion

Changes in ultrastructure of the posterior lobe pituicytes during six phases (Spring premigratory, Spring postmigratory, breeding, moulting, Fall premigratory and Fall postmigratory) of the annual life cycle of the migratory Canada goose, were studied. Pituicyte nuclear volume in females was significantly greater than that in males during the Spring premigratory and moulting phases. In the Fall postmigratory phase, nuclear volume in males was greater than that in females. During Spring premigratory phase, the axonal endings in males contained fewer neurosecretory granules (NSG) than those in the Fall postmigratory phase. In females, however, the number of NSG was relatively more than that in males in the Spring premigratory phase but fewer in the moulting phase. Nuclear volume is correlated with pituicyte function in releasing NSG containing arginine vasotocin (AVT) from axonal endings. The significance of AVT release is discussed in relation to initiation of reproductive and migratory behaviour, and to osmotic and metabolic regulation.     

Seasonal immunocytochemical and ultrastructural changes in the neurohypophysis of the migratory Canada goose

Immunocytochemical localization of arginine vasotocin (AVT) in the neurohypophysis of the migratory Canada goose was studied during six periods (Spring premigratory, Spring postmigratory, breeding, moulting, Fall or Autumn premigratory, and Fall postmigratory) of its annual life cycle. Ultrastructural changes in the pars nervosa, with emphasis on the axonal endings, were also examined during the above periods. In the Spring premigratory period, the staining intensity for AVT was greater in the anterior median eminence than in the pars nervosa where the density of axonal secretory granules was lower than that in the preceding period, in both sexes. During moulting females seemed to be under greater osmotic stress than males as indicated by low AVT staining intensity and secretory granule concentration in the pars nervosa. The occurrence of considerable amounts of lipofuscin bodies in the pars nervosa of both sexes during moulting was indicative of high lysosomal activity in the pituicytes. AVT release is recognized as a significant event in initiating Spring migration.     

Ultrastructure and staining properties of aortic microfibrils (oxytalan)

Microfibrils are the insoluble, 10- to 12-nm components of the extracellular matrix that are involved in elastogenesis. Reports of their ultrastructure vary: they have been described as tubular and beaded and as nontubular filaments that are devoid of any periodicity. Ultrastructurally, microfibrils resemble oxytalan fibers that have been observed in peridontal membranes, skin, and other locations. Whether microfibrils have the staining characteristics of oxytalan is difficult to determine in tissues because available light microscopic stains also stain elastin. Calf aortic smooth muscle cells grown in media without added ascorbate provide a unique model for examining the ultrastructure and staining characteristics of chemically defined microfibrils. Microfibrils are the predominant insoluble extracellular protein in such cultures, which do not deposit collagen or elastin. These studies demonstrate that microfibrils are tubular structures with 10- and 12-nm striations and have the same staining characteristics as oxytalan, reacting with aldehyde fuchsin and orcein after oxidation. Microfibrillar protein is enriched in glutamic and aspartic acids and the electron density of microfibrils is enhanced by fixation in the presence of cationic dyes. In such preparation, microfibrils are made visible within the core of amorphous elastin as well as in regions that are free of elastin. The widespread distribution of microfibrils (oxytalan) indicates that their function extends beyond elastogenesis. Their localization within tissues suggests that they serve as an elastic attachment protein in sites that are subject to mechanical stress.     

A new deformation model of hard alpha-keratin fibers at the nanometer scale: implications for hard alpha-keratin intermediate filament mechanical properties

The mechanical behavior of human hair fibers is determined by the interactions between keratin proteins structured into microfibrils (hard alpha-keratin intermediate filaments), a protein sulfur-rich matrix (intermediate filaments associated proteins), and water molecules. The structure of the microfibril-matrix assembly has already been fully characterized using electron microscopy and small-angle x-ray scattering on unstressed fibers. However, these results give only a static image of this assembly. To observe and characterize the deformation of the microfibrils and of the matrix, we have carried out time-resolved small-angle x-ray microdiffraction experiments on human hair fibers stretched at 45% relative humidity and in water. Three structural parameters were monitored and quantified: the 6.7-nm meridian arc, which is related to an axial separation between groups of molecules along the microfibrils, the microfibril's radius, and the packing distance between microfibrils. Using a surface lattice model of the microfibril, we have described its deformation as a combination of a sliding process and a molecular stretching process. The radial contraction of the matrix is also emphasized, reinforcing the hydrophilic gel nature hypothesis.     

The protein components of the 12-nanometer microfibrils of elastic and nonelastic tissues

A procedure has been developed which is much more specific for the solubilization of the elastin-associated microfibrils from fetal bovine nuchal ligament using treatment with reductive saline in place of reductive guanidine hydrochloride buffer. When analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, reductive saline extracts were shown to contain only five major protein bands with Mrs of 340,000, 78,000, 70,000, 31,000, and 25,000. The 31-kDa species was identified immunologically as the previously described macromolecule named microfibril-associated glycoprotein (MAGP) (Gibson, M. A., Hughes, J. L., Fanning, J. C., and Cleary, E. G. (1986) J. Biol. Chem. 261, 11429-11436). The proteins were purified by gel permeation, ion exchange, and affinity chromatography. Amino acid analyses showed that each protein had a profile which was distinct from that of MAGP although each was also high in acidic amino acids and cystine. The 340- and 78-kDa species were each demonstrated by immunoelectron microscopy with affinity-purified antibodies to be derived from the elastin-associated microfibris, and these were provisionally named microfibrillar protein 340 (MP340) and microfibrillar protein 78 (MP78), respectively. Each of the above antibodies gave a tissue distribution identical to that of anti-MAGP antibodies, and thus MP340 and MP78 also were identified with the 12-nm microfibrils of nonelastic tissues. MP340 was shown to absorb out completely the microfibrillar immunoreactivity of anti-(reductive guanidine hydrochloride extract) antibodies, indicating that MP340 was (a) the major microfibrillar constituent in these extracts and (b) the second unidentified microfibrillar antigen described previously. The relationship of the 70- and 25-kDa proteins to microfibrils is yet to be established. Immunoblot and immunoabsorption studies showed that MAGP and MP78 were immunologically related to MP340 but not to each other. Cyanogen bromide peptide mapping indicated that MAGP was structurally related to MP340. It is postulated that MAGP and MP78 are constituents of MP340 which in turn is the subunit of which the 12-nm microfibrils are composed.     

Differentiation of murine premigratory primordial germ cells in culture.

In the mouse embryo, primordial germ cells first appear in the extraembryonic mesoderm and divide rapidly while migrating to the fetal gonad. Shortly after their arrival in the gonad, germ cells sexually differentiate as proliferation ceases. Previous studies have established that primordial germ cells proliferate and migrate in feeder layer culture. To explore cellular regulation of fetal germ cell development, we have used germ cell nuclear antigen 1 (GCNA1), a marker normally expressed only in postmigratory germ cells, to investigate the developmental potency of both pre- and postmigratory cells in this culture system. We found that explanted premigratory germ cells will initiate expression of this marker and are, therefore, capable of undertaking some aspects of gonocyte differentiation without intimate exposure to the fetal gonad. We have also tested whether postmigratory gonocytes are stable in culture. As detected by either alkaline phosphatase or GCNA1, we did not detect long-term survival of either prospermatogonia or oogonia under conditions that support the survival, proliferation, and differentiation of earlier premigratory cells. These observations are consistent with an autonomous cellular mechanism governing the initial stages of gonocyte differentiation, and suggest that differentiation towards gonocytes is accompanied by a change in requirements for cell survival.     

AN ELECTRON MICROSCOPE STUDY OF THE FINE STRUCTURE OF FEATHER KERATIN

Thin sections of the rachis of regenerating follicles of pigmented fowl feathers and of mature non-pigmented seagull feather rachis, embedded in methacrylate and Araldite respectively, were studied in the electron microscope. The late stages of development of keratin fibrils were examined in OsO₄-fixed follicle material, and after poststaining with lead hydroxide the keratin aggregates were found to be composed of fine microfibrils approximately 30 A in diameter apparently embedded in a matrix material which had absorbed the lead stain. The centre-to-centre separation of the microfibrils was of the order of 35 A. After bulk treatment by reduction with thioglycollic acid, OsO₄ staining, and poststaining with lead hydroxide, a similar microfibrillar fine structure was observed in mature rachis. Only after lead staining could the microfibrils be delineated, and their diameter and separation were similar to that found in the keratin of the follicle. It is suggested that feather keratin resembles α-keratins in consisting of microfibrils embedded in an amorphous protein matrix. However, in comparison with α-keratins, the microfibrils are much smaller in diameter, their arrangement is less orderly, and on the basis of the reactions towards the electron staining procedures, the cystine content of the matrix appears to be not greatly different from that of the microfibrils. The significance of a microfibrillar constitution of feather keratin is discussed in relation to current structural models for this fibrous protein deduced from x-ray diffraction studies. The boundaries between the component cells of feather rachis are desmosomal in character and similar to those of related keratinous structures and a number of different types of cells; the melanin granules are dissimilar to those of mammalian epidermis in their apparent lack of melanin-protein lamellae.     

The cell substratum modulates skeletal muscle differentiation

During chick embryogenesis, massive alterations occur in the migrating cell's substratum, or extracellular matrix. The possibility that some of the components of this milieu play a regulatory role in cell differentiation was explored in a cell-culture system derived from embryonic chick skeletal muscle tissue. In particular, the effects of collagen and the glycosaminoglycans were studied. Collagen is required for muscle cell attachment and spreading onto plastic and glass tissue-culture dishes. A major constituent of the early embryonic extracellular space, hyaluronate (HA), while having no significant effect on collagen-stimulated cell attachment and spreading, was found to inhibit myogenesis. The muscle-specific M subunit of creatine kinase was preferentially inhibited. Control experiments indicated that the inhibition was specifically caused by HA and not by other glycosaminoglycans. A general metabolic inhibition of the cultures was not observed. Muscle cells could bind to HA-coated beads at all stages of differentiation but were inhibited only when HA was added within the first 24 h of culture. Endogenous GAG in the culture is normally degraded during the first 24 h after plating as well; this may parallel the massive degradation of HA that occurs in the early embryo in vivo. These findings suggest a regulatory role for HA in modulating skeletal muscle differentiation, with degradation of an inhibitory component of the cell substratum a requirement for myogenesis.     

Distinctive fibroblastic subpopulations in skin and oral mucosa demonstrated by differences in glycosaminoglycan content

Summary The glycosaminoglycan (GAG) content of rabbit skin, oral mucosa, and cultured [³H]-glucosamine-labeled dermal and submucosal fibroblasts was compared. Skin contained predominantly dermatan sulfate (DS) and a small amount of hyaluronic acid (HA), whereas mucosa contained primarily keratan sulfate (KS) and smaller quantities of HA and DS. Culture medium from dermal and submucosal fibroblasts contained GAGs co-electrophoresing with DS, HA, and chondroitin sulfate (CS), although the relative proportions of these GAG differed. CS isolated from dermal and mucosal fibroblast culture medium co-electrophoresed with chondroitin 4-sulfate (C4-S) on cellulose acetate, whereas dermal medium CS was resistant to digestion by chondroitinase ABC, and mucosal medium CS was chondroitinase ABC-susceptible. The pericellular matrix of dermal fibroblasts contained primarily DS and C4-S/C6-S, as confirmed by chondroitinase ABC digestion; the corresponding fraction of mucosal fibroblasts contained HS and a GAG co-electrophoresing with a C6-S standard, yet resistant to digestion by chondroitinase ABC. Thus the GAG content of dermal and mucosal fibroblasts differed both qualitatively in terms of the type of GAG secreted into the culture medium and pericellular matrix, and quantitatively, in terms of the relative proportions of these GAGs in both fractions. These differences support the concept of distinctive fibroblastic subpopulations in skin and mucosal tissue, inasmuch as the cells were subjected to identical culturing conditions. This work was supported by research grant 15878 (C.N.B.) from the Shriners Hospitals for Crippled Children and DE 07803 (C.N.B.) from the National Institute of Dental Research, National Institutes of Health, Bethesda, MD.     

Atomic force microscopy and modeling of natural elastic fibrillin polymers

A central issue in the understanding of Marfan syndrome deals with the functional architecture of fibrillin-containing microfibrils. Fibrillin-rich microfibrils are long extracellular matrix fibrillar components exhibiting a 50 nm periodic beaded-structure with a width of around 20–25 nm after rotary shadowing and a 10–12 nm diameter when observed in ultra-thin sections. They are composed of fibrillin monomers more or less associated with many other components which are, for the most part, poorly characterized up to date. They are known to be elastic but few data have been accumulated to understand their properties. Atomic force microscopy (AFM) allowed us to morphologically differentiate fibrillin-rich microfibrils from other fibrillar components and to investigate the thin structure of these beaded filaments in their native state. They showed, in AFM, a periodic beaded structure ranging from 50 to 60 nm and a width of about 40 nm. The different sizes of fibrillin-containing microfibrils previously observed after rotary shadowing and in ultra-thin sections was resolved with our technique and is revealed to be 10 nm in diameter. Each beaded microfibril appears to be composed of heterogeneous beads connected by 2–3 arms. An orientation of the microfibrils has been shown, and allows us to propose a complementary model of microfibrillar monomer association.     

Calcium Determines the Supramolecular Organization of Fibrillin-rich Microfibrils

Microfibrils are ubiquitous fibrillin-rich polymers that are thought to provide long-range elasticity to extracellular matrices, including the zonular filaments of mammalian eyes. X-ray diffraction of hydrated bovine zonular filaments demonstrated meridional diffraction peaks indexing on a fundamental axial periodicity (D) of ~56 nm. A Ca²⁺-induced reversible change in the intensities of the meridional Bragg peaks indicated that supramolecular rearrangements occurred in response to altered concentrations of free Ca²⁺. In the presence of Ca²⁺, the dominant diffracting subspecies were microfibrils aligned in an axial 0.33-D stagger. The removal of Ca²⁺ caused an enhanced regularity in molecular spacing of individual microfibrils, and the contribution from microfibrils not involved in staggered arrays became more dominant. Scanning transmission electron microscopy of isolated microfibrils revealed that Ca²⁺ removal or addition caused significant, reversible changes in microfibril mass distribution and periodicity. These results were consistent with evidence from x-ray diffraction. Simulated meridional x-ray diffraction profiles and analyses of isolated Ca²⁺-containing, staggered microfibrillar arrays were used to interpret the effects of Ca²⁺. These observations highlight the importance of Ca²⁺ to microfibrils and microfibrillar arrays in vivo.     

The elastic fiber. I. The separation and partial characterization of its macromolecular components

The two morphologically different constituents of the mature elastic fiber, the central amorphous and the peripheral microfibrillar components, have been separated and partially characterized. A pure preparation of elastic fibers was obtained from fetal bovine ligamentum nuchae by extraction of the homogenized ligament with 5 M guanidine followed by digestion with collagenase. The resultant preparation consisted of elastic fibers which were morphologically identical with those seen in vivo. The microfibrillar components of these elastic fibers were removed either by proteolytic enzymes or by reduction of disulfide bonds with dithioerythritol in 5 M guanidine. The microfibrils solubilized by both methods were rich in polar, hydroxy, and sulfur-containing amino acids and contained less glycine, valine, and proline than the amorphous component of the elastic fiber. In contrast, the amino acid composition of the amorphous component was identical with that previously described for elastin. This component demonstrated selective susceptibility to elastase digestion, but was relatively resistant to the action of other proteolytic enzymes and to reduction. These observations establish that the microfibrils consist of a different connective tissue protein (or proteins) that is neither collagen nor elastin. During embryologic development the microfibrils form an aggregate structure before the amorphous component is secreted. These microfibrils may therefore play a primary role in the morphogenesis of the elastic fiber.     

Electron microscope study of ribonucleoprotein polyparticles and their relation to perichromatin granules

Nuclear ribonucleoprotein particles were studied in the central nervous system of the rat after fixation by perfusion with hypotonic-detergent formaldehyde. Beads-on-a-string structures (polyparticles or chains) formed of 14-nm granules related by a 7-nm thick filament were found. These polyparticles are positively contrasted by a preferential method for ribonucleoproteins and they are sensitive to RNase. Perichromatin granules are loosened by this fixation and their internal structure is clearly depicted. They are composed of a tangled mass of 3-nm thick filaments, but lack 14-nm granules. The internal filaments of the perichromatin granules display frequent continuities with polyparticles. These results show that polyparticles correspond to perichromatin fibrils visualized in nuclei by standard fixation procedures.     

Further cytochemical studies on the perichromatin granules

Summary The perichromatin granules were studied in hepatocytes of experimental rats injected with cycloheximide because the increased number of these nuclear components after such treatment facilitated their cytochemical investigation. Most perichromatin granules were sensitive to the digestion with pepsin and ribonuclease. In contrast, small population of perichromatin granules was resistent to such digestion under conditions which remove known RNA containing components such as ribosomes, nucleolar RNP components and interchromatin granules. The size of these resistent perichromatin granules was reduced and they consisted of filaments the width of which was similar to that of filaments in the chromatin. Moreover, a small population of perichromatin granules was sensitive to the digestion with pepsin and deoxyribonuclease. The size of these granules was only slightly reduced. All these observations indicate that most perichromatin granules contain the RNA and some the DNA. A possibility also exists that the perichromatin granules might contain both RNA and DNA but in various proportions. In addition, partial digestion with pepsin followed by a complete digestion with ribonuclease and deoxyribonuclease removed perichromatin granules as well as other nucleoprotein structures. On the other hand, such digestion facilitated the visualization of the nuclear and cytoplasmic skeleton (matrix) in situ.Dedicated to the memmory of Dr. W. Bernhard     

Ruthenium red staining and tannic acid fixation of dental basement membrane

Summary Ruthenium red staining and tannic acid fixation were used to analyse the fine structure of embryonic mouse dental basement membrane in intact first mandibular molars or in EDTA-isolated dental papillae. Preameloblasts are separated from extracellular matrix proper by a basal lamina that contains regularly arranged proteoglycan granules of about 10 nm in diameter. This distribution pattern is particularly evident in the inner and outer lamina rara of the basal lamina associated with EDTA-isolated dental papillae. The plasmalemma of preameloblasts demonstrates electron dense plaques on the inner leaflet. Ruthenium red positive granules (50 nm in diameter) coat non-striated and striated fibrils of the matrix. Hyaluronidase treatment digested the ruthenium red positive granules. Tannic acid fixation allowed the demonstration of filaments within the lamina rara interna, connecting the lamina densa with plasmalemma of preameloblasts. These observations are discussed in the context of the terminal differentiation of odontoblasts.These studies were supported by INSERM, grant n 537785 and DGRST     

Regulation of UDP‐glucose dehydrogenase is sufficient to modulate hyaluronan production and release,control sulfated GAG synthesis,and promote chondrogenesis

Glycosaminoglycans (GAGs) are critical for extracellular matrix (ECM) integrity in cartilage but mechanisms regulating their synthesis are not defined. UDP‐glucose dehydrogenase (UGDH) catalyses UDP‐glucose oxidation to UDP‐glucuronic acid, an essential monosaccharide in many GAGs. Our previous studies in articular surface (AS) cells from embryonic joints have established pivotal roles for mitogen‐activated protein kinases (MAPK) in synthesis of the unsulfated GAG, hyaluronan (HA). We investigated the functional significance of UGDH in GAG production and chondrogenesis, and determined roles for MEK–ERK and p38^MAPK pathways in regulating UGDH expression and function. Inhibitors of MEK and p38^MAPK reduced UGDH protein in AS cells. Treatment with TGF‐β (archetypal growth factor) increased UGDH expression, sulfated (s)‐GAG/HA release and pericellular matrix formation in a p38^MAPK‐dependent manner. Retroviral overexpression of UGDH augmented HA/sGAG release and pericellular matrix elaboration, which were blocked by inhibiting MEK but not p38^MAPK. UGDH overexpression increased cartilage nodule size in bone marrow culture, promoted chondrogenesis in limb bud micromass culture and selectively suppressed medium HA levels and modified GAG sulfation, as assessed by FACE analysis. Our data provide evidence that: (i) TGF‐β regulates UGDH expression via p38^MAPK to modulate sGAG/HA secretion, (ii) MEK–ERK, but not p38^MAPK facilitates UGDH‐induced HA and sGAG release, and (iii) increased UGDH expression promotes chondrogenesis directly and differential modifies GAG levels and sulfation. These results indicate a more diverse role for UGDH in the support of selective GAG production than previously described. Factors regulating UGDH may provide novel candidates for restoring ECM integrity in degenerative cartilage diseases, such as osteoarthritis.Arthritis Research Campaign. J. Cell. Physiol. 226: 749–761, 2011. © 2010 Wiley‐Liss, Inc.