Localization of hyaluronan in mouse embryos during implantation, gastrulation and organogenesis

Abstract. Hyaluronan was localized in postimplantation mouse embryos using CD44, the principal hyaluronan receptor. The specificity of CD44 receptor-globulin labelling was confirmed using Streptomyces hyaluronidase, anti-chondroitin sulfate antibody, and other receptor globulins. Our major findings are summarized as follows:

• 1. 

  Implantation of the blastocyst into the uterine wall triggers a rapid loss of hyaluronan from the extracellular matrix of decidual cells on the anti-mesometrial side of the uterus.
• 2. 

  Hyaluronan appears early in development in the yolk cavity, and the basement membranes of primitive ectoderm and primitive endoderm.
• 3. 

  During gastrulation, mesodermal cells enter a hyaluronan-rich environment, but lack a pericellular hyaluronan coat themselves.
• 4. 

  In limb bud embryos, hyaluronan is present throughout the cranial mesenchyme, but is generally not present in the branchial bars, somites, or limb buds.
• 5. 

  At mid-gestation, hyaluronan is present in the axial skeleton, craniofacial mesenchyme, endocardial cushions of the heart, smooth muscle of the gastrointestinal tract, and connective tissue throughout the body.

The pattern of hyaluronan expression in the day 13 fetus is nearly identical to the published distribution of transforming growth factor β (TGF β ), suggesting a close functional relationship between these molecules. Together, the results suggest that hyaluronan is involved in the formation of early mesoderm, differentiation of craniofacial mesenchyme, and morphogenesis of the axial skeleton.     

Hyaluronan Disappears Intercellularly and Appears at the Basement Membrane Region during Formation of Embryonic Epithelia

The distribution of tissue hyaluronan has been assessed in the neuraxial region of 8.5 to 10.5 day mouse embryos using a fragment of bovine nasal cartilage proteoglycan that binds specifically to hyaluronan. Hyaluronan is abundant in all mesenchymal tissues, predominantly intercellularly, but markedly diminishes when mesenchymal cells organize into epithelia, as in the formation of somites. Hyaluronan reappears in abundance when epithelia (e.g. sclerotome) disperse into mesenchyme. Hyaluronan is present between cells of early epithelia (e.g. neural plate), but is lost during their subsequent development when it becomes abundant at their basement membrane regions. These results show for the first time changes in hyaluronan distribution during the development of embryonic epithelia. The hyaluronan distribution found is consistent with the functions proposed for hyaluronan in embryonic mesenchyme: intercellular hyaluronan would allow the epithelial cells to move and reduced hyaluronan would allow the cells to associate. The absence of intercellular hyaluronan in later epithelia would allow increased membrane contacts that lead to the formation of intercellular junctions. The restriction of hyaluronan to basement membrane regions in later epithelia further substantiates the suggestion that hyaluronan is a bona fide component of the basal lamina and that it is involved in maintaining epithelial morphology.     

Hyaluronate and CD44 expression patterns in the human placenta throughout pregnancy

Hyaluronan (HA) and CD44 are involved in several processes such as cell migration and differentiation. In the present study, we examined the expression and distribution of both hyaluronan and its cell surface receptor (CD44) in the human placenta, which is a rapidly growing and differentiating organ that plays a fundamental role in fetal life. Hyaluronan was detected by a specific biotinylated binding probe, termed b-PG. In the first half of gestation, HA was strongly expressed in the stroma of the mesenchymal villi which have been previously identified as responsible for the growth and differentation of the villous trees. The other villous types showed an intense staining only in the fetal vessel walls and in the connective tissue closely underlying the trophoblastic cover. In addition, hyaluronan positive staining was also apparent in a restricted rim of villous stroma directly apposed to extravillous cytotrophoblastic cell islands and cell columns. In full term placentas, all villi expressed HA in their stromal tissue with a more homogenous staining than in the first half of gestation. In contrast to hyaluronan, in the first trimester CD44 was restricted to some of the Hofbauer cells which may be able to internalize hyaluronan, thus playing a significant role in its removal in early pregnancy. CD44 was primarily expressed starting from the 16th week of gestation. At the end of pregnancy it was expressed in the various villous types, especially in stem villi. Moreover, the plasma membrane of some extravillous cytotrophoblastic cells in the basal plate and the large majority of the decidual cells showed a positive immunostaining for this receptor. Taken together, these data suggest that HA is strongly involved in early villous morphogenesis, whereas CD44 seem to be play an important role in tissue remodelling later in gestation.     

CD44 expression is developmentally regulated in the mouse lens and increases in the lens epithelium after injury

Hyaluronan is an oligosaccharide found in the pericellular matrix of numerous cell types and hyaluronan-induced signaling is known to facilitate fibrosis and cancer progression in some tissues. Hyaluronan is also commonly instilled into the eye during cataract surgery to protect the corneal endothelium from damage. Despite this, little is known about the distribution of hyaluronan or its receptors in the normal ocular lens. In this study, hyaluronan was found throughout the mouse lens, with apparently higher concentrations in the lens epithelium. CD44, a major cellular receptor for hyaluronan, is expressed predominately in mouse secondary lens fiber cells born from late embryogenesis into adulthood. Surgical removal of lens fiber cells from adult mice resulted in a robust upregulation of CD44 protein, which preceded the upregulation of α-smooth muscle actin expression typically used as a marker of epithelial–mesenchyma transition in this model of lens epithelial cell fibrosis. Mice lacking the CD44 gene had morphologically normal lenses with a response to lens fiber cell removal similar to wildtype, although they exhibited an increase in cell-associated hyaluronan. Overall, these data suggest that lens cells have a hyaluronan-containing pericellular matrix whose structure is partially regulated by CD44. Further, these data indicate that CD44 upregulation in the lens epithelium may be an earlier marker of lens injury responses in the mouse lens than the upregulation of α-smooth muscle actin.     

Internalization of the Hyaluronan Receptor CD44 by Chondrocytes

Chondrocytes express CD44 as a primary receptor for the matrix macromolecule hyaluronan. Hyaluronan is responsible for the retention and organization of proteoglycan within cartilage, and hyaluronan-chondrocyte interactions are important for the assembly and maintenance of the cartilage matrix. Bovine articular chondrocytes were used to study the endocytosis and turnover of CD44 and the effects of receptor occupancy on this turnover. Matrix-intact chondrocytes exhibit approximately a 6% internalization of cell surface CD44 by 4 h. Treatment with Streptomyces hyaluronidase to remove endogenous pericellular matrix increased internalization to approximately 20% of cell surface CD44 at 4 h. This turnover could be partially inhibited by the addition of exogenous hyaluronan to these matrix-depleted chondrocytes. Cell surface biotin-labeled CD44 was internalized by chondrocytes and this internalization was decreased in the presence of hyaluronan. Colocalization of internalized CD44 and fluorescein-labeled hyaluronan in intracellular vesicles correlates with the previous results of receptor-mediated endocytosis pathway for the degradation of hyaluronan by acid hydrolases. Taken together, our results indicate that CD44 is internalized by chondrocytes and that CD44 turnover is modulated by occupancy with hyaluronan.     

Hyaluronan is essential for the expansion of the cranial base growth plates

Exquisite control of chondrocyte function in the zone of hypertrophy results in expansive growth of cartilaginous growth plates, and is a prerequisite for normal skeletal lengthening. We hypothesize that hyaluronan-mediated hydrostatic pressure causes lacunae expansion in the zone of hypertrophy; an important mechanism in cartilaginous growth plate and associated skeletal expansion. The role of hyaluronan and CD44 in this mechanism was studied using organ culture of the bipolar cranial base synchondroses. Hyaluronan was present in the hypertrophic zones, pericellular to the hypertrophic chondrocytes, while no hyaluronan was detected in the resting, proliferating and maturing zones. This localization of hyaluronan was associated with increased lacunae size, suggesting that chondrocytes deposit and retain pericellular hyaluronan as they mature. In comparison, Toluidine Blue staining was associated with the territorial matrix. Hyaluronidase, the hyaluronan-degrading enzyme, and CD44, the receptor for hyaluronan which also participates in the uptake and degradation of hyaluronan, were co-localized within the zone of ossification. This pattern of expression suggests that cells in the early zone of ossification internalize and degrade hyaluronan through a CD44-mediated mechanism. Treatment of the cultured segments with either Streptomyces hyaluronidase or hyaluronan hexasaccharides inhibited lacunae expansion. These observations demonstrate that hyaluronan-mediated mechanisms play an important role in controlling normal skeletal lengthening.     

Temporal expression of CD44 during embryonic chick limb development and modulation of its expression with retinoic acid.

Hyaluronan-cell interactions are initiated co-ordinately with mesenchymal condensation during chondrogenic differentiation in the limb bud. Hyaluronan is responsible for the retention and organization of proteoglycan within the cartilage matrix. Hyaluronan-CD44 binding also retains proteoglycan aggregates to the chondrocyte plasma membrane. A sequence for CD44 protein in chick has recently been reported, but never evaluated in chick chondrocytes. Total RNA was isolated from embryonic chick limb buds, stages 18, 19, 24, 25 and 30. Using semi-quantitative RT-PCR, expression of aggrecan, this chick CD44 orthologue and GAPDH mRNA was analyzed. Aggrecan expression was detected at all stages, but was increased at stage 30. CD44 mRNA was detected at extremely low levels at stage 18 to higher levels in the latter stages. Thus, the temporal expression of CD44 mRNA correlated with the onset of pre-cartilage condensation. The full-length chick chondrocyte CD44 cDNA was obtained following RT-PCR using RNA derived from tibial chondrocytes from stage 37 chick embryos. The nucleotide sequence was used to generate an amino acid sequence and analyses revealed homologies of 44.4% with mouse, 47.8% with bovine and 46.3% with human CD44. Tibial chondrocytes were cultured in the presence or absence of retinoic acid for 36 or 72 h. By RT-PCR, expression of aggrecan and the CD44 mRNA by chick chondrocytes was decreased after retinoic acid treatment, while GAPDH expression showed no change. As expected, control chondrocytes exhibited a round morphology while retinoic acid-treated chondrocytes were elongated. The retinoic acid-treated chondrocytes also exhibited reduced hyaluronan binding. This functional assay indicates a role for a CD44 receptor in matrix retention by chick chondrocytes.     

Differential use of chondroitin sulfate to regulate hyaluronan binding by receptor CD44 in Inflammatory and Interleukin 4-activated Macrophages

CD44 is a cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan and is involved in processes ranging from leukocyte recruitment to wound healing. In the immune system, the binding of hyaluronan to CD44 is tightly regulated, and exposure of human peripheral blood monocytes to inflammatory stimuli increases CD44 expression and induces hyaluronan binding. Here we sought to understand how mouse macrophages regulate hyaluronan binding upon inflammatory and anti-inflammatory stimuli. Mouse bone marrow-derived macrophages stimulated with tumor necrosis factor α or lipopolysaccharide and interferon-γ (LPS/IFNγ) induced hyaluronan binding by up-regulating CD44 and down-regulating chondroitin sulfation on CD44. Hyaluronan binding was induced to a lesser extent in interleukin-4 (IL-4)-activated macrophages despite increased CD44 expression, and this was attributable to increased chondroitin sulfation on CD44, as treatment with β-d-xyloside to prevent chondroitin sulfate addition significantly enhanced hyaluronan binding. These changes in the chondroitin sulfation of CD44 were associated with changes in mRNA expression of two chondroitin sulfotransferases, CHST3 and CHST7, which were decreased in LPS/IFNγ-stimulated macrophages and increased in IL-4-stimulated macrophages. Thus, inflammatory and anti-inflammatory stimuli differentially regulate the chondroitin sulfation of CD44, which is a dynamic physiological regulator of hyaluronan binding by CD44 in mouse macrophages.     

Hyaluronan synthesis induces microvillus-like cell surface protrusions

Hyaluronan synthases (HASs) are plasma membrane enzymes that simultaneously elongate, bind, and extrude the growing hyaluronan chain directly into extracellular space. In cells transfected with green fluorescent protein (GFP)-tagged Has3, the dorsal surface was decorated by up to 150 slender, 3-20-microm-long microvillus-type plasma membrane protrusions, which also contained filamentous actin, the hyaluronan receptor CD44, and lipid raft microdomains. Enzymatic activity of HAS was required for the growth of the microvilli, which were not present in cells transfected with other GFP proteins or inactive GFP-Has3 mutants or in cells incubated with exogenous soluble hyaluronan. The microvilli induced by HAS3 were gradually withered by introduction of an inhibitor of hyaluronan synthesis and rapidly retracted by hyaluronidase digestion, whereas they were not affected by competition with hyaluronan oligosaccharides and disruption of the CD44 gene, suggesting independence of hyaluronan receptors. The data bring out the novel concept that the glycocalyx created by dense arrays of hyaluronan chains, tethered to HAS during biosynthesis, can induce and maintain prominent microvilli.     

Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44

CD44 on lymphocytes binding to its carbohydrate ligand hyaluronan can mediate primary adhesion (rolling interactions) of lymphocytes on vascular endothelial cells. This adhesion pathway is utilized in the extravasation of activated T cells from the blood into sites of inflammation and therefore influences patterns of lymphocyte homing and inflammation. Hyaluronan is a glycosaminoglycan found in the extracellular matrix and is involved in a number of biological processes. We have shown that the expression of hyaluronan on the surface of endothelial cells is inducible by proinflammatory cytokines. However, the manner through which hyaluronan is anchored to the endothelial cell surface so that it can resist shear forces and the mechanism of the regulation of the level of hyaluronan on the cell surface has not been investigated. In order to characterize potential hyaluronan receptors on endothelial cells, we performed analyses of cell surface staining by flow cytometry on intact endothelial cells and ligand blotting assays using membrane fractions. Hyaluronan binding activity was detected as a major species corresponding to the size of CD44, and this was confirmed to be the same by Western blotting and immunoprecipitation. Moreover, alterations in the surface level of hyaluronan after tumor necrosis factor-alpha stimulation is regulated primarily by changes in the cell surface levels of the hyaluronan-binding form of CD44. In laminar flow assays, lymphoid cells specifically roll on hyaluronan anchored by purified CD44 coated on glass tubes, indicating that the avidity of the endothelial CD44/hyaluronan interaction is sufficient to support rolling adhesions under conditions mimicking physiologic shear forces. Together these studies show that CD44 serves to anchor hyaluronan on endothelial cell surfaces, that activation of CD44 is a major regulator of endothelial surface hyaluronan expression, and that the non-covalent interaction between CD44 and hyaluronan is sufficient to provide resistance to shear under physiologic conditions and thereby support the initial steps of lymphocyte extravasation.     

Hyaluronan enters keratinocytes by a novel endocytic route for catabolism.

Hyaluronan synthesized in the epidermis has an exceptionally short half-life, indicative of its catabolism by epidermal keratinocytes. An intracellular pool of endogenously synthesized hyaluronan, from 1 to 20 fg/cell, inversely related to cell density, was observed in cultured rat epidermal keratinocytes. More than 80% of the intracellular hyaluronan was small (<90 kDa). Approximately 25% of newly synthesized hyaluronan was endocytosed by the keratinocytes and had a half-life of 2-3 h. A biotinylated aggrecan G(1) domain/link protein probe demonstrated hyaluronan in small vesicles of approximately 100 nm diameter close to the plasma membrane, and in large vesicles and multivesicular bodies up to 1300 nm diameter around the nucleus. Hyaluronan did not co-localize with markers of lysosomes. However, inhibition of lysosomal acidification with NH(4)Cl or chloroquine, or treating the cells with the hyaluronidase inhibitor apigenin increased intracellular hyaluronan staining, suggesting that it resided in prelysosomal endosomes. Competitive displacement of hyaluronan from surface receptors using hyaluronan decasaccharides, resulted in a rapid disappearance of this endosomal hyaluronan (t(12) approximately 5 min), indicating its transitory nature. The ultrastructure of the hyaluronan-containing vesicles, co-localization with marker proteins for different vesicle types, and application of specific uptake inhibitors demonstrated that the formation of hyaluronan-containing vesicles did not involve clathrin-coated pits or caveolae. Treatment of rat epidermal keratinocytes with the OX50 monoclonal antibody against the hyaluronan receptor CD44 increased endosomal hyaluronan. However, no CD44-hyaluronan co-localization was observed intracellularly unless endosomal trafficking was retarded by monensin, or cultivation at 20 degrees C, suggesting CD44 recycling. Rat epidermal keratinocytes thus internalize a large proportion of their newly synthesized hyaluronan into non-clathrin-coated endosomes in a receptor mediated way, and rapidly transport it to slower degradation in the endosomal/lysosomal system.     

Hyaluronidases and CD44 undergo differential modulation during chondrogenesis

Hyaluronan, a high-molecular-weight glycosaminoglycan of cartilage, is deposited directly into the extracellular space by hyaluronan synthases, while hyaluronan catabolism is mediated by the hyaluronidases. An in vitro cell culture system has been established in which human dermal fibroblasts are induced to undergo chondrogenesis. Here, we describe the differential modulation of the hyaluronidases and the up-regulation of the hyaluronan receptor, CD44, during such chondrogenesis. Dermal fibroblasts, plated in micromass cultures in the presence of lactic acid and staurosporine for 24 h, were then placed in serum-free, chemically defined medium. At 3 days, RNA was extracted and RT-PCR performed using primers for the hyaluronidase genes. Marked increase in HYAL1 expression was observed, with only moderate increases occurring in HYAL2 and HYAL3. No expression of HYAL4 and PH-20, the sperm-associated hyaluronidase, was detected. RNA levels correlated well with changes in hyaluronidase enzyme activity. Finally, greater expression and staining for the hyaluronan receptor, CD44s, the standard form, were detected. Differential expression of the somatic hyaluronidases and CD44-mediated hyaluronan turnover play a critical role in cartilage development from mesenchymal precursors.     

Hyaluronan oligosaccharides perturb lymphocyte slow rolling on brain vascular endothelial cells: Implications for inflammatory demyelinating disease

Inflammatory demyelinating diseases like multiple sclerosis are characterized by mononuclear cell infiltration into the central nervous system. The glycosaminoglycan hyaluronan and its receptor, CD44, are implicated in the initiation and progression of a mouse model of multiple sclerosis, experimental autoimmune encephalomyelitis (EAE). Digestion of hyaluronan tethered to brain vascular endothelial cells by a hyaluronidase blocks the slow rolling of lymphocytes along activated brain vascular endothelial cells and delays the onset of EAE. These effects could be due to the elimination of hyaluronan or the generation of hyaluronan digestion products that influence lymphocytes or endothelial cells. Here, we found that hyaluronan dodecasaccharides impaired activated lymphocyte slow rolling on brain vascular endothelial cells when applied to lymphocytes but not to the endothelial cells. The effects of hyaluronan dodecasaccharides on lymphocyte rolling were independent of CD44 and a receptor for degraded hyaluronan, Toll-like receptor-4. Subcutaneous injection of hyaluronan dodecasaccharides or tetrasaccharides delayed the onset of EAE in a manner similar to subcutaneous injection of hyaluronidase. Hyaluronan oligosaccharides can therefore act directly on lymphocytes to modulate the onset of inflammatory demyelinating disease.     

Chronic UVR Causes Increased Immunostaining of CD44 and Accumulation of Hyaluronan in Mouse Epidermis

Chronic intense UV radiation is the main cause of epidermal tumors. Because hyaluronan (HA), a large extracellular polysaccharide, is known to promote malignant growth, hyaluronan expression was studied in a model in which long-term UV radiation (UVR) induces epidermal tumors. Mouse back skin was exposed three times a week for 10.5 months to UVR corresponding to one minimal erythema dose, processed for histology, and stained for hyaluronan and the hyaluronan receptor CD44. This exposure protocol caused epidermal hyperplasia in most of the animals; tumors, mainly squamous cell carcinomas (SCCs), were found in ~20% of the animals. Specimens exposed to UVR showed increased hyaluronan and CD44 staining throughout the epidermal tissue. In hyperplastic areas, hyaluronan and CD44 stainings correlated positively with the degree of hyperplasia. Well-differentiated SCCs showed increased hyaluronan and CD44 staining intensities, whereas poorly differentiated tumors and dysplastic epidermis showed areas where HA and CD44 were locally reduced. The findings indicate that HA and CD44 increase in epidermal keratinocytes in the premalignant hyperplasia induced by UV irradiation and stay elevated in dysplasia and SCC, suggesting that the accumulation of hyaluronan and CD44 is an early marker for malignant transformation and may be a prerequisite for tumor formation.     

Hyaluronan.

Hyaluronan (hyaluronic acid) is a high-molecular-mass polysaccharide found in the extracellular matrix, especially of soft connective tissues. It is synthesized in the plasma membrane of fibroblasts and other cells by addition of sugars to the reducing end of the polymer, whereas the nonreducing end protrudes into the pericellular space. The polysaccharide is catabolized locally or carried by lymph to lymph nodes or the general circulation, from where it is cleared by the endothelial cells of the liver sinusoids. The overall turnover rate is surprisingly rapid for a connective tissue matrix component (t1/2 0.5 to a few days). Hyaluronan has been assigned various physiological functions in the intercellular matrix, e.g., in water and plasma protein homeostasis. Hyaluronan production increases in proliferating cells and the polymer may play a role in mitosis. Extensive hyaluronidase-sensitive coats have been identified around mesenchymal cells. They are either anchored firmly in the plasma membrane or bound via hyaluronan-specific binding proteins (receptors). Such receptors have now been identified on many different cells, e.g., the lymphocyte homing receptor CD 44. Interaction between a hyaluronan receptor and extracellular polysaccharide has been connected with locomotion and cell migration. Hyaluronan seems to play an important role during development and differentiation and has other cell regulatory activities. Hyaluronan has also been recognized in clinical medicine. A concentrated solution of hyaluronan (10 mg/ml) has, through its tissue protective and rheological properties, become a device in ophthalmic surgery. Analysis of serum hyaluronan is promising in the diagnosis of liver disease and various inflammatory conditions, e.g., rheumatoid arthritis. Interstitial edema caused by accumulation of hyaluronan may cause dysfunction in various organs.     

Regulation of hyaluronan binding by F-actin and colocalization of CD44 and phosphorylated ezrin/radixin/moesin (ERM) proteins in myeloid cells

Proinflammatory cytokines such as TNF-alpha up-regulate the expression of the cell adhesion molecule, CD44, and induce hyaluronan (HA) binding in peripheral blood monocytes (PBM). Here we show that in PBM, TNF-alpha induced cytoskeletal rearrangement, increased threonine phosphorylation of ERM proteins, and induced the redistribution and colocalization of phospho-ERM proteins (P-ERM) with CD44. In the myeloid progenitor cell line, KG1a, hyaluronan binding occurred in the pseudopod where CD44, P-ERM, and F-actin were highly localized. Hyaluronan binding correlated with high expression of both CD44 and P-ERM clustered in a single pseudopod. Disruption of polymerized actin reduced hyaluronan binding in both PBM and KG1a cells and abolished CD44 clustering and the pseudopod in KG1a cells. The pseudopod was not required for the clustering of CD44, the colocalization with P-ERM, or hyaluronan binding. However, treatment with a kinase inhibitor abolished ERM phosphorylation and reduced hyaluronan binding. Furthermore, expression of CD44 lacking the putative ERM binding site resulted in reduced hyaluronan binding. Taken together, these data suggest that CD44-mediated hyaluronan binding in human myeloid cells is regulated by P-ERM and the actin cytoskeleton.     

CD44-mediated uptake and degradation of hyaluronan.

Hyaluronan turnover occurs systemically from the lymph and serum as well as locally by the same cells responsible for its synthesis. Local turnover involves receptor-mediated uptake and delivery to lysosomes. Of the many hyaluronan binding proteins/receptors known, the participation of CD44 in the internalization of hyaluronan has been best characterized. Some fraction of the hyaluronan bound to CD44 becomes internalized and delivered to lysosomes by a mechanism that is not dependent on clatherin, caveolae or pinocytosis. In cells such as chondrocytes, anabolic and catabolic cytokines can alter the activity of CD44 toward hyaluronan internalization. However, the mechanism of cellular regulation remains unclear. Regulation may involve the participation of alternatively spliced isoforms of CD44, changes in CD44 phosphorylation, changes in cytoskeletal binding proteins or, the activity or extracellular proteolytic activity.     

Ontogeny of an Extracellular Matrix Component of Sea Urchins and its Role in Morphogenesis

A monoclonal antibody, Sp14, recognizes fibers that form a complex meshwork within the blastocoel of embryos of the sea urchin, Strongylocentrotus purpuratus . The fibers first appear as the blastocoel begins to form and increase in density throughout development. Ultrastructural localizations using the immunoperoxidase method show bundles of 20 nm fibers that are continuous with the basal lamina and have an indistinct axial periodicity. Embryos treated with tunicamycin, β-D-xylopyranoside, β-aminoproprionitrile, proline analogues, or deprived of sulfate all form immunoreactive fibers although in some treatments the pattern formed is abnormal. Immunoreactivity of extracted fibers is not affected by digestion with chondroitinase ABC, hyaluronidase, collagenase or heparinase. However, proteinase K readily destroys immunoreactivity. Fibers will form in cultures of micromeres or mesenchyme 24 to 48 hr after plating with or without horse serum. In embryos in which the blastocoelar matrix has been altered by injection with Sp14, there is inhibition of the release of secondary mesenchyme from the tip of the archenteron and in some embryos supernumerary skeletal elements are formed. It is proposed that Sp14 recognizes a component of the blastocoelar extracellular matrix that is required for the migration of mesenchyme.     

Hyaluronan Controls the Deposition of Fibronectin and Collagen and Modulates TGF-β1 Induction of Lung Myofibroblasts

The contribution of hyaluronan-dependent pericellular matrix to TGF-β1-driven induction and maintenance of myofibroblasts is not understood. Hyaluronan is an extracellular matrix (ECM) glycosaminoglycan important in cell adhesion, proliferation and migration, and is implicated in myofibroblast formation and maintenance. Reduced turnover of hyaluronan has been linked to differentiation of myofibroblasts and potentiation of lung fibrosis. Fibronectin is a fibril forming adhesive glycoprotein that is also upregulated following induction with TGF-β1. Although they are known to bind each other, the interplay between hyaluronan and fibronectin in the pericellular matrix during myofibroblast induction and matrix assembly is not clear. This study addresses the role of hyaluronan and its interaction with fibrillar matrix components during myofibroblast formation. Hyaluronan and fibronectin were increased and co-localized in the ECM following myofibroblast induction by TGF-β1. Inhibition of hyaluronan synthesis in TGF-β1-induced lung myofibroblasts over a 4 day period with 4-methyl umbelliferone (4-MU) further enhanced myofibroblast morphology, caused increased deposition of fibronectin and type I collagen in the ECM, and increased expression of alpha-smooth muscle actin and hyaluronan synthase 2 (HAS2) mRNA. Hyaluronan oligosaccharides or hyaluronidase treatment, which more effectively disrupted the pericellular matrix, had similar effects. CD44 and β1 integrins co-localized in the cell membrane and along some stress fibers. However, CD44 and hyaluronan were specifically excluded from focal adhesions, and associated primarily with cortical actin. Time-lapse imaging of the immediate effects of hyaluronidase digestion showed that hyaluronan matrix primarily mediates attachment of membrane and cortical actin between focal contacts, suggesting that surface adhesion through hyaluronan and CD44 is distinct from focal adhesion through β1 integrins and fibronectin. Fluorescein-labeled hyaluronan bound regularly along fibronectin fibers and co-localized more with β1 integrin and less with CD44. Therefore, the hyaluronan matrix can interfere with the assembly of fibrillar ECM components, and this interplay regulates the degree of myofibroblast formation. These data also suggest that adhesion through hyaluronan matrix impacts cytoskeletal organization, and is potentially part of a clutch mechanism that regulates stick and slip of myofibroblasts by affecting the adhesion to and organization of fibronectin and collagen.     

Hyaluronan in human and rat muscle spindles

 Previous data on the composition of the periaxial fluid of muscle spindles have relied on indirect histochemical methods. We used a biotinylated hyaluronan-binding protein as a specific probe for the detection of hyaluronan in sections of human and rat limb muscles. Hyaluronan was identified in the axial and periaxial space of the muscle spindles as well as in the endoneurium and in the space in between individual axons. Hyaluronan was also present in the innermost layer of the spindle capsule in the A region and in all layers of the capsule in the B region. Accepted: 12 February 1998