Vertebral Cartilage Research Articles

Two domains in vertebral development: antagonistic regulation by SHH and BMP4 proteins.

It has previously been shown that the notochord grafted laterally to the neural tube enhances the differentiation of the vertebral cartilage at the expense of the derivatives of the dermomyotome. In contrast, the dorsomedial graft of a notochord inhibits cartilage differentiation of the dorsal part of the vertebra carrying the spinous process. Cartilage differentiation is preceded by the expression of transcription factors of the Pax family (Pax1/Pax9) in the ventrolateral domain and of the Msx family in the dorsal domain. The proliferation and differentiation of Msx-expressing cells in the dorsal precartilaginous domain of the vertebra are stimulated by BMP4, which acts upstream of Msx genes. It has previously been shown that the SHH protein arising from the notochord (and floor plate) is necessary for the survival and further development of Pax1/Pax9-expressing sclerotomal cells. We show here that SHH acts antagonistically to BMP4. SHH-producing cells grafted dorsally to the neural tube at E2 inhibit expression of Bmp4 and Msx genes and also inhibits the differentiation of the spinous process. We present a model that accounts for cartilage differentiation in the vertebra.

The ultrastructure of chondrocytes in the cartilage of Sepia officinalis and Octopus vulgaris (Mollusca, Cephalopoda)

Thin sections of cartilage from the chondrocranium of cuttle fish and octopus were examined using the transmission electron microscope. It was found that cephalopod chondrocytes differed considerably from the chondrocytes of vertebrate cartilage; in particular they possessed many long and ramifying cytoplasmic processes and had an ultrastructure typical of protein-secreting cells. They did not, however, contain secretory granules; while vesicles and rough endoplasmic reticulum cisternae seemed to open directly to the cell surface. The cell body and processes contained cytoskeletal structures: microtubules were easily recognized, but intermediate and thin filaments were difficult to make out as they were frequently clumped into bundles. Some chondrocytes contained conspicuous accumulations of hemocyanin. The cytoplasmic processes possessed intercellular contacts similar to gap junctions. Also present on processes and the cell body were cell-extracellular matrix focal adhesions. The chondrocytes were not polarized or arranged in any preferential spatial order, however, with their processes they formed a three-dimensional network throughout the cartilage tissue. Ultrastructural findings are discussed in relation to the singular morphofunctional characteristics of cephalopod cartilage which shares features with both the cartilage and bone of vertebrates.

Use of rotary shadowing electron microscopy to investigate the collagen fibrils in the extracellular matrix of cuttle-fish ( Sepia officinalis) and chicken cartilage

Collagen fibrils isolated from sternal cartilage of chick embryo and chondrocranium of cuttle-fish ( Sepia officinalis) were examined with the electron microscope after rotary shadowing. The aim was to determine whether collagen fibrils from S. officinalis cartilage contained collagen molecules similar to the type IX collagen of vertebrate cartilage. Cartilage from both sources presented a highly variable appearance and only occasionally did preparations contain fibrils having the structure described by Vaughan et al. (1988) for vertebrate cartilage. Subsequent electron microscope investigation of collagen samples during the various stages of fibril preparation showed that the method did not yield reproducible results, and that it altered the morphology of the isolated structures. It was not, therefore, possible to confirm the hypothesis that collagen molecules with a morphology similar to that of type IX vertebrate collagen are a component of the extracellular matrix of cephalopod cartilage.

Possible roles for Wnt genes in growth and axial patterning during regeneration of the tail in urodele amphibians.

Urodele amphibians are nearly the only adult vertebrates able to regenerate their missing or amputated tail. An interesting aspect of this biological model lies in the ability of regenerates to differentiate the spinal cord (SC), the vertebral cartilage, and muscles. The main questions addressed in this study concern the possible roles of Wnt genes in these regenerative processes. We have previously reported the expression pattern of a Pleurodeles Waltl wnt-10a gene (Pwnt-10a) in tail blastema (Caubit et al. [1997] Dev. Dyn. 208:139-148). We report here the cloning and tissue distribution of three additional Wnt genes (Pwnt-5a, Pwnt-5b, and Pwnt-7a) in adult and regenerating tail tissues and in the central nervous system (CNS) of adult newt. In adult and regenerating tails, Pwnt-5a and Pwnt-5b transcripts exhibit a graded distribution along the antero-posterior (A-P) axis, the maximal accumulation of these transcripts being detected in the mesenchyme within the subectodermal apical region of the normal tail and blastema. In contrast to Pwnt-5a and Pwnt-5b, Pwnt-7a is expressed in adult normal tail skin and in the epidermis of the regenerating tail. In the adult CNS, Pwnt-5a, Pwnt-5b, Pwnt-7a, and Pwnt-10a genes are expressed in sharp overlapping but not identical domains along the A-P axis. The sustained expression of Wnt genes in the adult newt and the spatial distribution of transcripts in adult and regenerating tail tissues suggest roles of these genes in continuous growth capacities in the urodeles and may explain the ability for CNS and tail regeneration.

Use of Calcein as a Fluorescent Marker for Elasmobranch Vertebral Cartilage

Abstract The effectiveness of calcein as a fluorescent marker of elasmobranch vertebrae was examined in the nurse shark Ginglvmostotna cirratum. Intramuscular injection of calcein at a standard dosage of 25 mg/kg body weight (BW) produced distinct fluorescent marks in nurse shark vertebrae; however, it also induced physiological stress and mortality. Lower dosages of 10 and 5 mg/kg BW also produced chemical marks suitable for age validation but were not associated with animal death. These data suggest that calcein dosage levels recommended for teleost age validation may be toxic to elasmobranchs. Future studies should focus on the toxicity of lower-dosage calcein injections.

The role of bone morphogenetic proteins in vertebral development.

This study first shows a striking parallel between the expression patterns of the Bmp4, Msx1 and Msx2 genes in the lateral ridges of the neural plate before neural tube closure and later on, in the dorsal neural tube and superficial midline ectoderm. We have previously shown that the spinous process of the vertebra is formed from Msx1- and 2-expressing mesenchyme and that the dorsal neural tube can induce the differentiation of subcutaneous cartilage from the somitic mesenchyme. We show here that mouse BMP4- or human BMP2-producing cells grafted dorsally to the neural tube at E2 or E3 increase considerably the amount of Msx-expressing mesenchymal cells which are normally recruited from the somite to form the spinous process of the vertebra. Later on, the dorsal part of the vertebra is enlarged, resulting in vertebral fusion and, in some cases (e.g. grafts made at E3), in the formation of a 'giant' spinous process-like structure dorsally. In strong contrast, BMP-producing cells grafted laterally to the neural tube at E2 exerted a negative effect on the expression of Pax1 and Pax3 genes in the somitic mesenchyme, which then turned on Msx genes. Moreover, sclerotomal cell growth and differentiation into cartilage were then inhibited. Dorsalization of the neural tube, manifested by expression of Msx and Pax3 genes in the basal plate contacting the BMP-producing cells, was also observed. In conclusion, this study demonstrates that differentiation of the ventrolateral and dorsal parts of the vertebral cartilage is controlled by different molecular mechanisms. The former develops under the influence of signals arising from the floor plate-notochord complex. These signals inhibit the development of dorsal subcutaneous cartilage forming the spinous process, which requires the influence of BMP4 to differentiate.

Chicken Pax-1 gene: structure and expression during embryonic somite development

Recent mouse genetic studies have implicated Pax-1, a paired-box-containing gene, in sclerotomal differentiation and vertebral body formation. To investigate Pax-1 function in somitic sclerotomal differentiation in the chick embryo, we have cloned the chicken Pax-1 gene, and its full length cDNA, and characterized its temporal and spatial expression pattern during somite development. Sequence analysis shows that chicken Pax-1 is highly homologous to murine and human Pax-1 genes with respect to the putative DNA-binding paired-box domain and the octapeptide domain. Northern analysis using probes derived from the paired-box domain and a unique non-paired box sequence of chicken Pax-1 detected 2-kb mRNA transcript. The expression profiles of Pax-1 were examined by in situ hybridization and Northern analysis. The first detectable expression of Pax-1 is seen in the most caudal epithelial somite. As the somite matures, Pax-1 expression takes on a medial distribution, thus corresponding to but preceding the emergence of the sclerotome. In the more mature, rostral somites (stage V and older), Pax-1 expression is found to be progressively localized first to the ventral-medial regions, and then to the caudal-ventral-medial quadrant of the mature somite. This pattern strongly supports the notion that Pax-1 expression is involved in somitogenesis and sclerotomal differentiation, and that it is subsequently a characteristic of the caudal half of the sclerotome, the presumptive precursor of vertebral cartilage. Northern analysis substantiated this expression profile and further revealed that the level of somitic Pax-1 expression increases as a function of embryonic development. Finally, we subjected chicken embryos to controlled heat shock treatment to perturb somite formation and segmentation. The pattern of Pax-1 expression in the anomalous somitic structures generated by controlled heat shock further supports a functional role for Pax-1 in somite development.

SPC4, SPC6, and the novel protease SPC7 are coexpressed with bone morphogenetic proteins at distinct sites during embryogenesis.

In the present study, we screened for subtilisin-like proprotein convertases (SPCs) that potentially regulate the activation of known growth factors during embryonic development. We isolated a novel protease, SPC7, as well as several known SPCs. SPC7, like SPC1, is expressed ubiquitously throughout development. In contrast, SPC4 and SPC6 exhibit dynamic expression patterns. SPC4 transcripts were initially detected in the granulosa cells of secondary follicles. Shortly after implantation, SPC4 transcripts are localized to extraembryonic cell populations, and at later stages are detected in discrete tissues including the primitive gut, heart, neural tube, and limb buds. Within the limb buds, SPC4 mRNA is most abundant in the apical ectodermal ridge (AER). At later stages of limb development, SPC4 mRNA is strongly expressed in cartilage and in the interdigital mesenchyme. In contrast, high SPC6 mRNA levels are detected in somites, the dorsal surface ectoderm, and in vertebral cartilage primordia. In limb buds, SPC6 is strongly expressed in the AER, and at later stages in dorsal mesenchyme. A comparison of these expression patterns with those of several bone morphogenetic proteins (BMPs) indicates that processing of these growth factors may be limited by the local availability of SPCs.

Proteoglycans from the vertebral cartilage of the clearnose skate, Raja eglanteria: Inhibition of hydroxyapatite formation

Studies were undertaken to examine the importance of organic proteoglycan matrices in the calcification of elasmobranch vertebral cartilage. Proteoglycans were extracted from the vertebral cartilage of the clearnose skate, Raja eglanteria Bosc, with a 3M guanidine hydrochloride/10% EDTA solution. Proteoglycan solutions (12 μg ml(-1)) were effective inhibitors of hydroxyapatite formation in vitro from high concentration calcium phosphate solutions. Inhibition of crystal formation appears to occur through the restriction of phase transformation from a calcium phosphate precursor to hydroxyapatite crystals. The concentration and/or degradation of proteoglycans in elasmobranch vertebral cartilage may have a physiological role in the production of alternating mineral-rich and mineral-poor growth zones, currently used in ageing studies.

Co-expression of collagen types II and X mRNAs in newly formed hypertrophic chondrocytes of the embryonic chick vertebral body demonstrated by double-fluorescence in situ hybridization.

Collagen types II and X mRNAs have been demonstrated simultaneously in newly formed hypertrophic chondrocytes of embryonic chick vertebral cartilage using a double-fluorescence in situ hybridization technique. Digoxigenin- and biotin-labelled type-specific collagen II and X cDNA probes were used. In the embryonic chick vertebra at stage 45, two different fluorescence signals (Fluorescein isothiocyanate and Rhodamine)--one for collagen type II mRNA, the other for type X mRNA--showed differential distribution of the two collagen mRNAs in the proliferating and hypertrophic chondrocyte zones. Several layers of newly formed hypertrophic chondrocytes expressing both collagen types II and X genes were identified in the same section as two different fluorescent colour signals. Low levels of fluorescent signals for collagen type II mRNA were also detected in the hypertrophic chondrocyte zone. Cytological identification of maturing chondrocyte phenotypes, expressing collagen mRNAs, is easier in sections processed by non-radioactive in situ hybridization than in those subjected to radioactive in situ hybridization using 3H-labelled cDNA probes. This study demonstrates that double-fluorescence in situ hybridization is a useful tool for simultaneously detecting the expression of two collagen genes in the same chondrocyte population.

Isolation and sequence of the vitamin K-dependent matrix Gla protein from the calcified cartilage of the soupfin shark

High levels of the vitamin K-dependent matrix Gla protein (MGP) have been found in the calcified costal cartilage of the cow and the calcified vertebral cartilage of the soupfin shark (Galeorhinus galeus). In both species, MGP accounts for 35-40% of the total protein in the acid demineralization extract of calcified cartilage, and the mineral content of calcified cartilage is comparable to that of bovine cortical bone. Shark and bovine MGP are both nearly insoluble in neutral buffers, a conserved property that indicates that self-aggregation could be important to the as yet unknown function of MGP. The complete amino acid sequence of shark MGP was determined to compare the structure of the elasmobranch protein to the several currently known mammalian MGP sequences. Shark MGP contains 4 residues of the vitamin K-dependent amino acid gamma-carboxyglutamic acid in its 102 residue sequence and has a calculated molecular weight = 12,770 daltons. The first 76 residues of shark MGP are homologous in sequence to mammalian MGPs, with 37% sequence identity, but the C-terminal 23 residues of the shark protein have no counterpart in the mammalian MGPs. This C-terminal segment of shark MGP contains 8 basic residues and no acidic residues. Among the features conserved in shark MGP, in all mammalian MGPs, and in all other currently known vitamin K-dependent mammalian proteins are a 15-residue region of sequence homology that has been shown to function as the gamma-carboxylase recognition sequence and an invariant sequence of unknown function, Gla-Xaa-Xaa-Xaa-Gla-Xaa-Cys.

Mice homozygous for a targeted disruption of Hoxd-3 (Hox-4.1) exhibit anterior transformations of the first and second cervical vertebrae, the atlas and the axis.

Gene targeting in embryo-derived stem (ES) cells was used to generate mice with a disruption in the homeobox-containing gene Hoxd-3 (Hox-4.1). Mice homozygous for this mutation show a radically remodeled craniocervical joint. The anterior arch of the atlas is transformed to an extension of the basioccipital bone of the skull. The lateral masses of the atlas also assume a morphology more closely resembling the exoccipitals and, to a variable extent, fuse with the exoccipitals. Formation of the second cervical vertebra, the axis, is also affected. The dens and the superior facets are deleted, and the axis shows 'atlas-like' characteristics. An unexpected observation is that different parts of the same vertebra are differentially affected by the loss of Hoxd-3 function. Some parts are deleted, others are homeotically transformed to more anterior structures. These observations suggest that one role of Hox genes may be to differentially control the proliferation rates of the mesenchymal condensations that give rise to the vertebral cartilages. Within the mouse Hox complex, paralogous genes not only encode very similar proteins but also often exhibit very similar expression patterns. Therefore, it has been postulated that paralogous Hox genes would perform similar roles. Surprisingly, however, no tissues or structures are affected in common by mutations in the two paralogous genes, Hoxa-3 and Hoxd-3.

Developmental Plasticity of the Prospective Dermatome and the Prospective Sclerotome Region of an Avian Somite: (somite/dermomyotome/sclerotome/determination/quail-chick chimera).

The ventro-medial wall of a somite gives rise to the sclerotome and then to cartilaginous axial skeleton, while the dorso-lateral wall differentiates into the dermomyotome to form dermal mesenchyme and muscle. Although previous studies suggested pluri-potency of somite cell differentiation, apparent pluri-potency may be the result of migration of predetermined cells. To investigate whether the developmental fate of any region is determined, I isolated fragments of a region of a quail somite and transplanted them into chick embryos. When a fragment of the ventral wall of a quail somite, the prospective sclerotome, was transplanted into a chick embryo between the ectoderm and a newly formed somite, the transplanted quail cells were shown to form myotome and mesenchyme in 4-day chimera embryos and to form muscle and dermal tissue in 9-day chimeras. On the other hand, when a fragment of the dorsal wall of a quail somite, the prospective dermomyotome, was transplanted into a chick embryo between the neural tube and a newly formed somite, the graft gave rise to mesenchyme around the neural tube and notochord and then to vertebral cartilage. Thus the developmental fate of a region of a somite was shown not to be determined at the time of somite segmentation, confirming previous observations.

A role for Quox-8 in the establishment of the dorsoventral pattern during vertebrate development.

We have been interested in the possible involvement of the gene Quox-8, a member of the Hox-7/msh family of homeobox-containing genes in the patterning of the neural tube and the somitic-derived mesoderm. We demonstrate that in the embryonic day 2-6 avian embryo, the expression of Quox-8 in the roof plate depends upon (i) the normal dorsoventral orientation of the neural primordium and (ii) interactions between the dorsal neural tube and the superficial ectoderm. We also show that Quox-8 is expressed in the dorsal mesenchyme located between the neural tube and the superficial ectoderm that yields the spinous processes of the vertebrae. Experimental inhibition of Quox-8 expression in these areas of the dorsal mesenchyme results in the failure of its differentiation into vertebral cartilage. We conclude, therefore, that this gene is involved in the patterning of vertebral structures through a cascade of tissue interactions primarily occurring between the dorsal neural tube and the overlying ectoderm.

Remodeling of the rough endoplasmic reticulum during stimulation of procollagen secretion by ascorbic acid in cultured chondrocytes. A biochemical and morphological study.

The cellular and molecular mechanisms regulating the reversible accumulation of nonhelical, underhydroxylated procollagen in the rough endoplasmic reticulum (RER) remain obscure. To clarify these mechanisms, we isolated chondrocytes from chick vertebral cartilage and kept them in scorbutic monolayer cultures. By Day 9 of culture, the chondrocytes had accumulated a large amount of underhydroxylated Type II procollagen in their RER. Within 1 h of ascorbate treatment, the accumulated procollagen was hydroxylated; this was accompanied by a slight stimulation of procollagen secretion and was followed by a marked stimulation starting between 2 and 3 h of treatment. Secretion of the accumulated procollagen was completed by about 24 h of treatment. Strikingly, the marked stimulation of procollagen secretion at 2-3 h of treatment was associated with marked remodeling of the RER. This organelle came to consist of a few, unusually large cisternae ("sacs") and many flat cisternae while the RER in untreated cells consisted of uniform, oval cisternae. The RER remodeling was accompanied by a comparable redistribution of the accumulated Type II procollagen stored in it. The RER sacs and flat cisternae invariably communicated directly and were still detectable by 8 h but not by 24 h of treatment. RER remodeling and procollagen redistribution also occurred in untreated chondrocytes that had been shifted to 23 degrees C for 2-3 h. Together, the data indicate that folding of the accumulated procollagen molecules into their normal helical configuration is followed by procollagen redistribution within, and remodeling of, the RER. These processes may have a role in stimulating procollagen export from the RER and secretion.

Intracellular avian type X collagen in situ and determination of its thermal stability using a conformation-dependent monoclonal antibody

The thermal stability of the helical domain of intracellular and matrix-associated type X̄ collagen was examined in situ within the hypertrophic region of embryonic chick vertebral cartilages. For this we employed indirect immunofluorescence histochemistry of unfixed tissue sections reacted at progressively higher temperatures ( Linsenmayer et al., J cell biol 99 (1984) 1405) [18] with a conformation-dependent monoclonal antibody ( X ̄ - AC9 ) ( Schmid & Linsenmayer, J cell biol 100 (1985) 598) [19]. The hypertrophic chondrocytes which had most recently initiated synthesis of type X̄ did not immediately secrete it, but instead retained it intracellularly within cytoplasmic organelles. This allowed for clear visualization of the intracellular type X̄. Within the pool of intracellular type X̄ collagen, the epitope recognized by the antibody was stable up to 55 °C, but was destroyed at 60 °C. This is 5–10 °C higher than the thermal stability of the epitope when the molecule is in neutral solution (as determined by competition ELISA). The matrix-associated type X̄ collagen is stable at least to 65–67.5 °C. We conclude that in situ the stability of the collagen helix in its normal intracellular environment is considerably greater than might be predicted from measurements made on molecules in solution.

Segmental appearance of type X collagen in the developing avian notochord

To determine whether short-chain cartilage collagen, collagen type X, is a component of notochordal matrix, we have performed immunohistochemistry with a monoclonal antibody (X-AC9) previously shown to be specific for this molecule ( Schmid and Linsenmayer (1984). J. Cell Biol. 100, 598–605). We have also examined different stages of embryos to establish the temporo-spatial appearance of the molecule. The data show that type X collagen is indeed a component of notochordal matrix, but that its developmental appearance is quite late compared to that of type II collagen, another cartilage matrix molecule found in notochord. It is detected only in embryos older than 12 days. The appearance of type X collagen within the notochord is preceded by its appearance within the surrounding hypertrophic cartilage matrix of the vertebral bodies. Spatially, within the notochord the appearance of type X collagen is initially restricted to sites at which the surrounding vertebral cartilage matrix is also reactive for type X. With subsequent development, the notochordal reactivity extends, in a decreasing gradient, anteriorly and posteriorly toward the intervertebral zones. However, the brightest immunofluorescence of notochordal type X is maintained at the midvertebral sites. The ultimate fate of the notochordal tissue at such midvertebral sites is to be removed during endochondral bone formation within the vertebrae. We have observed that type II collagen is also found within notochordal tissue, but this molecule has a distribution which is the converse of that of the type X collagen. The type II collagen is preferentially deposited at intervertebral sites (i.e., the locations of future intervertebral discs). That the type X collagen within the notochord is preferentially deposited at sites destined to be replaced is consistent with one of the hypotheses we previously raised for the function of the type X collagen molecule—it may “target” skeletal tissues for eventual removal.

Effects of hydrocortisone on the vertebral cartilage plate in mice. A light and electron microscopic study.

Together with other side effects, the clinical use of steroid appears to produce disorders in the spinal column, especially in young patients. However, morphologic details of steroid induced changes in the spinal column are little known. In this study, mice were treated with daily intramuscular injections of hydrocortisone at doses of 1, 5, and 10 mg/kg body weight for 1, 2, or 4 weeks after 2 weeks of age, and the cartilage plates of the lumbar vertebrae were examined, compared with controls, by light and electron microscopy. Cartilage plates consist of an outer zone, abutting the nucleus pulposus and an inner zone oriented toward the vertebral body. The outer zone is divided into a superficial layer and a deep calcified layer. With steroid therapy, chondrocytes in the inner zone and the superficial layer of the outer zone became degenerative or necrotic. In mice treated with 5 and 10 mg hydrocortisone/kg body weight, ossification appeared earlier in the deep calcified layer than in control animals. After 2 weeks of treatment, the cartilage tissue of the inner zone in mice treated with 10 mg hydrocortisone contained ossification gaps, columnar bone tissue, connecting the bony vertebral body, and the deep calcified layer of the outer zone. The thickness of each layer was measured by light microscopy. By hydrocortisone treatment, the whole of the cartilage plate decreased in thickness, the two cartilage layers of the inner zone and the superficial layer of the outer zone became thinner, and the calcified or ossified layer of the outer zone became widened.(ABSTRACT TRUNCATED AT 250 WORDS)

Expression of hyaluronic acid-binding glycoprotein, hyaluronectin, in the developing rat embryo

Immunological and histological methods have been applied to the developing rat embryo to study the distribution of hyaluronectin (HN, a glycoprotein with hyaluronic acid-binding properties) previously shown to be present in the nervous system and in desmoplasias. HN was absent in the morula and the blastula and was first detected in the mesenchyme bordering the neural tube and somites on Day 10, i.e., at a time when hyaluronic acid is already widely dispersed in the mesenchyme. At this stage HN appeared to be closely associated with the basement membrane around the epithelial structures (somites, notochord, ectoderm) whereas the intercellular areas of mesenchyme were less strongly stained. The delineation of basement membranes decreased progressively, while the accumulation of HN increased in the cell-free areas of mesenchyme, giving a continuous, diffuse pattern. Differentiation of mesenchyme into vertebral cartilage and gut smooth muscle was accompanied by a progressive disappearance of HN. Even after streptomyces hyaluronidase or chondroitinase digestion the antigen was not unmasked in these tissues. The results are in agreement with the few observations made in the human. They suggest that HN could play a role, in association with fibronectin and glycosaminoglycans (hyaluronic acid), in the physiology of the embryonic extracellular matrix. HN appeared at a later stage in the embryonic nervous tissue; its distribution was extracellular in areas where both cell migration and proliferation occur.

Presence of sulfated glycans in Ascidian tunic and in the body wall of a sea cocumber

The tunic of several Ascidian species, and the body wall of a sea cucumber, contain high amounts of sulfated glycans, at a concentration which is similar to that of sulfated glycosaminoglycans in vertebrate cartilales. The glycans from the different species are distinguished by the pattern of electrophoretic migration and by the molar proportion of amino sugars, hexoses and sulfate. The possibility that these compounds be involved in the maintenance of the integrity of these tissues, resembling the strutural function of the glycosaminoglycans in vertebrate cartilages, is discussed.