Modulation of chondrogenesis by the cytoskeleton and extracellular matrix.
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Actins↗
Biomedical subjects
M Solursh
Publications and source records attributed to M Solursh.
Bone acidic glycoprotein-75 is a major synthetic product of osteoblastic cells and localized as 75- and/or 50-kDa forms in mineralized phases of bone and growth plate and in serum.
Anti-peptide and anti-protein antisera were produced which both recognize bone acidic glycoprotein-75 (Mr = 75,000) and an apparent fragment or biosynthetic intermediate (Mr = 50,000) in calcified tissues and/or serum. A fragment-precursor relationship is suggested from the fact that closely spaced doublet polypeptides of Mr = 50,000 could be produced by proteolysis of the purified protein upon long term storage. No reactivity was detected with osteopontin, bone sialoprotein, or small bone proteoglycans. Bone acidic glycoprotein-75 represents 0.5-1% of the total radiolabeled proteins synthesized by explant cultures of neonatal calvaria or growth plate, by calvarial outgrowth cultures, and by rat osteosarcoma cells. Amounts produced by explant cultures and calvarial outgrowth cultures were similar to that for osteopontin, a major product of osteoblasts. In osteosarcoma cultures, 80% of labeled antigens were associated with the cell layer fraction wherein specific immunoprecipitation pelleted Mr = 50,000 and 75,000 sized antigens. Bone acidic glycoprotein-75 (Mr = 75,000) is enriched in 4 M guanidine HCl/0.5 EDTA extracts of neonatal rat bone and growth plate tissues, whereas largely absent from heart, lung, spleen, liver, brain, and kidney. Explant cultures of these noncalcifying tissues also synthesized bone acidic glycoprotein-75 antigen, but the quantities produced were only 5% or less that obtained with calvaria. By immunohistochemistry, antigenicity is associated with the bony shaft and calcified cartilage of long bones, but is absent from associated soft tissues. These finding demonstrate that bone acidic glycoprotein-75 is antigenically distinct, predominantly localized to calcified tissues, represents a major product of normal osteoblastic cells and may undergo a characteristic fragmentation in vivo and in vitro.
Animals↗
Cloning of a fibrillar collagen gene expressed in the mesenchymal cells of the developing sea urchin embryo.
We have cloned and characterized several overlapping cDNAs that specify a large portion of a Paracentrotus lividus fibrillar collagen molecule. Our conclusions are based on sequencing data, which showed that the clones code for a 786-amino acid collagenous domain composed of an uninterrupted series of Gly-X-Y repeats and for a 265-amino acid carboxyl-terminal globular extension. The latter domain exhibits features highly reminiscent of those of the vertebrate counterparts, notably a putative carboxyl-peptidase cleavage site, a series of similarly arranged cysteinyl residues, and an N-linked glycosylation attachment site. In situ and Northern blot hybridizations have established the size, time of appearance, and tissue localization of the collagen mRNA during sea urchin development. The collagen transcript, 9 kilobases in length, is first detected in the primary and, more predominantly, in the secondary mesenchyme cells of late gastrulae where it progressively accumulates thereafter. This and other work (D'Alessio, M., Ramirez, F., Suzuki, H.R., Solursh, M., and Gambino, R. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 9303-9307) provide evidence of a genetic heterogeneity of fibrillar collagens in the sea urchin embryo and suggest that the two genes are activated in the same cell lineages at distinct developmental stages.
Amino Acid Sequence↗
The distribution of mesenchyme proteoglycan (PG-M) during wing bud outgrowth.
This study utilizes immunofluorescence to describe the distribution of several extracellular matrix molecules in the chick embryo during the process of limb outgrowth and the formation of precartilage condensations. A large chondroitin sulfate proteoglycan (PG-M) is detected at the wing level at Hamburger and Hamilton stage 14 in and under the dorsal ectoderm, and is associated with the basement membranes around the neural tube, notochord and pronephros, but not with other basement membranes. The galactose-specific lectin, peanut agglutinin (PNA), has a similar distribution except that it also binds to the dorsal side of the neural tube. PG-M is not detected in the limb mesenchyme until after stage 17, when it is present in the distal region, as is PNA-binding material. With further development of the wing bud, PG-M is present in the subectodermal mesenchyme, the mesenchyme at the distal tip and in the prechondrogenic core. After stage 22 PNA-binding material becomes localized in the prechondrogenic core, the basement membranes under the apical ectodermal ridge, and the ventral sulcus. The distribution of these components (PG-M and PNA binding material) overlaps, but differs from that of type I collagen and fibronectin and basement membrane components, such as laminin, basement membrane heparan sulfate proteoglycan, and type IV collagen. Tenascin, on the other hand, is not detected in the limb bud until stage 25, after the appearance of cartilage matrix components such as type II collagen and cartilage proteoglycan (PG-H). These results are considered in relation to the formation of precartilage aggregates, and indicate that PNA binds to components in precartilage aggregates other than PG-M or tenascin.
Aggrecans↗
The formation of premuscle masses during chick wing bud development.
The skeletal musculature of chick limb buds is derived from somitic cells that migrate into the somatopleure of the future limb regions. These cells become organized into the earliest muscle primordia, the dorsal and ventral premuscle masses, prior to myogenic differentiation. Therefore, skeletal-muscle specific markers cannot be used to observe myogenic cells during the process of premuscle mass formation. In this study, an alternative marking method was used to determine the specific stages during which this process occurs. Quail somite strips were fluorescently labeled and implanted into chick hosts. Paraffin sections of the resulting chimeric wing buds were stained with the monoclonal antibody QH1 in order to identify graft-derived endothelium. Non-endothelial graft-derived cells present in the wing mesenchyme were assumed to be myogenic. At Hamburger and Hamilton stage 20, myogenic cells were distributed throughout the central region of the limb, including the future dorsal and ventral premuscle mass regions and the prechondrogenic core region. By stage 21, the myogenic cells were present at greater density in dorsal and ventral regions than in the core. By stage 23, nearly all myogenic cells were located in the dorsal and ventral premuscle masses. Therefore, the two premuscle masses become established by stage 21 and premuscle mass formation is not complete until stage 23 or later. Premuscle mass formation occurs concurrently with early chondrogenic events, as observed with the marker peanut agglutinin. To facilitate the investigation of possible underlying mechanisms of premuscle mass formation, the micromass culture system was evaluated, to determine whether or not it can serve as an accurate in vitro model system. The initially randomly distributed myogenic cells were observed to segregate from prechondrogenic regions prior to myogenic differentiation. This is similar to myogenic patterning in vivo.
Animals↗
Precocious appearance of cardiac troponin T pre-mRNAs during early avian embryonic skeletal muscle development in ovo.
Cardiac troponin T (cTNT), a component of the muscle contractile apparatus, is transiently expressed in skeletal muscle during avian limb development. While cTNT was first detected immunohistochemically in limb buds undergoing overt myogenic differentiation (Hamburger and Hamilton stage 26, about 5 days in ovo), RNA blot analyses of early, predifferentiated wing buds have revealed the presence of cTNT transcripts in limb buds as early as stage 23 (4 days in ovo). Steady-state cTNT poly(A) RNAs of stage 22 through stage 37 fore- and hindlimbs were compared using both cTNT cDNA and cTNT intron-specific probes. In the predifferentiated state, two incompletely processed RNAs (3.8 and 2.4 kb) were expressed in the absence of the mature cTNT transcript, while a third pre-mRNA (3.5 kb) appeared concomitantly with the mature mRNA as differentiation and development proceeded. In addition, a population of unique cTNT transcripts were expressed in a proximal to distal manner in wing buds which had undergone initial overt myogenic differentiation (stage 26). Some of the cTNT pre-mRNAs observed in premyogenic limbs appeared to accumulate stably in a tissue-specific manner, based on their absence from the cardiac poly(A) RNA population. These results suggest that the appearance of cardiac troponin T mRNA, as well as the polypeptide, may be regulated at multiple levels including RNA processing, stability, and/or translation during early skeletal muscle myogenesis.
Animals↗
Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development.
Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid outgrowth, and in preventing the onset of chondrogenesis.
Animals↗
Comparison between ectoderm-conditioned medium and fibronectin in their effects on chondrogenesis by limb bud mesenchymal cells.
Limb bud ectoderm inhibits chondrogenesis by limb bud mesenchymal cells cultured at high density or on collagen gels. This ectodermal antichondrogenic influence has been postulated to function in vivo in regulating the spatial patterning of cartilage and soft connective tissue in the limb. We have developed a method for preparing ectoderm-conditioned medium containing antichondrogenic activity. Using a simple bioassay, we have investigated some characteristics of the ectodermal products and their effects on limb bud mesenchymal cells. Inhibition of chondrogenesis by ectoderm-conditioned medium was tested on limb bud mesenchymal cells cultured on collagen gels. The antichondrogenic influence involves enhanced cell spreading and is alleviated by agents, such as cytochalasin D, that induce cell rounding. Fibronectin resembles ectoderm-conditioned medium in its ability to inhibit chondrogenesis and promote cell spreading in collagen gel cultures of limb bud mesenchymal cells. However, Western blot analysis shows that the antichondrogenic activity of ectoderm-conditioned medium is not due to fibronectin in the medium. Peptides related to the fibronectin cell-binding domain block the antichondrogenic effect of fibronectin, but not that of ectoderm-conditioned medium. On the other hand, an antibody to an integrin, as well as heparan sulfate, alleviates the antichondrogenic effects of both fibronectin and ectoderm-conditioned medium. The antichondrogenic effect of ectoderm-conditioned medium may be mediated by an integrin and by a cell surface heparan sulfate proteoglycan, but it does not depend directly upon fibronectin-mediated cell spreading.
Amino Acid Sequence↗
Formation of chondrous and osseous tissues in micromass cultures of rat frontonasal and mandibular ectomesenchyme.
Rat frontonasal and mandibular mesenchyme was isolated from day-12 1/2 (stage-22) rat embryos and cultured at high density for up to 12 days. The stage chosen was based on the observation that mandibular mesenchyme at this stage became independent of its epithelium with respect to the production of both cartilage and bone. Frontonasal cultures developed aggregates of anastomosing columns of cells within 2 days. These grew as the cells enlarged, laying down an Alcian-blue-positive matrix by day 3 of culture. Significant mineral was detected by von Kossa staining by day 5 at which time the aggregates covered a large portion of the culture, eventually covering the entire micromass by day 10-12. Mandibular cultures developed centrally located nodular aggregates by 3 days of culture. These nodules increased in number, spreading outwards as the cells enlarged, laying down an Alcian-blue-positive matrix by day 4 and mineral by days 6-7. At this time the nodules began to elongate and coalesce, but never covered the entire culture over the 12-day period. Antibody staining revealed that in both cultures the cells were initially positive for type I collagen. Subsequently, the aggregates began expressing type II collagen, followed by type X, which coincided with the onset of mineralization. At this time some cells were negative for these cartilage markers, but positive for osteoblast markers, bone sialoprotein II, osteocalcin and type I collagen. In addition osteonectin and alkaline phosphatase were demonstrable in all of the aggregate cells late in the culture period. This provided clear evidence that chondroblast and osteoblast differentiation was proceeding within these cultures. The culture of rat facial mesenchyme should prove very useful, not only for the analysis of bone and cartilage induction and lineage relationships, but also in furthering our knowledge of craniofacial differentiation, growth and pattern formation by extending our analysis to a mammalian system.
Animals↗
Extracellular matrix and cell surface as determinants of connective tissue differentiation.
This paper reviews in vitro studies, largely from the author's laboratory, concerning the conditions that are permissive for the differentiation of limb bud mesenchymal cells into chondrocytes. In high-density cell culture, even in a defined medium, the same normal sequence of events that is found in vivo in developing cartilage is also observed. This system can be used to study heritable disorders in model systems such as in mutant mouse embryos. In addition, single mesenchymal cells can differentiate into hypertrophic chondrocytes in hydrated collagen gel or agarose cultures. A rounded cell shape promotes chondrogenesis, while a flattened cell shape promotes fibroblast differentiation. The actin cytoskeleton is shown to play a central role in regulating connective tissue cell differentiation. By use of such cell culture manipulations, it is now possible to grow large numbers of fibroblastic cells from human biopsy material for storage and to carry out experimental studies after re-expression of chondrogenesis in gel cultures. It is suggested that cytoskeletal-extracellular matrix interactions play a fundamental role in connective tissue differentiation. Matrix receptors might be developmentally regulated and modify epithelial effects on mesenchymal cells. In this way mesenchymal cells differentiate in a highly organized manner in spatial and temporal terms.
Animals↗
Degradation of hyaluronic acid does not prevent looping of the mammalian heart in situ.
It is currently proposed that accumulation of hyaluronic acid (HA) and subsequent hydration of the cardiac extracellular matrix is required for normal looping of the vertebrate heart. To test this hypothesis, we cultured Wistar rat embryos (Gestational Day 9.5) in rat serum plus 20 TRU/ml of Streptomyces hyaluronidase (treated embryos) or rat serum alone (control embryos). Despite degradation of HA as documented by Alcian blue staining at pH 2.5, 57 of 59 treated embryos developed normally looped hearts after 36 hr in culture. These experiments suggest that the accumulation of HA is not required for normal looping of the mammalian heart in situ.
Animals↗
Differentiation of cartilage and bone.
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Animals↗
Widespread distribution of type II collagen during embryonic chick development.
Type II collagen is a major component of hyaline cartilage, and has been suggested to be causally involved in promoting chondrogenesis during embryonic development. In the present study we have performed an immunohistochemical analysis of the distribution of type II collagen during several early stages of embryonic chick development. Unexpectedly, we have found that type II collagen is widely distributed in a temporally and spatially regulated fashion in basement membranes throughout the trunk of the embryo at stages 14 through 19, including regions with no apparent relationship to chondrogenesis. Immunohistochemical staining with two different monoclonal antibodies against type II collagen, as well as with an affinity-purified polyclonal antibody, is detectable in the basement membranes of the neural tube, notochord, auditory vesicle, dorsal/lateral surface ectoderm, lateral/ventral gut endoderm, mesonephric duct, and basal surface of the splanchnic mesoderm subjacent to the dorsal aorta, and at the interface between the epimyocardium and endocardium of the developing heart. In contrast, immunoreactive type IX collagen is detectable only in the perinotochordal sheath in the trunk of the embryo at these stages of development. Thus type II collagen is much more widely distributed during early development than previously thought, and may be fulfilling some as yet undefined function, unrelated to chondrogenesis, during early embryogenesis.
Animals↗
Possible recruitment of osteoblastic precursor cells from hypertrophic chondrocytes during initial osteogenesis in cartilaginous limbs of young rats.
The appearance of the bone phenotype during rat embryogenesis was studied by in situ hybridization using a cDNA clone to osteopontin. Radiolabeled sense and antisense RNA probes were prepared from the osteopontin cDNA by in vitro transcription. The probes were used to hybridize paraffin sections of the cartilaginous diaphysis from embryonic rats at day 17 of gestation. The hybridization pattern was analyzed by autoradiography. Hybridization with the antisense probe gave patterns of silver grain labeling, indicating the presence of osteopontin mRNA among the hypertrophic chondrocytes. No silver grains could be detected in the corresponding region following hybridization of consecutive sections with the sense probe, showing the specificity of the technique being used. Whether these results indicate that the osteopontin gene is transiently expressed by hypertrophic chondrocytes or that osteopontin is an early marker for osteoblastic precursor cells will have to be explored further.
Animals↗
Structure and developmental expression of a sea urchin fibrillar collagen gene.
We have isolated and characterized cDNA and genomic clones that specify a Paracentrotus lividus procollagen chain. The cDNAs code for 160 uninterrupted Gly-Xaa-Yaa triplets and a 252-amino acid carboxyl propeptide. Analysis of the deduced amino acid sequences indicated that the sea urchin polypeptide exhibits structural features that are characteristic of the fibril-forming class of collagen molecules. Partial characterization of two genomic recombinants revealed that the 3' end of the echinoid gene displays a complex organization that closely resembles that of a prototypical vertebrate fibrillar collagen gene. In situ and Northern (RNA) blot hybridizations established the size, time of appearance, and tissue distribution of the collagen transcripts in the developing sea urchin embryo. Collagen mRNA, approximately equal to 6 kilobases in size, is first detected in the forming primary mesenchyme cells of late blastulae where it progressively accumulates until the free swimming/feeding pluteus larval stage. Interestingly, collagen transcripts are also detected in the forming secondary mesenchyme cells of late gastrulae, and by the prism stage, their derivatives appear to be the most intensively labeled cells.
Amino Acid Sequence↗
Differential effects of physiological concentrations of retinoic acid in vitro on chondrogenesis and myogenesis in chick craniofacial mesenchyme.
Recent studies have shown that in the developing limb bud retinoic acid is a skeletal morphogen at physiological levels, but a potent teratogen at higher levels. Retinoic acid has also been shown to be teratogenic during facial development, but very low levels may have an as yet unspecified role in normal development. In the present study the effects of retinoic acid on chondrogenesis and myogenesis by craniofacial cells grown in micromass cell culture were investigated. Retinoic acid, at concentrations of 0.01-100 ng/ml, was supplied to cells derived from day-4 (H.H stage 23/24) chick embryo mandibular, maxillary and frontonasal processes, grown in micromass cultures for 4 days in both serum-containing and defined media. Based on Alcian-blue-staining, concentrations of retinoic acid of 0.1-1 ng/ml were found to enhance chondrogenesis by mandibular cells grown in defined medium, while greater concentrations up to 100 ng/ml inhibited chondrogenesis. By contrast, chondrogenesis was generally retarded by all concentrations of retinoic acid applied to frontonasal cells grown in defined medium and when applied to both mandibular and frontonasal cells when grown in serum-containing medium. Cells from stage-23/24 maxillae did not display any significant chondrogenic activity in either medium under these culture conditions. Unlike chondrogenesis, myogenesis in mandibular, frontonasal and maxillary cultures was greater in defined than serum-containing medium, based on the appearance of immunologically detectable muscle myosin, and was reduced considerably less in defined medium by all concentrations of retinoic acid tested. In the presence of serum however, myogenesis was retarded with increasing concentrations of retinoic acid beyond 1 ng/ml in micromass cultures from all three facial regions.(ABSTRACT TRUNCATED AT 250 WORDS)
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Cartilage stem cells: regulation of differentiation.
The developing limb bud is a useful source of cartilage stem cells for studies on the regulation of chondrogenesis. In high density cultures these cells can progress through all stages of chondrogenesis to produce mineralized hypertrophic cartilage. If the cells are maintained in a spherical shape, single stem cells can progress through a similar sequence. The actin cytoskeleton is implicated in the regulation of chondrogenesis since conditions that favor its disruption promote chondrogenesis and conditions that favor actin assembly inhibit chondrogenesis. Since a number of extracellular matrix receptors mediate effects of the extracellular matrix on cytoskeletal organization and some of these receptors are developmentally regulated, it is proposed that matrix receptor expression plays a central role in the divergence of connective tissue cells during development.
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Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis.
Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.
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