Chick Limb Bud Mesenchymal Cells Research Articles

Effect of 4-methylumbelliferyl-β- d-xyloside on collagen synthesis in chick limb bud mesenchymal cell cultures

Previous studies showed that cultures of chick limb bud mesenchymal cells plated at high density, to maximize chondrogenic expression, had a much reduced extracellular matrix around chondrocytes when exposed to 4-methyl-, umbelliferyl-β- d-xyloside. The majority of newly synthesized chondroitin sulfate chains were found in the culture medium presumably bound to the xyloside as opposed to their normal deposition on the core protein of proteoglycan. The question remained open as to whether the development of an abnormal matrix affected the synthesis of extracellular deposition of other cartilage-specific macromolecules. We have analyzed, both morphologically and biochemically, the synthesis and deposition of Type I and Type II collagen by β- d-xyloside-treated cultures of limb mesenchymal cells. While the rate of collagen synthesis per plate and its extracellular accumulation after 8 days in culture were reduced to some extent, the ratios of Type II to Type I collagen and the morphological distribution of these macromolecules were not affected by exposure to β- d-xyloside. We conclude that the expression of the cartilage-specific Type II collagen during chondrogenic differentiation is, although reduced, qualitatively not dependent on the amount of extracellular chondroitin sulfate chains attached to matrix-associated proteoglycan core protein. However, prolonged exposure of limb bud cells to xylosides leads to the formation of a chondroitin sulfate- and collagen-deficient matrix which, in turn, reduces the capacity of limb bud cells to synthesize Types I and II collagen.

Effects of 4-methyl umbelliferyl-beta-D-xylopyranoside on chondrogenesis and proteoglycan synthesis in chick limb bud mesenchymal cell cultures.

When chick limb bud mesenchyme cells from stage 23 to 24 embryos are plated at high density, they rapidly divide and a large proportion initiate chondrogenic expression during the first 2 to 3 days in culture. Between Days 4 and 8, the emergent chondrocytes mature and elaborate a cartilaginous matrix. The proteoglycans synthesized by the newly emergent Day 3 to 4 chondrocytes differ from those synthesized by either the prechondrogenic mesenchyme cells or the mature Day 8 chondrocytes. Cultures were grown from initial plating (Day 0) or from Day 2 in the continuous presence of 1 mM 4-methyl umbelliferyl-beta-D-xyloside, which acts intracellularly as a competitive acceptor with the endogenous core protein of proteoglycans for chondroitin sulfate synthesis. The proteoglycans synthesized by Day 8 cultures which had been maintained on xyloside or to which xyloside was added only 1 h prior to labeling were essentially identical. They were able to form aggregates, and they contained the same number of keratan sulfate chains, but only about 40% as many chondroitin sulfate chains, as normal. Additionally, both the chondroitin sulfate and keratan sulfate chains were 25% shorter than in the normal proteoglycans. The proteoglycans synthesized by cells in a culture maintained on xyloside until Day 8, and then switched to medium with no xyloside 1 h prior to labeling, were characteristic of those synthesized by normal mature Day 8 chondrocytes. These data suggest that stage 23 to 24 mesenchyme cells undergo normal chondrogenic maturation in culture in the presence of xylosides even though (a) most of the polysaccharides are synthesized onto the exogenously supplied xyloside substrate and released into the medium, (b) the proteoglycans that are synthesized are greatly reduced in polysaccharide content, and (c) the extracellular matrix as a consequence is greatly depleted in chondroitin sulfate content and, therefore, is abnormal in general morphology.

The sorting out of embryonic cells in monolayer, the differential adhesion hypothesis and the non-specificity of cell adhesion.

It has been reported previously that sorting out of chick embryonic liver parenchyma and limb bud mesenchymal cells would take place in monolayer culture. The distribution of cell types obtained (liver formed the internal, discontinuous phase) was interpreted in terms of the differential adhesion hypothesis. It was suggested that, in monolayer, liver cells were more cohesive than limb bud cells. In this paper we set out to extend the previous observations with 2 particular questions in mind: (i) Is sorting out in monolayer a general phenomenon occurring between a wider range of cell types? (ii) Can evidence be provided for or against the interpretation of results in terms of the differential adhesion hypothesis? Sorting-out experiments were conducted on circular hydrophilic islands, on an otherwise hydrophobic substratum. Under these conditions, sorting-out in monolayer was obtained with binary combinations of 4 chick embryonic tissue types: liver parenchyma, limb bud mesenchyme, pigmented epithelium of the eye and corneal epithelium. With every combination but one, the cells of one type surrounded the cells of the other type, generating what we have called a 'circle-within-a-circle' configuration. With the remaining combination, liver parenchyma and corneal epithelium, only localized sorting was obtained. The 'circle-within-a-circle' configuration is consistent with an interpretation in terms of the differential adhesion hypothesis, according to which the distribution of cells is determined by the relative strengths of cohesions between their lateral surfaces. In direct support of this is the finding from plating the different cell types at sub-confluent density on hydrophilic substrata that limb bud is the cell tye having the weakest lateral cohesion in monolayer. Limb bud surrounded the other 3 tissues on hydrophilic island. A hierachy of lateral cohesiveness between the 4 cell types has been constructed. It is unlikely that the results can be explained in terms of specific cohesion. When plated together at subconfluent density, the 3 epithelial cell types aggregate together to form mixed monolayered islands, suggesting that they share common adhesive mechanisms.

Electron microscopic and biochemical studies of proteoglycan polydispersity in chick limb bud chondrocyte cultures

Proteoglycan aggregates from bovine nasal cartilage, from Day 8 and Day 16 chondrocyte cultures derived from chick limb bud mesenchymal cells, and from em- bryonic chick epiphyseal cartilage were visualized as monolayers with electron microscopy. The lengths of the monomers in the aggregates and the average spac- ing of monomers on the central filament of hyaluronic acid were determined. The Gaussian distribution of core lengths for the bovine cartilage sample (343 + 95 nm) was significantly different from the distributions observed for the chick samples. With the exception of the sample derived from Day 16 cultures, all the chick samples gave statistically identical Gaussian distribu- tions (-310 f 60 nm). The Day 16 monomer core length distribution was somewhat skewed toward shorter lengths (297 + 58 nm). The average spacing of mono- mers on hyaluronic acid was larger for a reconstituted aggregate sample from Day 8 culture than for a prep- aration isolated from a similar culture by a procedure which did not involve dissociation of the aggregate structure. In all samples, the number of monomers per aggregate was linearly correlated with the length of hyaluronic acid in the central filament. Chondrocyte cultures were double-labeled with [3H]serine and [14C]serine on Day 8, or on Day 16, or both. Sepharose 2B analyses of the monomer fractions indicated that monomers synthesized on Day 16 had significantly smaller average sizes than those synthe- sized on Day 8. Further, although only 50% of the monomers synthesized on Day 8 remained in the tissue matrix on Day 16, the remaining molecules exhibited an identical size distribution to that of proteoglycans synthesized by Day 8 cultures and isolated directly. Differences in the average sizes of the chondroitin sul- fate chains in the monomer samples account in large part for the smaller sizes of the Day 16 macromolecules. Cartilage proteoglycan monomers consist of a protein core with covalently bound polysaccharide chains of chondroitin sulfate and keratan sulfate. The protein core of these macro- molecules can be divided into three regions; a chondroitin sulfate-enriched region, an intermediate keratan sulfate-en- riched region, and a hyaluronic acid binding region which is located at one end of the molecule (l-4). This noncovalent and highly specific interaction between the protein core of the * This work was supported in part (at Case Western Reserve University) by an Institutional Grant from the American Cancer Society, National Institutes of Health Grants HD-07209, 5T3-6M- 07225,HD-00020, HD-35609, AM-17110, and grants from the National Foundation (March of Dimes). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked