Limb Bud Mesenchymal Cells Research Articles

Placental transfer and developmental effects of 9‐cisretinoic acid in mice

9-cis retinoic acid (RA) is a naturally occurring isomer of all-trans RA. While both isomers can bind with high affinity and activate RA receptors, only 9-cis RA is the specific ligand for the retinoid X receptors. 9-cis RA has also been shown to be much more potent than all-trans RA in inducing digit duplication in the chick embryo wing bud. To gain further insight into its mechanisms, here we investigated the teratogenic activity in pregnant mice of 9-cis RA and compared it with those of all-trans RA and 13-cis RA. Using frequency and severity of limb reduction defects as well as palatal clefts in the resultant fetuses as indicators, we found that orally administered 9-cis RA was one-half as potent a teratogen as all-trans RA. That 9-cis RA was intrinsically less active than all-trans RA was deduced by comparing the inhibitory activities of the two retinoids in the limb bud mesenchymal cell micromass cultures using chondrogenesis as an end-point. Since placental transfer of cis isomers of RA is generally poor, we monitored the identities and amounts of retinoids in the embryo after administration of 9-cis RA to the mother. We found that 9-cis RA undergoes extensive metabolism and isomerization during absorption resulting in a number of metabolites in the maternal circulation within 30 min after administration. Although some of these metabolites remain to be identified, the most abundant RA isomers in the plasma coeluted with 13-cis RA.(ABSTRACT TRUNCATED AT 250 WORDS)

Fibroblast growth factors induce additional limb development from the flank of chick embryos

Fibroblast growth factors (FGFs) act as signals in the developing limb and can maintain proliferation of limb bud mesenchyme cells. Remarkably, beads soaked in FGF-1, FGF-2, or FGF-4 and placed in the presumptive flank of chick embryos induce formation of ectopic limb buds, which can develop into complete limbs. The entire flank can produce additional limbs, but generally wings are formed anteriorly and legs posteriorly. FGF application activates Sonic hedgehog in cells with polarizing potential to make a discrete polarizing region. Hoxd-13 is also expressed in the ectopic bud, and an apical ectodermal ridge forms. A limb bud is thus established that can generate the appropriate signals to develop into a complete limb. The additional limbs have reversed polarity. This can be explained by the distribution of cells in the flank with potential polarizing activity. The results suggest that local production of an FGF may initiate limb development.

Interactive Cellular Modulation of Chondrogenic Differentiation in Vitro by Subpopulations of Chick Embryonic Calvarial Cells

The embryonic chick calvarium normally develops into an intramembranous bone without an intermediate cartilage stage, although cartilage-like calvarial cells have been observed in calcium-deficient chick embryos ( Dev. Biol. 115, 215, 1986; Dev. Biol. 133, 221, 1989). To analyze the cellular basis of calvarial development, Incubation Day 14 embryonic chick calvarial cells were fractionated by Percoll gradient isopycnic centrifugation; after 12 days in monolayer culture, a subpopulation of cells (fraction F) was observed to differentiate into a rounded cellular morphology with refractile extracellular matrix. The cartilaginous nature of the extracellular matrix produced by fraction F cells was strongly suggested by the immunodetection of aggrecan and type II collagen, and Alcian blue staining. The other calvarial cell fractions (C, D, E) showed predominantly osteoblastic morphology, expressed alkaline phosphatase activity, and elaborated a collagen type I extracellular matrix. These findings suggest that a "chondrocyte-like" subpopulation of cells exist in the embryonic calvarium. To investigate how cellular interactions may influence the expression of osteogenic versus chondrogenic phenotypes by calvarial cells in vitro, the following cell type combinations were tested in high-density (20 × 10 6 cells/ml) micromass cultures: (1) total, unfractionated calvarial cells; (2) Percoll fractions (C to F) of calvarial cells; and (3) a highly chondrogenic cell type, Hamburger-Hamilton stage 23/24 chick limb bud mesenchymal cells. The micromass cocultures were set up either by mixing and plating two cell types to form the initial, single micromass or by plating the two cell types as separate, side-by-side cultures in the same culture well. The effects of interactions between cocultured calvarial and limb mesenchymal cells upon their respective diffrentiation fates were separately analyzed, on the basis of the number of Alcian blue-staining cartilage nodules in limb mesenchymal cells and [ 35S]-sulfate incorporation in both cell types. In cocultures with unfractionated or "osteoblast-like" fraction C and D calvarial cells, limb mesenchymal cells had decreased chondrogenesis. In separate cocultures with "chondrocyte-like" fraction F cells, limb mesenchymal cells exhibited enhanced chondrogenesis. Conditioned media from fraction C and D cells, and from fraction F cells, inhibited and enhanced limb mesenchymal cell chondrogenesis, respectively. Q 35S]Sulfate incorporation was greater in (1) unfractionated and fractionated calvarial cells cocultured separately with limb mesenchymal cells, compared to calvarial cells cultured alone, and (2) fraction F cells, compared to other fractions or unfractionated calvarial cells. Interestingly, [ 35S]sulfate incorporation in fraction F cells was decreased when cocultured with fraction C, D, and E cells. Taken together, these findings show that (1) chondrogenesis in limb mesenchymal cells was inhibited by total, unfractionated and fraction C and D calvarial cells and enhanced by chondrocyte-like fraction F calvarial cells, and (2) the chondrocyte-like phenotype of the fraction F calvarial cells could be modulated by their interaction with chondrogenic (limb mesenchyme) or osteogenic (calvarial osteoblastic fractions) cells. It is proposed that in unfractionated calvarial cells in vitro, or in the calvaria developing in vivo, osteogenic differentiation predominates as a result of regulated inhibition of potential chondrogenic differentiation. As a corollary, aberrant chondrogenesis in the developing calvarium probably results from perturbation of such regulatory mechanisms.

Expression of Transforming Growth-Factor-β 1-4 in the Chick Limb Bud Mesenchymal Cells: Regulation by 1,25-Dihydroxyvitamin-D3

When mesenchymal cells from the early chick limb bud (stage 23-24) are plated at high cell density they spontaneously undergo chondrogenesis, implicating extensive cell-to-cell interactions. In the past it has been shown that TGF-β or 1,25 dihydroxyvitamin D3 influences this process. In the present study we investigated the expression of TGF-β isoforms in the mesenchymal cells after treatment with 1,25-dihydroxyvitamin D3. We found that TGF-β2 was down-regulated due to the treatment. The size of the transcripts was the expected as described for sternum cartilage cells but with differential use preference.

Adenosine 5'-triphosphate promotes mineralization in differentiating chick limb-bud mesenchymal cell cultures.

When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells. In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

Expression of N-Cadherin and Alkaline Phosphatase in Chick Limb Bud Mesenchymal Cells: Regulation by 1,25-Dihydroxyvitamin D 3 or TGF-β1

When mesenchymal cells from the early chick limb bud (stage 23-24) are plated at high cell density they spontaneously undergo chondrogenesis implicating extensive cell-to-cell interactions. In the past it has been shown that TGF-β and vitamin D can influence this process and can stimulate chondrogenesis. Given the importance of cell adhesion molecules during cellular interactions we decided to examine the effects of 1,25-dihydroxyvitamin D 3 or TGF-β on the expression of molecules involved in cell-to-cell adhesion (N-cadherin) or cell-substrate adhesion (alkaline phosphatase). Immunofluorescence demonstrated that N-cadherin was expressed in the mesenchymal cells and in the very early cartilage nodules but it was down-regulated in the older nodules. As shown by Western blotting, the expression of N-cadherin declined as chondrogenesis proceeded and was affected in cultures treated with 1,25-dihydroxyvitamin D 3 and TGF-β1. Alkaline phosphatase was also expressed in the mesenchymal cells; these cells preferentially use an alternative transcript compared to the cartilage cells of the sternum. Thus, our data suggest that the involvement of 1,25-dihydroxyvitamin D 3 in chondrogenesis could be mediated via regulation of cell adhesion.

Strontium alters the complexed acidic phospholipid content of mineralizing tissues

High dietary strontium induces rickets in both calcium-replete and calcium-deficient animals. To test the hypothesis that strontium directly perturbs complexed acidic phospholipid (CPLX) metabolism and thus, mineralization, the effect of strontium treatment on CPLX formation was studied in solution, in culture, and in growing rats. Synthetic CPLX containing calcium or strontium were found to be similar in composition. Strontium, however, appeared to incorporate into CPLX less avidly than calcium. Mineralizing chick limb bud mesenchymal cell cultures treated with strontium demonstrated a significantly increased CPLX content and decreased 45Ca uptake compared to calcium-treated cultures. Long bones from young growing rats fed a diet supplemented with strontium demonstrated defective mineralization based on radiologic and histologic analyses. Metaphyseal bone of strontium-fed rats contained significantly greater amounts of CPLX and had significantly lower ash weights compared with control bone. Thus, treatment of mineralizing tissues with strontium both in vitro and in vivo resulted in defective mineralization and an accumulation of CPLX. Strontium appears to perturb mineralization, in part, by a direct effect on the cells of mineralizing tissues.

Stable, position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures.

Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA's effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21-22 and 23-24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA's effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.

NuSerum, a synthetic serum replacement, supports chondrogenesis of embryonic chick limb bud mesenchymal cells in micromass culture

In an effort to establish a more chemically defined culture system to study the regulation of chondrogenic differentiation in vitro, two commercially available serum replacements, NuSerum and NuSerum IV, were tested on embryonic limb mesenchyme. Limb bud (LB) mesenchymal cells were isolated from Hamilton-Hamburger stage 23-24 chick embryos and plated at various densities (1, 5, 10, or 20 x 10(6) cells/ml) in micromass culture for 4 days in media supplemented with 10% fetal bovine serum (FBS), NuSerum or NuSerum IV. Cell growth was assessed by the incorporation of [3H]leucine and [3H]thymidine. Chondrogenesis was determined by the incorporation of [35S]sulfate and by the number of Alcian blue-staining cartilage nodules. In high density (20 x 10(6) cells/ml) cultures, which favored chondrogenic differentiation, both serum replacements supported protein synthesis and chondrogenesis equally well as FBS. In cultures plated at 5 x 10(6) cells/ml, a cell density in which was chondrogenesis-limiting, both NuSerum and NuSerum IV significantly enhanced incorporation of [35S]sulfate (2.6-fold), [3H]leucine (1.4-fold), and [3H]thymidine (1.9-fold), compared to FBS. Enhancement of chondrogenesis was also apparent by the increases in the number of Alcian blue-staining cartilage nodules and the ratio of sulfate: leucine incorporation in cultures plated at 5 x 10(6) cells/ml. Interestingly, the localization of cartilage nodules was extended out to the periphery of micromass cultures fed with NuSerum or NuSerum IV.(ABSTRACT TRUNCATED AT 250 WORDS)

Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells

Abstract To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 × 10 −8 M) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (2 × 10 6 cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 × 10 7 cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.

Vimentin Expression Pattern is Different between the Flank Region and Limb Regions of Somatopleural Mesoderm in the Chicken Embryo: (vimentin/chicken embryo/limb bud/limbness/mesoderm).

It is known that the chicken flank somatopleure also has a limb-forming potential at early stages of development, but loses this potential later. Molecular changes during this process is, however, not well known. We obtained a monoclonal antibody which reacts to the flank somatopleure, but not to the wing bud, the leg bud and the neck somatopleure in the stage 22 chicken embryo. Further study revealed that this antibody is specific to vimentin. Time course of vimentin expression in the somatopleural mesoderm during the development was studied. It was revealed to be biphasic. Somatopleural mesoderm expressed vimentin at stage 10, but not at stage 16. Flank somatopleural mesoderm began to express vimentin again at stage 18, whereas limb bud mesenchymal cells did not until stage 27. The earlier re-expression of vimentin at the flank somatopleure suggests that certain physiological changes take place in cells at this region.

Gallium nitrate inhibits alkaline phosphatase activity in a differentiating mesenchymal cell culture

The effect of gallium nitrate on alkaline phosphatase activity in a differentiating chick limb-bud mesenchymal cell culture was monitored in order to gain insight into the observation that rachitic rats treated with gallium nitrate failed to show the expected increase in serum alkaline phosphatase activity. Cultures maintained in media containing 15 microM gallium nitrate showed drastically decreased alkaline phosphatase activities in the absence of significant alterations in total protein synthesis and DNA content. However, addition of 15 microM gallium nitrate to cultures 18 h before assay for alkaline phosphatase activity had little effect. At the light microscopic and electron microscopic level, gallium-treated cultures differed morphologically from gallium-free cultures: with gallium present, there were fewer hypertrophic chondrocytes and cartilage nodules were flatter and further apart. Because of altered morphology, staining with an antibody against chick cartilage alkaline phosphatase appeared less extensive; however, all nodules stained equivalently relative to gallium-free controls. Histochemical staining for alkaline phosphatase activity was negative in gallium-treated cultures, demonstrating that the alkaline phosphatase protein present was not active. The defective alkaline phosphatase activity in cultures maintained in the presence of gallium was also evidenced when cultures were supplemented with the alkaline phosphatase substrate, beta-glycerophosphate (beta GP). The data presented suggest that gallium inhibits alkaline phosphatase activity in this culture system and that gallium causes alterations in the differentiation of mesenchymal cells into hypertrophic chondrocytes.

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

Effects of ofloxacin on chondrogenesis in murine cartilage organoid culture

The fluoroquinolone, ofloxacin, a widely used antimicrobial agent, has been shown to cause athropathogenic syndromes in juvenile animals. In the present study, the effect of ofloxacin on chondrogenesis in cartilage organoid cultures of limb-bud mesenchymal cells obtained from day-12 mouse embryos was investigated. Cultures treated with increasing concentrations of ofloxacin (10, 30 and 100 μg/ml medium) for 6 days showed no significant changes in overall protein content and dry weight. Collagen type II, as a specific marker for cartilage, and collagen type I, as a marker protein in the internodular loose connective tissue in this culture system, were estimated by an inhibition-ELISA after cyanogen-bromide digestion. The collagen type I content of treated cultures remained constant, but the collagen type II level decreased in a dose-dependent manner to about 40% of that of the controls. There was no detectable increase in the concentration of collagen type II in the medium suggesting that ofloxacin inhibits synthesis rather than stimulate degradation of collagen type II. Cultures treated with 100 μg ofloxacin/ml were further investigated by indirect immunofluorescence and electron microscopy. Anticollagen type II antibodies demonstrated irregularities, several defects in the cartilage nodules, and much weaker staining in the treated cultures compared with controls. Similar results were obtained with antibodies directed against the core protein of large chondroitin sulphate proteoglycan monomers. Using monoclonal antibodies specific for unsulphated, 4-sulphated and 6-sulphated disaccharide “stubs” that remain attached to the core protein after chondroitinase ABC digestion of proteoglycans, different changes in these glycosaminoglycans were observed. While unsulphated chondroitin seemed to disappear nearly completely from the cartilage matrix, the level of chondroitin 4-sulphate remained unchanged and in the case of chondroitin 6-sulphate a slight increase in staining intensity was observable in the ofloxacin-treated cultures. Ultrastructurally, there was a reduction in the number of collagenous fibrils in the cartilage matrix of treated cultures and necrotic chondroblasts could be demonstrated. The present results resemble, in some aspects, observations that have been made in vivo after ofloxacin treatment, indicating that this in vitro model may provide a suitable system for examining the mechanism of quinolone-induced athropathia.

Position-dependence of retinoic acid receptor-β gene expression in the chick limb bud

Retinoic acid and 3,4-didehydroretinoic acid are metabolites of vitamin A that can induce duplications and other malformations when locally applied to the anterior margin of the chick limb bud. There is evidence that they may be natural signaling substances in the limb bud. Both compounds are thought to act by binding to ligand-dependent transcription factors that belong to the steroid/thyroid hormone nuclear receptor superfamily. In situ hybridization analyses show that in the mesenchyme of the chick wing bud between embryonic stages 20 and 27, retinoic acid receptor-β (RAR-β) transcripts are restricted to the proximal region of the bud and are present at highest levels in the region of the limb bud mesenchyme that contributes to the shoulder. We have performed grafting experiments in order to examine whether RAR-β gene expression in limb bud mesenchyme cells is cell-autonomous or whether it is dependent upon the cell's position within the limb bud. When tissue from the proximal region of the stage 22 wing bud, which contains high levels of RAR-β transcripts, was grafted to the distal tip of the bud, RAR-β transcripts were undetectable in the graft 6 hr later. When tissue from the distal tip of the bud was grafted to a proximal site, most of the grafts exhibited a slight increase in the level of RAR-β transcripts, which was detectable 6 hr after grafting. However, the levels of RAR-β transcripts in these grafts never approached those found in the proximal core of the bud. These data indicate that RAR-β gene expression in the chick wing bud is position-dependent in that it is repressed at the distal tip of the bud and partially activated by grafting distal tissue to a proximal site. However, accumulation of RAR-β transcripts to high levels appears to be a characteristic of mesenchyme that was initially specified to form proximal structures.

Studies of mineralization in tissue culture: optimal conditions for cartilage calcification

The optimal conditions for obtaining a calcified cartilage matrix approximating that which exists in situ were established in a differentiating chick limb bud mesenchymal cell culture system. Using cells from stage 21-24 embryos in a micro-mass culture, at an optimal density of 0.5 million cells/20 microliters spot, the deposition of small crystals of hydroxyapatite on a collagenous matrix and matrix vesicles was detected by day 21 using X-ray diffraction, FT-IR microscopy, and electron microscopy. Optimal media, containing 1.1 mM Ca, 4 mM P, 25 micrograms/ml vitamin C, 0.3 mg/ml glutamine, no Hepes buffer, and 10% fetal bovine serum, produced matrix resembling the calcifying cartilage matrix of fetal chick long bones. Interestingly, higher concentrations of fetal bovine serum had an inhibitory effect on calcification. The cartilage phenotype was confirmed based on the cellular expression of cartilage collagen and proteoglycan mRNAs, the presence of type II and type X collagen, and cartilage type proteoglycan at the light microscopic level, and the presence of chondrocytes and matrix vesicles at the EM level. The system is proposed as a model for evaluating the events in cell mediated cartilage calcification.

Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes

The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18–180 μ m; acrolein, 50–500 μ m) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 m m) or GSH (1 m m) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

The Extracellular Matrix in Cartilage Organoid Culture: Biochemical, Immunomorphological and Electron Microscopic Studies

Limb bud mesenchymal cells obtained from day-12 mouse embryos were grown at high density on a membrane filter (pore size 0.2μm) at the medium/air interphase. Chondrogenesis in this so-called cartilage organoid culture was monitored quantitatively by immunological estimation of type I and type II collagen and qualitatively by indirect immunofluorescence and electron microscopy in the course of a 36 days culture period. Three stages of cartilage development could be substantiated: 1. Formation of cartilage between days 2 and 7; 2. maturation of cartilage between days 9 and 13; 3. degeneration of cartilage beginning at day 20. Differentiation in cell aggregates and a loose mesenchymal tissue occurred during the first two days of the culture period. Type II collagen synthesis started in cell aggregates two days after plating and after 6 days in culture distinct cartilage nodules had developed which were embedded in loose connective tissue that contained type I collagen. During this period the type II collagen content increased progressively from 2.3 μg (day 3) to nearly 40 μg (day 7) per mg dry weight, whereas the type I collagen level increased more linearly from 2.7 to 21.3 ltg/mg dry weight. The second period was characterized by enlargement and fusion of cartilage nodules and a diminished increase in type II collagen content from 45 to 60 μg/mg dry weight. Enlargement and fusion occurred by matrix production as well as by transformation of perichondrial cells into chondroblasts. Type I collagen synthesis enhanced from 29 to 54 μg/mg. Hypertrophic chondrocytes could be demonstrated ultra structurally. At the third stage a nearly continuous layer of cartilage on the membrane filter covered by noncartilagenous tissue had developed. To some extent chondrocytes lost their matrix capsule and changed into fibroblast-like cells accompanied by a switch of collagen synthesis from type II to type I collagen. Quantitative studies yielded a constant level of about 60 Vg/mg type II collagen and a further increase in type I collagen from 77 to 116 μg/mg dry weight. This study reveals an in vitro model of a prolonged, but almost identical image of chondrogenesis in vivo prior to endochondral mineralization which may be useful for investigations on cartilage differentiation, maturation and degeneration.

A Possible Role of Protein Kinase C in the Chondrogenesis of Chick Limb Bud Mesenchyme Cells

A Possible Role of Protein Kinase C in the Chondrogenesis of Chick Limb Bud Mesenchyme Cells

Identification of a cis-acting sequence in the collagen II enhancer required for chondrocyte expression and the binding of a chondrocyte nuclear factor.

Collagen II is synthesized at high levels by differentiated chondrocytes. A 620-base pair DNA sequence in the first intron of the rat collagen II gene was previously determined to have chondrocyte-specific enhancer activity. Using mobility shift assays, we have defined a decamer sequence, 5'-CACAATGCAT-3', in the middle of the enhancer that binds a protein or protein complex expressed by chondrocytes but not by NIH3T3 cells or L2 rat fibroblasts. This protein was also induced during the differentiation of limb bud mesenchymal cells into chondrocytes. Mutational analyses, coupled with both in vitro binding studies and direct assays of enhancer activity following transfection into cells, demonstrated that this sequence is involved in the binding of the chondrocyte nuclear protein(s) and is necessary for the enhancer activity. This sequence has homology to the consensus binding sequence for helix-loop-helix transcription factors.