Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.     

Changes in endogenous cell surface galactosyltransferase activity during in vitro limb bud chondrogenesis

When the subridge mesoderm of the embryonic chick limb bud is cultured in the absence of the apical ectodermal ridge and adjacent ectoderm, the cells rapidly progress through the various stages of chondrogenesis. During the first day of culture, the cells initiate condensation, and during subsequent days, deposit a cartilage matrix. In the present study, we show that early in the first day there is a progressive 2-fold increase in cell surface galactosyltransferase activity towards endogenous acceptors. Later in the first day, although the cells are still in condensation, endogenous galactosyltransferase activity has decreased, suggesting in situ galactosylation of surface acceptors. During subsequent development, when cartilage matrix is being deposited, surface galactosyltransferase activity remains low. All controls have been performed to insure cell surface localization of enzyme activity. Two other surface glycosyltransferases show very low levels of activity, which do not change significantly during culture. We suggest that during cellular condensation, an interaction between surface galactosyltransferases and acceptors on adjacent cells occurs, and this interaction may be causally related to subsequent chondrogenic differentiation.     

In vitro histogenic capacities of limb mesenchyme from various stage mouse embryos

Mesenchyme cells isolated from mouse embryo forelimb buds (stages 15 through 21) and placed in high-density micromass cultures are compared with respect to their in vitro histogenic capacities. Particular emphasis is placed on changes in in vitro chondrogenic capacity. Stage 15 mouse limb cultures form numerous aggregates which uniformly fail to differentiate into cartilage nodules. On the other hand, cartilage nodules are observed in cultures prepared from all subsequent stage limbs, although there is a linear decrease in the size of nodules between stage 16–17 and middle-late stage 21 cultures. This decrease correlates with simultaneous decreases in both the proportion of aggregating cells and the extent of dibutyryl cyclic AMP-stimulated cartilage formation. At the same time, observations indicate that the proportions of nonaggregating and nonchondrogenic mesenchyme, myogenic cells, and, perhaps, fibrogenic mesenchyme are increasing. The only exceptions to these patterns are observed in cultures from middle-late stage 21 limbs, when cartilage differentiation in situ is already extensive. Unlike earlier stage cultures, which form nearly identical numbers of aggregates and nodules, middle-late stage 21 cultures form variable numbers of aggregates, only a few of which differentiate into cartilage nodules. Middle-late stage 21 cultures also contain unexpectedly low numbers of myogenic cells/unit area of culture. Based on changes in the in vitro histogenic capacities, it is concluded that concurrent with a progression of morphogenic events in the limb, there is a progression of changes in the relative proportions of cell subpopulations. Both the existence of the different subpopulations and the changes in their relative proportions can be detected in vitro. Furthermore, it is concluded that cartilage formation in the limbs of both mouse and chick embryos probably occurs according to very similar developmental programs.     

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.     

The effect of avian retroviruses on limb bud chondrogenesis in vitro

Mesenchymal cells isolated from stage 24 embryonic chicken limb buds were infected with the temperature-sensitive transformation mutants of Rous sarcoma virus tsNY68, tsNY10 and tsLA25 at the nonpermissive temperature for transformation (41 degrees C). Virus infection greatly inhibited subsequent limb bud chondrogenesis under nontransforming conditions, as indicated by a reduction in the rate of 35SO4 incorporation into cell-associated proteoglycans. The inhibition of chondrogenesis was directly related to the percentage of cells infected with tsNY68 at 41 degrees C. The observed inhibition of chondrogenesis was independent of src gene expression since this effect was also caused by many viruses which lack the src gene, including the leukosis viruses RAV-1, RAV-2 and MAV-2(0); the src deletion mutant RSVtd107; and the reticuloendotheliosis viruses REV-T and SNV. Infection of mesenchymal cells with tsNY68 under nontransforming conditions did not cause changes in parameters such as the rate of thymidine incorporation, total cell DNA and total cell protein. Infection with tsNY68 at 41 degrees C resulted in altered kinetics of 35SO4 incorporation into cell-associated proteoglycans and a corresponding reduction in 35SO4-labeled proteoglycans extracted from the cell layer. There were no apparent quantitative effects on the rate of accumulation of proteoglycans in the culture medium. The proteoglycans extracted from the cells and the collected medium of tsNY68-infected cultures were smaller than those of uninfected cultures, as shown by agarose gel chromatography.     

Immunological and biochemical studies of collagen type transition during in vitro chondrogenesis of chick limb mesodermal cells

This work describes an approach to monitor chondrogenesis of stage-24 chick limb mesodermal cells in vitro by analyzing the onset of type II collagen synthesis with carboxymethyl-cellulose chromatography, immunofluorescence, and radioimmunoassay. This procedure allowed specific and quantitative determination of chondrocytes in the presence of fibroblasts and myoblasts, both of which synthesize type I collagen. Chondrogenesis was studied in high-density cell preparations on tissue culture plastic dishes and on agar base. It was found that stage-24 limb mesenchymal cells initially synthesized only type I collagen. With the onset of chondrogenesis, a gradual transition to type II collagen synthesis was observed. In cell aggregates formed over agar, type II collagen synthesis started after 1 day in culture and reached levels of 80-90 percent of the total collagen synthesis at 6-8 days. At that time, the cells in the center of the aggregates had acquired the typical chondrocyte phenotype and stained only with type II collagen antibodies, whereas the peripheral cells had developed into a "perichondrium" and stained with type I and type II collagen antibodies. On plastic dishes plated with 5 X 10(6) cells per 35mm dish, cartilage nodules developed after 4-6 days, but the type II collagen synthesis only reached levels of 10-20 percent of the total collagen. The majority of the cells differentiated into fibroblasts and myoblasts and synthesized type I collagen. These studies demonstrate that analysis of cell specific types of collagen provides a useful method for detailing the specific events in the differentiation of mesenchymal cells in vitro.     

Dual roles of Wnt signaling during chondrogenesis in the chicken limb

Long bones of the appendicular skeleton are formed from a cartilage template in a process known as endochondral bone development. Chondrocytes within this template undergo a progressive program of differentiation from proliferating to postmitotic prehypertrophic to hypertrophic chondrocytes, while mesenchymal cells immediately surrounding the early cartilage template form the perichondrium. Recently, members of the Wnt family of secreted signaling molecules have been implicated in regulating chondrocyte differentiation. We find that Wnt-5a, Wnt-5b and Wnt-4 genes are expressed in chondrogenic regions of the chicken limb: Wnt-5a is expressed in the perichondrium, Wnt-5b is expressed in a subpopulation of prehypertrophic chondrocytes and in the outermost cell layer of the perichondrium, and Wnt-4 is expressed in cells of the joint region. Misexpression experiments demonstrate that two of these Wnt molecules, Wnt-5a and Wnt-4, have opposing effects on the differentiation of chondrocytes and that these effects are mediated through divergent signaling pathways. Specifically, Wnt-5a misexpression delays the maturation of chondrocytes and the onset of bone collar formation, while Wnt-4 misexpression accelerates these two processes. Misexpression of a stabilized form of beta-catenin also results in accelerated chondrogenesis, suggesting that a beta-catenin/TCF-LEF complex is involved in mediating the positive regulatory effect of Wnt-4. A number of the genes involved in Wnt signal tranduction, including two members of the Frizzled gene family, which are believed to encode Wnt-receptors, show very dynamic and distinct expression patterns in cartilaginous elements of developing chicken limbs. Misexpression of putative dominant-negative forms of the two Frizzled proteins results in severe shortening of the infected cartilage elements due to a delay in chondrocyte maturation, indicating that an endogenous Wnt signal does indeed function to promote chondrogenic differentiation.     

Stage-related capacity for limb chondrogenesis in cell culture.

Cells from wing buds of varying-stage chick embryos were dissociated and grown in culture to test their capacity for cartilage differentiation. Micro-mass cultures were initiated with a cell layer greater than confluency, which occupied a restricted area of the culture dish surface (10–13 mm²). Cells from stage 24 chick embryo wing buds (prior to the appearance of cartilage in vivo) undergo cartilage differentiation in such cultures. Typically, during the first 1–2 days of culture, cells form aggregates (clusters of cells with a density 1.5 times greater than that of the surrounding nonaggregate area). By Day 3, virtually all aggregates differentiate into cartilage nodules which are easily recognized by their Alcian blue staining (pH 1.0) extracellular matrix. Subsequently, nodules increase in size, and adjacent nodules begin to coalesce. Micro-mass cultures were used to test the chondrogenic capacity of wing bud cells from chick embryos representing the different stages of limb development up to the appearance of cartilage in vivo (stages 17–25). Cells from embryo stages 21–24 form aggregates which differentiate into cartilage nodules in vitro with equal capacity (scored as number of nodules per culture). In contrast, cells from embryo stages 17–19 form aggregates in similar numbers, but these aggregates never differentiate into nodules under routine conditions. However, aggregates which form in cultures of stage 19 wing bud cells do differentiate into cartilage nodules if exposed to dibutyryl cyclic AMP and theophylline. Cells from stage 20 embryos manifest a varying capacity to form cartilage nodules; apparently, this is a transition stage. Cells from stage 25 embryos produce cartilage in vitro without forming either aggregates or nodules. Based on the results presented in this paper, the authors propose a model for cartilage differentiation from embryonic mesoderm cells involving: (1) aggregation, (2) acquisition of the ability to respond to the environment in the aggregate, (3) elevated intracellular cyclic AMP levels, and (4) stabilization and expression of cartilage phenotype.     

Platelet-derived growth factor A modulates limb chondrogenesis both in vivo and in vitro

Cartilage formation in the chick limb follows rapid proliferation, condensation and differentiation of limb mesenchyme. The control of these early events is poorly understood. Platelet-derived growth factor receptor alpha (PDGFR-alpha) is present throughout the mesenchyme of early chick limb buds, while its ligand, PDGF-A, is expressed in the surrounding epithelium. PDGFR-alpha is down-regulated in areas that will not give rise to cartilage and is then lost from cartilage forming areas after they begin to differentiate. PDGF-A increases chondrogenesis in micromass cultures of stage-20-24 limb buds, but not stage 25, where it inhibits chondrogenesis. Ectopic PDGF-A in the chick wing can lead to either a localized increase in cartilage formation, or an inhibition. Inhibition of PDGF signalling in the chick limb results in the loss of cartilage. These data demonstrate that PDGF-A functions to promote chondrogenesis at early stages of limb development and suggest that it inhibits chondrogenesis at later stages.     

Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development

Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.     

Inhibition of limb chondrogenesis by fibronectin

Abstract. This study compares the chondrogenic capacity of high density cultures prepared from either the develop-mentally younger, distal region or more advanced proximal region of stage 23/24 limb mesenchyme in high density cultures. Distal cultures undergo extensive chondrogenesis whether in F₁₂ medium supplemented with 10% fetal calf serum, 5% fetal calf serum, or fibronectin. On the other hand, proximal cultures fail to undergo chondrogenesis in medium containing 10% fetal calf serum or fibronectin, but do form cartilage in medium containing a decreased serum concentration or no serum. Furthermore, if the cells are cultured at low densities in native type I collagen gels, proximal cells have a reduced chondrogenic capacity in the presence of fibronectin, while chondrogenesis by distal cells is unaffected by the addition of fibronectin. The results demonstrate that proximal and distal cells respond differentially to serum and to fibronectin, and they suggest that the response of the cell to prevalent components of the extracellular matrix might change with development.     

Inhibition of limb chondrogenesis in vitro by vitamin A: alterations in cell surface characteristics

Mesenchyme cells derived from embryonic mouse limb buds were cultured at high cell density. During the first 24 h in culture, groups of mesenchyme cells condensed and formed cell contacts and specialized junctions. These condensations were the nodule primordia which gave rise to cartilage nodules. The cell contacts were lost as the mesenchyme cells in the primordia developed into cartilage nodules. The mature nodules contained chondrocytes isolated from one another by an extensive extracellular matrix consisting of cartilage type collagen fibrils and proteoglycan granules. The differentiation of the mesenchyme cells to chondrocytes was also characterized by the loss of a 240,000-MW cell surface glycoprotein and the appearance of an 80,000-MW surface protein. The addition of vitamin A to the medium on Day 1 inhibited chondrogenesis. The cells were closely packed together, and the limited extracellular space contained thick, banded collagen fibrils with no proteoglycan granules. The cells exhibited extensive areas of close membrane contact and specialized junctions. Vitamin A-treated cultures also retained the 240,000-MW surface glycoprotein and retarded the appearance of the 80,000-MW cell surface protein. The results of this study suggest that cell surface features normally present on mesenchyme cells are maintained and exaggerated by vitamin A.     

Alterations in cell surface galactosyltransferase activity during limb chondrogenesis in brachypod mutant mouse embryos

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb-bud. We have previously shown that this defect is localized to the expression of terminal N-acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild-type (+/+), heterozygote (+/bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High-Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild-type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild-type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene-dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb-bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb-buds.     

The enhancement of in vitro survival and chondrogenesis of limb bud cells by cartilage conditioned medium

Dissociated stage 21–28 chick embryo limb bud cells showed an increasing ability to produce cartilage colonies in vitro with in vivo maturation. In addition dissociated stage 21–28 chick embryo limb bud cells exposed to cartilage conditioned medium continuously or only for 48 hr prior to subculture showed an enhanced (as much as 15-fold) ability to form differentiated cartilage colonies. By this criterion, cells were more responsive to conditioned medium prior to stage 25. Conditioned medium from fibroblast cultures caused an inhibition of cartilage colony formation, suggesting that the effect is cell-type specific. Besides increasing cartilage colony formation by enhanced cell survival, the incorporation of S³⁵O₄ into isolated glycosaminoglycans is also stimulated when limb bud cells are exposed to cartilage conditioned medium. The results support a model for cell differentiation which involves the enhancement of a particular differentiated capacity by a diffusible cell-type-specific macromolecule.     

Evaluation of the sensitive step of inhibition of chondrogenesis by retinoids in limb mesenchymal cells in vitro

The sensitive step of inhibition of chondrogenesis in vitro by retinoids was investigated in modified micromass cultures of limb bud mesenchymal cells from mouse embryos of day 11 and 12. Evaluation of chondrogenesis was performed after alcian blue staining, using a simple random hit counting of cartilage nodules. All-trans-retinoic acid, 13-cis-retinoic acid, and a newly developed arotinoid, RO 13-6298, were tested for their ability to inhibit chondrogenesis. We found that inhibition of chondrogenesis depended on the dosage and the duration of treatment with the different retinoids. Further analysis showed that chondrogenesis in limb bud mesenchymal cells from the proximal part was irreversibly inhibited after one hour of treatment, whereas distal cells showed a reduction of cartilage development only after a treatment period of 12 and more hours. In respect to the doses of the retinoids, proximal cells were about one magnitude more vulnerable than distal cells. These proximo-distal differences were obtained with 13-cis-retinoic acid at 10 micrograms/ml, with all-trans-retinoic acid at 1 microgram/ml and with arotinoid RO 13-6298 with 10 ng/ml. It is supposed that the late blastemal stage of chondrogenic differentiation before the onset of matrix synthesis is the step which is most vulnerable to retinoid treatment.     

Cyclic nucleotides during chondrogenesis: concentration and distribution in vivo and in vitro

This study correlates endogenous levels of cAMP and cGMP with their immunohistochemical localization during chondrogenic differentiation of C57B1/6J mouse limb mesenchyme in vivo and in vitro. A transient decrease in cGMP but not cAMP was found from days 12 to 13 in vivo correlating with early stages of chondrogenesis in the developing limb. Intracellular levels of both cAMP and cGMP in high density limb mesenchyme cultures increased 25% after 24 hr in culture when aggregate and nodule formation was detectable. When cells were seeded at different initial plating densities to delay the onset of aggregate and nodule formation, increased levels of intracellular cAMP correlated temporally with the appearance of nodules. Both cyclic AMP and cGMP were immunohistochemically localized in perichondrial cells and chondrocytes in vivo and in vitro. Therefore, (1) cAMP levels correlated temporally with the appearance of chondrogenic cells and (2) cAMP and cGMP were immunohistochemically localized to chondrogenic cells. These data indicate that fluctuations of both cAMP and cGMP levels may be involved in limb cartilage differentiation. Although increases in both nucleotides were found to correlate with the onset of chondrogenesis in vitro, in vivo data suggest that the amount of cAMP relative to cGMP rather than the absolute amount of an individual cyclic nucleotide may be more significant in modulating differentiation.