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.     

Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant two- to fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to cAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes.     

Ethanol Exposure Stimulates Cartilage Differentiation by Embryonic Limb Mesenchyme Cells

Studies of neural, hepatic, and other cells have demonstrated thatin vitroethanol exposure can influence a variety of membrane-associated signaling mechanisms. These include processes such as receptor-kinase phosphorylation, adenylate cyclase and protein kinase C activation, and prostaglandin production that have been implicated as critical regulators of chondrocyte differentiation during embryonic limb development. The potential for ethanol to affect signaling mechanisms controlling chondrogenesis in the developing limb, together with its known ability to promote congenital skeletal deformitiesin vivo,prompted us to examine whether chronic alcohol exposure could influence cartilage differentiation in cultures of prechondrogenic mesenchyme cells isolated from limb buds of stage 23–25 chick embryos. We have made the novel and surprising finding that ethanol is a potent stimulant ofin vitrochondrogenesis at both pre- and posttranslational levels. In high-density cultures of embryonic limb mesenchyme cells, which spontaneously undergo extensive cartilage differentiation, the presence of ethanol in the culture medium promoted increased Alcian-blue-positive cartilage matrix production, a quantitative rise in³⁵SO₄incorporation into matrix glycosaminoglycans (GAG), and the precocious accumulation of mRNAs for cartilage-characteristic type II collagen and aggrecan (cartilage proteoglycan). Stimulation of matrix GAG accumulation was maximal at a concentration of 2% ethanol (v/v), although a significant increase was elicited by as little as 0.5% ethanol (approximately 85 mM). The alcohol appears to directly influence differentiation of the chondrogenic progenitor cells of the limb, since ethanol elevated cartilage formation even in cultures prepared from distal subridge mesenchyme of stage 24/25 chick embryo wing buds, which is free of myogenic precursor cells. When limb mesenchyme cells were cultured at low density, which suppresses spontaneous chondrogenesis, ethanol exposure induced the expression of high levels of type II collagen and aggrecan mRNAs and promoted abundant cartilage matrix formation. These stimulatory effects were not specific to ethanol, since methanol, propanol, and tertiary butanol treatments also enhanced cartilage differentiation in embryonic limb mesenchyme cultures. Further investigations of the stimulatory effects of ethanol onin vitrochondrogenesis may provide insights into the mechanisms regulating chondrocyte differentiation during embryogenesis and the molecular basis of alcohol's teratogenic effects on skeletal morphogenesis.     

Subepithelial type II collagen deposition during embryonic chick limb development

Summary Type II collagen is a major component of hyaline cartilage but recent studies have demonstrated the presence of this protein in a variety of interfaces that separate epithelia from mesenchyme, particularly in early stages of embryonic chick development. In the present study an immunohistochemical analysis of the distribution of type II collagen was performed on closely staged wing buds of early chick embryo. This report describes how using two different monoclonal antibodies against type II collagen and the peroxidase or fluorescence staining technique reveals that deposition of type II collagen at the ectoderm-mesenchyme interface occurs in the proximal part of the limb coincidentally with the appearance of this protein in the proximal core region, where chondrogenesis begins (stage 25). Then the staining in the subepithelial region spreads distallly with time, following the progression of the formation of cartilage rudiments. At about 7 days of development type II collagen is present under the apical ectoderm ridge and surrounds completely the wing bud underneath the epithelium. At the same time, another antibody directed against the cartilage-specific proteoglycan core protein only stains the chondrogenic central core of the limb and not the subepithelium. Although type II collagen and cartilage-specific proteoglycan are closely associated in the cartilage, the observations presented here suggest that the deposition of these proteins can be regulated independently during limb formation. The role of type II collagen at the epithelium-mesenchyme interface during limb formation is still to be determined.     

Promotion of embryonic chick limb cartilage differentiation by transforming growth factor-beta

This study represents a first step in investigating the possible involvement of transforming growth factor-beta (TGF-beta) in the regulation of embryonic chick limb cartilage differentiation. TGF-beta 1 and 2 (1-10 ng/ml) elicit a striking increase in the accumulation of Alcian blue, pH 1-positive cartilage matrix, and a corresponding twofold to threefold increase in the accumulation of 35S-sulfate- or 3H-glucosamine-labeled sulfated glycosaminoglycans (GAG) by high density micromass cultures prepared from the cells of whole stage 23/24 limb buds or the homogeneous population of chondrogenic precursor cells comprising the distal subridge mesenchyme of stage 25 wing buds. Moreover, TGF-beta causes a striking (threefold to sixfold) increase in the steady-state cytoplasmic levels of mRNAs for cartilage-characteristic type II collagen and the core protein of cartilage-specific proteoglycan. Only a brief (2 hr) exposure to TGF-beta at the initiation of culture is sufficient to stimulate chondrogenesis, indicating that the growth factor is acting at an early step in the process. Furthermore, TGF-beta promotes the formation of cartilage matrix and cartilage-specific gene expression in low density subconfluent spot cultures of limb mesenchymal cells, which are situations in which little, or no chondrogenic differentiation normally occurs. These results provide strong incentive for considering and further investigating the role of TGF-beta in the control of limb cartilage differentiation.     

Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes

We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences, probably inhibitory in nature, must regulate chondrogenesis in mesectodermal derivatives. (ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Gap junctional communication during limb cartilage differentiation

The onset of cartilage differentiation in the developing limb bud is characterized by a transient cellular condensation process in which prechondrogenic mesenchymal cells become closely apposed to one another prior to initiating cartilage matrix deposition. During this condensation process intimate cell-cell interactions occur which are necessary to trigger chondrogenic differentiation. In the present study, we demonstrate that extensive cell-cell communication via gap junctions as assayed by the intercellular transfer of lucifer yellow dye occurs during condensation and the onset of overt chondrogenesis in high density micromass cultures prepared from the homogeneous population of chondrogenic precursor cells comprising the distal subridge region of stage 25 embryonic chick wing buds. Furthermore, in heterogeneous micromass cultures prepared from the mesodermal cells of whole stage 23/24 limb buds, extensive gap junctional communication is limited to differentiating cartilage cells, while the nonchondrogenic cells of the cultures that are differentiating into the connective tissue lineage exhibit little or no intercellular communication via gap junctions. These results provide a strong incentive for considering and further investigating the possible involvement of cell-cell communication via gap junctions in the regulation of limb cartilage differentiation.     

MEK-ERK signaling plays diverse roles in the regulation of facial chondrogenesis

The extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase pathway, also known as the MEK-ERK cascade, has been shown to regulate cartilage differentiation in embryonic limb mesoderm and several chondrogenic cell lines. In the present study, we employed the micromass culture system to define the roles of MEK-ERK signaling in the chondrogenic differentiation of neural crest-derived ectomesenchyme cells of the embryonic chick facial primordia. In cultures of frontonasal mesenchyme isolated from stage 24/25 embryos, treatment with the MEK inhibitor U0126 increased type II collagen and glycosaminoglycan deposition into cartilage matrix, elevated mRNA levels for three chondrogenic marker genes (col2a1, aggrecan, and sox9), and increased expression of a Sox9-responsive collagen II enhancer-luciferase reporter gene. Transfection of frontonasal mesenchyme cells with dominant negative ERK increased collagen II enhancer activation, whereas transfection of constitutively active MEK decreased its activity. Thus, MEK-ERK signaling inhibits chondrogenesis in stage 24/25 frontonasal mesenchyme. Conversely, MEK-ERK signaling enhanced chondrogenic differentiation in mesenchyme of the stage 24/25 mandibular arch. In mandibular mesenchyme cultures, pharmacological MEK inhibition decreased cartilage matrix deposition, cartilage-specific RNA levels, and collagen II enhancer activity. Expression of constitutively active MEK increased collagen II enhancer activation in mandibular mesenchyme, while dominant negative ERK had the opposite effect. Interestingly, MEK-ERK modulation had no significant effects on cultures of maxillary or hyoid process mesenchyme cells. Moreover, we observed a striking shift in the response of frontonasal mesenchyme to MEK-ERK modulation by stage 28/29 of development.     

The influence of epithelia on cartilage and loose connective tissue formation by limb mesenchyme cultures

In order to explain the observation that normally nonchondrogenic limb mesenchyme forms extensive cartilage in culture, the possibility that limb ectoderm inhibits chondrogenesis is examined. Small pieces of quail or chick ectoderm are grafted onto micromass cultures of wing mesenchyme from stage 23–34 chick embryos. The presence of nonridge wing or several other epithelia results in increased collagen accumulation in the underlying mesenchyme, a delay in cell differentiation, and the eventual formation of loose connective tissue, as determined by transmission electron microscopy. The influence can occur across Nuclepore filters having 0.1-μm-diameter pores and ultrathin Millipore filters, but not across Millipore filters of standard thickness. The influence is not contact dependent since cell processes do not cross these filters. The apical ectodermal ridge has the additional effect of stimulating mesenchymal outgrowth. These results support the idea that the ectoderm plays a direct but negative role in the formation of a chondrogenic core within the developing limb.     

Two distinct regulatory steps in cartilage differentiation

The effect of developmental stage on chondrogenic capacity in high-density cell cultures of chick embryonic wing bud mesenchyme is examined. Mesenchyme from stage 19 embryos forms aggregates of closely associated cells which do not form cartilage matrix, nor contain significant levels of type II collagen that are detectable by immunofluorescence, unless they are treated with dibutyryl cyclic AMP. Mesenchyme from stage 24 embryonic wing buds in high-density cell cultures will spontaneously form cartilage, as defined by electron microscopy and immunofluorescence with antibody to type II collagen. Cultures prepared from stage 26 wings form numerous aggregates which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells morphologically. However, because these cells show considerable intracellular immunofluorescence for type II collagen, they are actually unexpressed cartilage cells. Several treatments, including exposure to dibutyryl cyclic AMP, ascorbic acid and an atmosphere of 5% oxygen, or mixture with small numbers of stage 24 wing mesenchyme cells, stimulate expression, as determined by the accumulation of an Alcian blue-staining matrix and an ultrastructurally recognizable cartilage matrix. Since the addition of similar numbers of differentiated cartilage cells does not stimulate expression of stage 26 cells, it is proposed that initial cartilage expression is dependent on a mesenchyme-specific influence which might be removed by cell dissociation. These studies demonstrate that there are at least two distinct transitions in cartilage differentiation: one involves the conversion of mesenchyme to unexpressed chondrocytes and the second involves mesenchyme-dependent expression of chondrogenic differentiation.     

The differentiation of normal and muscle-free distal chick limb bud mesenchyme in micromass culture

Distal chick wing bud mesenchyme from stages 19 to 27 embryos has been grown in micromass culture. The behavior of cultures comprising mesenchyme located within 350 microns of the apical ectodermal ridge (distal zone mesenchyme) was compared to that of cultures of the immediately proximal mesenchyme (subdistal zone cultures). In cultures of the distal mesenchyme from stages 21-24 limbs, all of the cells stained immunocytochemically for type II collagen within 3 days, indicating ubiquitous chondrogenic differentiation. At stage 19 and 20, this behavior was only observed in cultures of the distal most 50-100 microns of the limb bud mesenchyme. Between stages 25 and 27, distal zone cultures failed to become entirely chondrogenic. At all stages, subdistal zone cultures always contained substantial areas of nonchondrogenic cells. The different behavior observed between distal zone and corresponding subdistal zone cultures appears to be a consequence of the presence of somite-derived presumptive muscle cells in the latter, since no such difference was observed in analagous cultures prepared from muscle-free wing buds. The high capacity of the distal zone for cartilage differentiation supports a view of pattern formation in which inhibition of cartilage is an important component. However, its consistent behavior in vitro indicates that micromass cultures do not reflect the in vivo differences between the distal zones at different stages. The subdistal region retains a high capacity of cartilage differentiation and the observed behavior in micromass reflects interactions with a different cell population.