The Chondroblast and the Chondrocyte*

3 The Chondroblast and the Chondrocyte* Robert A. Kosher

I. II. III. IV. V.

Introduction The Chondrocytic Phenotype Acquisition of the Chondrocytic Phenotype Precartilaginous Mesenchymal Cells Regulation of Cartilage Differentiation A. The Role of the AER in Limb Cartilage Differentiation B. The Role of Cyclic AMP in Limb Cartilage Differentiation C. The Role of Cellular Condensation in Limb Cartilage Differentiation D. The Role of Hyaluronate in Limb Cartilage Differentiation E. The Regulation of Somite Chondrogenesis VI. Maintenance of the Chondrocyte Phenotype References

59 60 64 69 71 71 72 73 74 75 76 78

I. INTRODUCTION We begin with a discussion of the major morphological and biochemical features that distinguish chondrocytes from other cell types. This is followed by a dis­ cussion of the properties of the precursor cells that give rise to chondrocytes and a description of the differentiation of the precursor cells into chondrocytes. Cells in the transition stage between precartilaginous mesenchymal cells and chon­ drocytes are designated chondroblasts. It should be emphasized, however, that the distinction between chondroblasts and chondrocytes is rather arbitrary and is based primarily on the relative maturity of the cells. Next, some studies on the regulation of the conversion of precartilaginous mesenchymal cells into chon­ droblasts and subsequently into chondrocytes are discussed. Finally, factors in­ volved in the maintenance of the chondrocytic phenotype are described briefly.

*Original research described in this chapter was supported, in part, by NSF grant PCM7925907.

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Copyright © 1983 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-319501-2

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II. THE CHONDROCYTIC PHENOTYPE Differentiated chondrocytes are typically rounded or polygonal cells with scal­ loped borders whose cytoplasm contains an extensive network of rough endo­ plasmic reticulum, large Golgi complexes, and secretory vacuoles (Godman and Porter, 1960; Goel, 1970; Searls etal., 1972; Thorogood and Hinchliffe, 1975). Although these structural features reflect the fact that chondrocytes actively synthesize and secrete extracellular matrix components, these cytological char­ acteristics are not unique or diagnostic. Therefore, chondrocytes are more reliably characterized by the extracellular matrix they secrete and which surrounds them. Ultrastructurally, hyaline cartilage matrix is characterized by the presence of numerous 2 0 - 7 0 nm diameter, densely staining granules and thin unhanded or faintly banded fibrils representing the proteoglycan and collagenous components of the matrix, respectively (Matukas et al.y 1967; Anderson and Sadjera, 1971; Searls etaL, 1972; Minor, 1973; Levitt et al., 1975; Pennypacker and Goetinck, 1976a). In the light microscope hyaline cartilage matrix appears relatively ho­ mogenous and structureless and is characterized by its ability to stain metachromatically with toluidine blue or positively with Alcian blue (at low pH). These staining characteristics reflect the high concentration of polyanionic sul­ fated glycosaminoglycans present in the matrix. The predominant glycosaminoglycans synthesized by chondrocytes are chon­ droitin 4- and 6-sulfates. Although the production of very high quantities of these sulfated glycosaminoglycans is characteristic of chondrocytes and has served as a very useful criterion in studies of chondrogenic differentiation (see, for example, Levitt and Dorfman, 1973; Kosher and Lash, 1975; Kosher et al., 1979a,b; Kosher and Savage, 1980), the synthesis of these molecules is not a qualitatively unique feature of chondrocytes. These glycosaminoglycans are synthesized at relatively low levels by precar­ tilaginous mesenchymal cells and also by a variety of nonchondrogenic tissues in the embryo and adult (Franco-Browder et al., 1963; Searls, 1965a,b; Lash, 1968; Kosher and Searls, 1973; Abrahamson et al., 1975). It is important to note, however, that sulfated glycosaminoglycans in situ are not free polysac­ charide chains; they are integral components of much larger proteoglycan mol­ ecules in which the glycosaminoglycan chains are covalently attached to a protein backbone or core. An average proteoglycan molecule (proteoglycan monomer) is thought to consist of a core protein (molecular weight 200,000) to which are covalently bound about 100 chondroitin sulfate glycosaminoglycan chains (each with molecular weight of about 20,000) and about 3 0 - 6 0 keratan sulfate chains (each with molecular weight of 4,000-8,000) (Hascall and Heinegard, 1975; Hascall, 1977). Furthermore, in cartilage matrix, proteoglycan molecules are associated with huge macromolecular aggregates in which individual proteogly­ can molecules (monomers) are noncovalently bound to a hyaluronate polymer via a region of the protein core at one end of the molecule (the hyaluronate-

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binding region) (see Hascall and Heinegard, 1975; Hascall, 1977; and, Hascall and Hascall, 1981 for reviews). The interaction of proteoglycan monomers with hyaluronate is stabilized by one or more link proteins. Differences may exist in the kinds of sulfated proteoglycans produced by chondrocytes compared to those produced by precartilaginous mesenchymal cells and nonchondrogenic cell types. It has been demonstrated that proteoglycans synthesized by embryonic chick chondrocytes can be separated into two major fractions by molecular sieve chromatography on either agarose (Palmoski and Goetinck, 1972; Levitt and Dorfman, 1974) or controlled-pore glass beads (Lever and Goetinck, 1976). The larger of these fractions comprises about 90% of the total proteoglycan produced by embryonic chondrocytes and consists of aggre­ gated and unaggregated proteoglycan monomers (Goetinck et al., 1974; Lever and Goetinck, 1976). Synthesis of this larger proteoglycan fraction by sternal and limb chondrocytes is selectively inhibited by 5-bromodeoxyuridine (Palmoski and Goetinck, 1972; Levitt and Dorfman, 1974) and is drastically reduced in chondrocytes of the cartilage-defective nanomelic mutant chick embryo (Pal­ moski and Goetinck, 1972; Pennypacker and Goetinck, 1976a; McKeown and Goetinck, 1979). Thus, it has been suggested that the larger proteoglycan fraction represents a cartilage-specific species and the smaller fraction, a ubiquitous nonspecific species (Palmoski and Goetinck, 1972; Levitt and Dorfman, 1973, 1974). Although precartilaginous limb mesenchymal tissue produces a small amount of a proteoglycan fraction that elutes in the same position as the large proteo­ glycan species during molecular sieve chromatography (Goetinck et al., 1974; Royal et al., 1980), the cartilage proteoglycan species can be separated from the limb mesenchymal tissue species by sucrose density gradient centrifugation in the presence of 4 M guanidinium chloride (Royal et al., 1980). Sucrose density gradient centrifugation in the presence of 4 M guanidinium chloride has also been used to demonstrate that embryonic chondrocytes synthesize a large pro­ teoglycan species produced neither by precartilaginous mesenchymal cells nor by a variety of nonchondrogenic cell types (Okayama et al., 1976; Kitamura and Yamagata, 1976). Even more compelling evidence supporting the existence of a cartilage-specific proteoglycan molecule derives from immunological studies. Antibodies prepared against the large proteoglycan monomers of 4-week old chick sternal cartilage do not cross-react with the proteoglycans produced by either precartilaginous limb mesenchymal cells (Royal et al., 1980) or chick skin (Sparks et al., 1980). The antibodies do, however, cross-react with proteoglycans produced by em­ bryonic chick and quail sternal cartilage, chick Meckel's cartilage, and chick limb cartilage (Sparks et al., 1980; Royal et al., 1980). Similarly, antibodies prepared against epiphyseal cartilage proteoglycan monomer from which most of the chondroitin sulfate glycosaminoglycan chains have been removed by hyaluronidase treatment show little, if any, cross-reactivity with the proteogly-

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cans of precartilaginous limb mesenchymal cells (Ho et al., 1977). Furthermore, immunohistochemical studies show that limb mesenchymal cells that have dif­ ferentiated into cartilage in high-density monolayer culture react with antibodies to hyaluronidase-digested cartilage proteoglycan, whereas noncartilage cells in the same cultures do not (Vertel and Dorfman, 1978). These immunological studies and the biochemical studies previously described indicate that chondrocytes produce a unique proteoglycan molecule not produced by precartilaginous mesenchymal cells or nonchondrogenic cell types. The im­ munological studies also suggest that the cartilage-specific proteoglycan mole­ cules may differ in their protein cores from those produced by prechondrogenic or nonchondrogenic cells. Genetic evidence that cartilage-specific proteoglycan possesses a unique protein core has been presented by Goetinck (1981). In addition, cartilage proteoglycan molecules appear to differ from those produced by precartilaginous mesenchymal cells in their possession of smaller chondroitin sulfate chains, higher ratio of chondroitin 4-sulfate to chondroitin 6-sulfate, and larger keratan sulfate chains (Hascall et al., 1976; DeLuca et al., 1977; Caplan, 1981). As previously described, a large proportion of the proteoglycan molecules of mature cartilage matrix interact with hyaluronate to form huge macromolecular complexes. During the course of cartilage differentiation in vitro (DeLuca et al., 1977) and in vivo (Vasan and Lash, 1977, 1979), there appears to be a progressive increase in the proportion of proteoglycan monomers that aggregate into such complexes. In fact, early studies indicated that proteoglycans of precartilaginous mesenchymal cells were not capable of interacting appreciably with hyaluronate (DeLuca et al., 1977; Vasan and Lash, 1979), suggesting the possibility that formation of proteoglycan aggregates might be cartilage-specific. However, Royal et al., (1980) have provided evidence indicating that a large proportion of the proteoglycan molecules of precartilaginous limb mesenchymal tissue is aggregated in vivo and that isolated proteoglycan monomers of prechondrogenic tissue are capable of interacting and forming aggregates with exogenous hyaluro­ nate. Furthermore, proteoglycans of several nonchondrogenic tissues are capable of interacting and forming aggregates with hyaluronate (Oegema et al., 1979; McMurtrey et al., 1979; Caterson and Baker, 1979), suggesting that this property is not a unique characteristic of chondrogenic or prechondrogenic tissue. Finally, it should be noted that Vasan and Lash (1977) have presented evidence indicating that precartilaginous limb mesenchymal tissue contains only one of the two link proteins that stabilize the binding of proteoglycan monomers to hyaluronate in mature cartilage. Whether or not the presence of both link proteins is unique to mature cartilage has not been thoroughly investigated, however. The second major component of extracellular cartilage matrix is collagen. The predominant collagenous component of hyaline cartilage matrix is a specific molecular species called type II collagen (Miller and Matukas, 1969; Miller,

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1976), although small amounts of other collagen species including type V may also be present (Rhodes and Miller, 1978; Burgeson and Hollister, 1979; Shellswell et al., 1980). Type II collagen is a triple helical molecule consisting of three identical polypeptide chains (designated α ϊ (II) chains), each with molec­ ular weight of about 95,000. The α ϊ (II) chains of type II collagen are distinct gene products differing in primary structure from the α chains that constitute the other four known collagen types (types I, III, IV, and V) (see Eyre, 1980, Bornstein and Sage, 1980, and Mayne and von der Mark, Chapter 7 in this volume, for reviews of the structure of different collagen species). Because of its widespread presence in hyaline cartilages from a variety of sources and its absence from tissues such as bone, tendon, and skin (Miller, 1976), type II collagen has frequently been referred to as cartilage-specific. Biochemical and immunological studies have shown that type II collagen is not produced by precartilaginous mesenchymal cells. Rather, they produce the more ubiquitous type I collagen (Linsenmayer et al., 1973a; von der Mark et al., 1976; von der Mark and von der Mark, 1977; Dessau et al., 1980). Also, type II collagen is not produced by myogenic and connective tissue cells that differentiate in association with limb cartilage in vivo and in vitro (Linsenmayer et al., 1973a; von der Mark et al., 1976; von der Mark and von der Mark, 1977; Bailey et al., 1979; Dessau et al., 1980), nor is it produced by limb mesenchymal cells whose differentiation into cartilage in vitro has been inhibited by 5bromodeoxyuridine (Levitt and Dorfman, 1974) or by low-density culture con­ ditions (von der Mark and von der Mark, 1977). It is important, however, to note that type II collagen is synthesized by some nonchondrogenic tissues. Type II collagen is produced by the embryonic notochord (Linsenmayer et al., 1973b; von der Mark et al., 1976; Miller and Mathews, 1974), embryonic chick corneal epithelium (Linsenmayer et al., 1977; von der Mark et al., 1977), and neural retina (Newsome etal., 1976; Smith etal., 1976; Linsenmayer and Little, 1978). Thus, strictly speaking, type II collagen is not a cartilage-specific molecule. However, it can certainly be considered cartilage-characteristic, because it is produced by only a limited number of nonchondrogenic cell types and is not produced by precartilaginous mesenchymal cells or the fibroblastic and myogenic cells that differentiate in association with cartilage. In summary, the major biochemical features that distinguish chondrocytes from precartilaginous mesenchymal cells and from most nonchondrogenic cells are their ability to synthesize a genetically distinct collagen species (type II) and a cartilage-specific sulfated proteoglycan that appears to differ from noncartilage sulfated proteoglycan in the protein core of the molecule as well as in other chemical characteristics. Double immunofluorescence reactions with specific antibodies have been used to locate cartilage proteoglycan and type II collagen intracellularly in the same chondrocyte, indicating that both molecules are syn­ thesized simultaneously in the same cell (Vertel and Dorfman, 1979). Because

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neither of these molecules is produced by precartilaginous mesenchymal cells, cartilage differentiation appears to involve selective activation of the genes re­ quired for the synthesis of these specific products.

III. ACQUISITION OF THE CHONDROCYTIC PHENOTYPE During embryonic development, chondrocytes differentiate from mesenchymal cells. The mesenchymal cells giving rise to limb and vertebral chondrocytes are derived from mesoderm, whereas the mesenchymal cells that differentiate into much of the cranial cartilage arise from the ectodermally derived neural crest. In general, during the course of chondrogenic differentiation, there is a period during which precursor cells have initiated a sequence of changes indicative of their differentiation into cartilage, but have not yet acquired all phenotypic characteristics of well differentiated chondrocytes nor become surrounded by a metachromatic cartilage matrix. Cells in this transition period of cartilage dif­ ferentiation are referred to as chondroblasts to distinguish them from the pre­ cartilaginous mesenchymal cells from which they arise and the mature chondrocytes into which they differentiate. The sequence of changes whereby precartilaginous mesenchymal cells differ­ entiate into chondroblasts and subsequently into chondrocytes has been most carefully studied in the embryonic chick limb bud. The morphological changes occurring during the process of cartilage differentiation in the limb bud are illustrated diagrammatically in Fig. 1. Following its initial formation during the third day of development, the embryonic chick limb bud essentially consists of a bulge of mesodermal cells surrounded by a thin rim of ectoderm. The ectoderm extending along the distal periphery of the limb bud is a thickened cap of epithelium called the apical ectodermal ridge (AER) (Fig. 1). As described in detail later in this chapter, the AER plays a critical role not only in limb mor­ phogenesis but also in the process of cartilage differentiation. The mesodermal cells constituting the bulk of the limb bud during the earliest stages of development (stages 16-22; Hamburger and Hamilton, 1951) appear to be unspecialized mesenchymal cells that are virtually identical both ultrastructurally and biochemically. Ultrastructurally, the mesodermal cells at these early stages constitute a loosely constructed network of cytologically unspecialized mesenchymal cells separated from one another by fairly extensive intercellular spaces (Searls etal., 1972; Thorogood and Hinchliffe, 1975; Singley and Solursh, 1981). Biochemically, the mesenchymal cells are characterized by low production of sulfated glycosaminoglycans (Searls, 1965a,b) and nonspecific type I collagen (Linsenmayer et al., 1973a; von der Mark et al., 1976; Dessau et al., 1980). The first morphological change marking the initiation of chondrogenic dif­ ferentiation and the transition from precartilaginous mesenchymal cell to chondroblast occurs in the proximal central core of the limb at late stage 22 to early

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21

65

22+/23

24

25

STAGE Fig. 1. Diagrammatic representation of the temporal and spatial progression of chondrogenic differentiation in the embryonic chick limb bud. Each drawing represents a mid-frontal section through a limb bud at the indicated stages of development (see Hamburger and Hamilton, 1951). Stippled areas represent chondroblasts in the proximal-central core of the limb that have initiated the process of cartilage differentiation but are not yet surrounded by metachromatic cartilage matrix. The darkened area at stage 25 indicates the ap­ proximate region in which a metachromatic matrix is first detectable. The dotted line represents the proximal limit of the undifferentiating subridge region. Note the apical ectodermal ridge extending along the distal periphery of this region. Adapted from Stark and Searls (1973).

stage 23 (Fig. 1), about 1 day prior to the time metachromatic cartilage matrix is first detectable in the limb. At this time, the cells in the central proximal core undergo a process that has been termed condensation, that is, the cells that were previously separated from one another by rather extensive intercellular spaces become closely packed and have large areas of close surface contact (Fell and Canti, 1934; Summerbell and Wolpert, 1972; Thorogood and Hinchliffe, 1975). A similar widespread cellular condensation or aggregation precedes overt car­ tilage formation by limb mesenchymal cells in high-density monolayer culture (see, for example, Ahrens et al., 1977; Ede et al., 1977; Hassell et al., 1978a; Lewis et al., 1978) and in organ culture (Kosher et al., 1979a,b). As discussed in detail later in this chapter, during this cellular condensation process a homotypic cell-cell interaction occurs that is necessary to trigger chondrogenic dif­ ferentiation. Following the initiation of condensation at late stage 22 to early stage 23, the closely packed cells gradually separate as the result of deposition of extracellular matrix until, at stage 25, the cells in the proximal central core are surrounded by metachromatic cartilage matrix (Fell and Canti, 1934; Searls, 1965a; 1973; Searls etal., 1972). As cells in the proximal central core of the limb (the so-called chondrogenic area) initiate cartilage differentiation at late stage 22 to early stage 23, cells in the proximal peripheral regions of the limb undergo a sequence of changes involved in muscle and connective tissue differentiation (Hilfer et al., 1973) (Fig. 1). However, it is important to note that as cells in the proximal regions of the limb initiate differentiation, those mesenchymal cells directly subjacent

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to the AER (i.e., cells extending approximately 0.4 mm from the AER) retain the characteristics of unspecialized mesenchymal cells and exhibit no signs of differentiation (Searls, 1965a; Stark and Searls, 1973) (Fig. 1). During subse­ quent stages of development, as the limb undergoes polarized outgrowth in a proximal-to-distal direction, the size of the nondifferentiating subridge region remains constant (Searls, 1965a, 1973; Stark and Searls, 1973), although cells in the subridge region double in number every 11-13 hours (Janners and Searls, 1970) (see Fig. 1). Apparently, therefore, when polarized limb growth and division in the subridge region causes cells to become located more than about 0.4 mm from the AER, the cells in the proximal central core initiate chondrogenic differentiation, the first morphological indication of which is condensation. It is also important to note that in virtually all early stages of limb development there is a gradation of differentiation along the proximodistal axis of the limb such that cartilage differentiation is more advanced proximally than distally. For example, at stage 25, cells in the central core of the limb immediately proximal to the unspecialized subridge region have initiated condensation (i.e., are closely packed and exhibit large areas of close surface contact), whereas cells in the central core regions of progressively more proximal segments of the limb lose close surface associations as they become separated by matrix (Thorogood and Hinchliffe, 1975; R. A. Kosher and J. A. Grasso, in preparation). In the extreme proximal central core at stage 25 the cells are surrounded by metachromatic cartilage matrix. The first detectable biochemical change marking the transition from precar­ tilaginous mesenchymal cells to chondroblast occurs in the proximal central core of the limb at stages 22 + /23 concomitant with the cellular condensation process 35 described above. At this time, cells in the proximal central core exhibit an autoradiographically detectable, greatly enhanced incorporation of [ S]sulfate into sulfated glycosaminoglycans compared to cells either in the unspecialized subridge region or in the nonchondrogenic proximal peripheral regions of the limb (Searls, 1965a; see also Hinchliffe and Ede, 1973). This enhanced accu­ mulation of sulfated glycosaminoglycans thus occurs about 1 day prior to the 35 time that metachromatic cartilage matrix is first detectable (i.e., at stage 25). It has been suggested that greater incorporation of [ S]sulfate that is detectable autoradiographically in the proximal central core may reflect a decreased rate of synthesis in the proximal peripheral regions of the limb rather than a greatly amplified synthesis in the proximal central core region (Cioffi et al., 1980). However, biochemical studies have demonstrated that the rate 3of accumulation of chondroitin sulfate during a 90-min labeling period with [ H] glucosamine 3-4-fold greater in the central core of the limb following initiation of conden­ sation than in either the undifferentiated subridge region or the proximal pe­ ripheral regions (Kosher et al., 1981). As previously described, one of the major biochemical features that distinguish well differentiated chondrocytes from precartilaginous mesenchymal cells and

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nonchondrogenic cells is their ability to synthesize a cartilage-specific proteo­ glycan molecule that appears to differ from non-cartilage sulfated proteoglycan in the protein core of the molecule as well as in other chemical characteristics. Whether or not the amplified synthesis of chondroitin sulfate occurring with condensation at stages 22 + /23 reflects the initiation of synthesis of this cartilagespecific sulfated proteoglycan has not yet been unequivocally determined, how­ ever. Royal et al., (1980) could not detect the presence of cartilage-specific sulfated proteoglycan at stages 2 3 - 2 4 in whole limb buds. However, this negative result must be interpreted very cautiously, because at these stages only a relatively small number of cells (i.e., those in the proximal central core) have initiated chondrogenic differentiation, and the bulk of the cells either have not yet dif­ ferentiated (i.e., those in the subridge region) or have initiated differentiation into nonchondrogenic tissues (i.e., those in the proximal peripheral regions). Thus, biochemical analyses of whole limbs at this early stage might not be sufficiently sensitive to detect production of cartilage-specific sulfated proteo­ glycan by a small percentage of cells in the total limb. Immunohistochemical studies with antibodies to cartilage-specific proteoglycans should greatly aid in determining precisely when synthesis is initiated during the course of chondro­ genic differentiation. The amplified synthesis of chondroitin sulfate that occurs with condensation and that marks the transition from precartilaginous mesenchymal cells to chon­ droblasts appears to be accompanied by depression of the synthesis of the nonsulfated glycosaminoglycan, hyaluronate. The first indication of this was in a study by Toole (1972) who demonstrated that the amount of hyaluronate syn­ thesized relative to chondroitin sulfate was higher in whole limb buds at stage 23 than at stage 26, when overt cartilage formation was detectable. The synthesis of hyaluronate in various well-defined regions of the limb in which the cells are 3 Rate of accumulation in different phases of differentiation has been examined. of hyaluronate during a 90-min. labeling period with [ H]glucosamine is 3 - 4 fold greater in the undifferentiated subridge region of the limb than in the con­ densing proximal central core region and 2-3-fold greater in the nonchondrogenic proximal peripheral regions than in the condensing central core (Kosher et al., 1981). The significance of the decline in hyaluronate synthesis in the condensing proximal central core to the regulation of chondrogenic differentiation is dis­ cussed later in this chapter. As previously described, well-differentiated chondrocytes are characterized by synthesis of cartilage-characteristic type II collagen, whereas precartilaginous mesenchymal cells synthesize type I collagen. Although morphological and bio­ chemical indications of cartilage differentiation first occur at late stage 22 in the proximal central core of the limb, the presence of type II collagen cannot be detected in this region until stage 24 (Dessau et al., 1980). However, it is noteworthy that type II collagen synthesis is initiated prior to the appearance of a metachromatic cartilage matrix which is first detectable at stage 25. Similarly,

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during the chondrogenesis of limb mesenchymal cells in high-density monolayer culture, the presence of type II collagen is detectable immunohistochemically a day or so prior to the appearance of histochemically detectable cartilage matrix (see Sasse and von der Mark, referred to in von der Mark, 1980). It should also be noted that initiation of type II collagen synthesis by chondroblasts in the proximal central core of the limb at stage 24 is apparently not accompanied by cessation of type I collagen synthesis (Dessau et al., 1980). In fact, the presence of both type I and type II collagen in the chondrogenic core of the limb is detectable immunohistochemically until at least stage 27 (Dessau et al., 1980; Shellswell et al., 1980). An extensive review on the transitions in collagen types during the course of chondrogenic differentiation in vivo and in vitro has been published by von der Mark (1980). In summary, during the process of chondrogenesis in the limb, there is a transition period extending from late stage 22 through stage 24 during which cells in the proximal central core of the limb (chondroblasts) have initiated a sequence of changes indicative of their differentiation into cartilage, but have not yet become surrounded by a metachromatic cartilage matrix. This transition period is initiated by precartilaginous limb mesenchymal cells undergoing a cellular condensation process that is accompanied by greatly amplified synthesis of chondroitin sulfate and a striking depression in the synthesis of the nonsulfated glycosaminoglycan, hyaluronate. The synthesis of type II collagen is initiated late in this transition period (i.e., at stage 24). In addition, the transition from precartilaginous mesenchymal cells to chondroblasts is accompanied by a decline in the rate of cell division (Janners and Searls, 1970). By late stage 22, the proliferative index of chondroblasts in the proximal central core is 25% compared to 75-100% observed in the nondifferentiating subridge region and in the non­ chondrogenic proximal peripheral regions of the limb (Janners and Searls, 1970). Furthermore, initiation of chondrogenic differentiation by chondroblasts consti­ tuting the proximal central core from late stage 22 through stage 24 is indicated by a strong bias toward cartilage formation in vitro compared to cells of the proximal peripheral regions of the limb (Ahrens et al., 1979). When cells from the core and peripheral regions of stage 23-24 limb buds are separately subjected to high-density monolayer culture, the cells from the core undergo extensive cartilage differentiation, but cells from the periphery form little cartilage (Ahrens etal., 1979). Although the chondroblasts constituting the proximal central core of stage 22 4- through stage 24 limb buds initiate the process of cartilage differentiation and exhibit a strong bias toward cartilage formation in vitro, most of the cells apparently are not irreversibly committed to cartilage formation. This contrasts strikingly to the chondrocytes surrounded by a metachromatic matrix that con­ stitute the proximal central core of stage 25 and older limb buds. When blocks of tissue from the central core regions of stage 22 through stage 24 limb buds are transplanted into the nonchondrogenic proximal peripheral regions of host

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stage 24 or younger limb buds, the transplanted pieces of tissue in the vast majority of cases do not continue cartilage differentiation but rather become relegated to their new position in the host limb and form nonchondrogenic tissues (Searls, 1967; Searls and Janners, 1969). In contrast, when proximal central core tissue from stage 25 or older limb buds is similarly transplanted, more than 90% of the implants form ectopic cartilage in the nonchondrogenic regions of the limb (Searls, 1967; Searls and Janners, 1969). It has been suggested that, at stage 25, cells in the central core of the limb have become stabilized as cartilage, a result of the acquisition of more than threshold amounts of some stabilizing material (Searls and Janners, 1969; Searls, 1973). Thus, the transition from chondroblasts to chondrocytes surrounded by a metachromatic matrix ap­ pears to be accompanied by the acquisition of stability, in that the tissue has acquired the ability to maintain its phenotype in the presence of influences from the periphery of the limb that normally elicit the formation of nonchondrogenic tissues.

IV. PRECARTILAGINOUS MESENCHYMAL CELLS As previously described, the mesodermal cells constituting the prechondrogenic limb bud prior to stage 22 and those constituting the subridge region of stage 22 and older limb buds appear to be a homogeneous population of seemingly unspecialized mesenchymal cells that are virtually identical ultrastructurally and biochemically. In addition, a variety of experimental studies indicate these mes­ enchymal cells are identical in their developmental potential (i.e., their ability to subsequently form either the cartilage, muscle, or connective tissue compo­ nents of the limb) and that the ultimate differentiative fate of the cells simply depends upon their location in the limb and the influences they are subjected to in that particular location. For example, it has been demonstrated that chon­ droblasts from the proximal central core of stage 22-24 limb buds differentiate into nonchondrogenic tissues when transplanted into the peripheral, myogenic areas of host limb buds (Searls, 1967; Searls and Janners, 1969). Conversely, tissue from the peripheral, myogenic regions of stage 22-24 embryos differentiate into cartilage when cultured in plasma clots or on the chorioallantoic membrane (Zwilling, 1966) or when grafted into the chondrogenic central core of the limb (Shellswell and Wolpert, 1977). In fact, even well-differentiated skeletal muscle cells respond to appropriate environmental cues by differentiating into cartilage (Nathanson et al., 1978; Nathanson and Hay, 1980a,b). In addition, when the undifferentiated subridge region of the limb is subjected to organ culture in the absence of the AER and dorsoventral ectodermal tissues that normally surround the tissue, virtually all of the cells of the expiant differentiate into cartilage, and nonchondrogenic tissues are not discernible (Kosher et al., 1979a). However, when the subridge mesoderm is cultured in the presence of the dorsoventral

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ectoderm, nonchondrogenic tissues form along the periphery of the expiant subjacent to the ectoderm, and cartilage differentiation only occurs in the central core of the expiant (Kosher et al., 1979a). All of these studies, although not unequivocal, indicate that prechondrogenic mesenchymal cells have the potential to differentiate not only into chondrocytes but also into the nonchondrogenic cell types present in the limb, and that their differentiative fate depends upon their location in the limb and influences they are subjected to in that location (see also Caplan and Koutroupas, 1973). An alternate possibility is that the prechondrogenic limb bud may consist of a heterogeneous group of discrete, predetermined subpopulations of mesenchy­ mal cells, which although phenotypically indistinguishable are restricted in their differentiative potential. One subpopulation might be capable of differentiating only into cartilage (and perhaps the connective tissue cells at the limb), but another subpopulation might ber capable of differentiating only into myogenic cells (see Dienstman et al., 191 4, and Newman et al., 1981, for detailed elab­ oration and defense of this hypothesis). The most compelling evidence consistent with this hypothesis derives from quail-chick transplantation studies demon­ strating that limb musculature is ultimately derived from somitic mesoderm, but limb cartilage and connective tissue are derived from somatic plate (somato­ pleural) mesoderm (Christ et al., 1977; Chevallier et al., 1977). The somitic cells ultimately giving rise to the limb musculature migrate into the limb-forming region of the somatopleural mesoderm at about stage 14, which is prior to the formation of the limb bud (Chevallier, 1978). Because cartilage- and muscleforming cells of the limb have distinct lineages, it has been argued that the prechondrogenic limb bud contains covertly differentiated subpopulations of mesenchymal cells that cannot be distinguished phenotypically (see, for example, Christ et al., 1977; Newman et al., 1981). It is important to note, however, that even if the mesenchymal cells of the limb giving rise to cartilage have an origin different from those giving rise to muscle, this does not in itself eliminate the possibility that all mesenchymal cells might have equivalent differentiative po­ tential. In fact, the limb-forming region of the somatopleural mesoderm can differentiate not only into cartilage and connective tissue but also into muscle when it is explanted as early as stage 10, which is well prior to the time somitic cells migrate into the region (McLachlan and Hornbruch, 1979). Thus, the mesodermal cells giving rise to limb cartilage apparently have the ability to differentiate into muscle even if they normally do not. Whether or not they have the ability to differentiate into other cell types, it is clear that the mesenchymal cells of the limb that differentiate into cartilage differ in several significant respects from the mesenchymal cells of the somites that differentiate into vertebral cartilage. For example, Zwilling (1961, 1968) demonstrated that when dissociated precartilaginous somite and limb mesodermal cells were mixed with one another and allowed to reaggregate, the two types of cells sorted out from one another, indicating that the two cell types possess

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distinctly different surface properties. Furthermore, Zwilling (1961, 1968) showed that when fragmented or dissociated precartilaginous limb mesodermal cells were placed subjacent to the AER in isolated limb-bud ectodermal jackets, the mesodermal cells responded to the AER by undergoing polarized proximalto-distal limb outgrowth. However, when somite cells were placed subjacent to the AER, they showed no indication of outgrowth, but rather formed cartilage nodules. That somite cells fail to respond to the influence of the AER, but limb mesodermal cells do, emphasizes the distinctly different properties of these two cell types. Finally, as described in detail in Section V of this chapter, the reg­ ulatory mechanisms controlling the conversion of limb mesenchymal cells into cartilage are of a distinctly different nature than those controlling the chondro­ genic differentiation of mesenchymal cells of the sclerotome of the somites. Therefore, although the prechondrogenic mesenchymal cells of the limb and somite both differentiate into phenotypically identical cartilage, the two types of mesenchymal cells are not equivalent but are clearly covertly, if not overtly, differentiated from one another.

V. REGULATION OF CARTILAGE DIFFERENTIATION Although development of cartilage is discussed in detail in Volume 2, in view of the preceding discussion of the conversion of precartilaginous limb mesen­ chymal cells into chondroblasts and chondrocytes it seems appropriate to at least briefly describe some studies of the regulation of this process. In addition to limb chondrogenesis, a brief discussion of the regulation of somite chondrogenic differentiation also is presented to emphasize the distinctly different regulatory mechanism operating during differentiation in the two systems. A.

The Role of the AER in Limb Cartilage Differentiation The AER capping the distal periphery of the limb bud is required for the outgrowth and formation of distal limb structures by the mesodermal cells of the limb. Surgical removal of the AER results in the formation of limbs with distal deficiencies (Saunders, 1948; Summerbell, 1974). Grafting an extra AER onto the mesodermal cells results in distal duplications (Saunders and Gasseling, 1968; Zwilling, 1956; Saunders etal., 1976). As previously described, through­ out early limb development mesenchymal cells extending about 0.4 mm from the AER retain the characteristics of unspecialized mesenchymal cells, whereas cells in the proximal central core located more than 0.4 mm or so from the AER have initiated cartilage differentiation (see Fig. 1 and Stark and Searls, 1973). Thus, it has been suggested that one of the major functions of the AER is to maintain the mesenchymal cells directly subjacent to it in a labile undifferentiated condition (Stark and Searls, 1973; Summerbell et al., 1973). Direct evidence supporting this suggestion has been obtained by studying the morphogenesis and differentiation of the subridge mesoderm in the presence and

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absence of the AER in an organ culture system. In the presence of the AER, subridge mesoderm expiants undergo quite normal morphogenesis characterized primarily by progressive polarized proximal-to-distal outgrowth and change in the contour of the developing expiant (Kosher et al., 1979a). As cells of the expiant become located greater than 0.4 mm or so from the AER as a result of polarized outgrowth, they concomitantly initiate chondrogenic differentiation, whereas cells remaining closer to the AER remain undifferentiated (Kosher et al. y1979a). In contrast, when the subridge mesoderm is cultured in the absence of the AER, the cells fail to undergo morphogenesis and rapidly and precociously initiate chondrogenic differentiation (Kosher et al., 1979a). These results indicate that the AER maintains limb mesenchymal cells directly subjacent to it in a labile undifferentiated condition, and that when mesenchymal cells are freed from the influence of the AER either artificially or as a result of polarized proximal-to-distal outgrowth, they are freed to commence cartilage differentia­ tion (Kosher et al., 1979a). Further evidence derives from the observation that when a limb bud ectodermal jacket including the AER is placed on top of and in close contact with a monolayer of stage 19 or stage 20 limb mesodermal cells, the mesodermal cells underlying the AER accumulate under it in an organized fashion, initiate proximal-to-distal outgrowth, but fail to differentiate into car­ tilage (Globus and Vethamany-Globus, 1976). In contrast, mesodermal cells in regions of the same monolayer not covered by AER-containing limb ectoderm fail to show indications of outgrowth, but rather differentiate into cartilage nod­ ules (Globus and Vethamany-Globus, 1976; see also Solursh et al., 1981b). Β. The Role of Cyclic AMP in Limb Cartilage Differentiation A variety of recent studies suggest cyclic AMP plays a key role in the regulation of limb cartilage differentiation. A variety of agents that elevate cyclic AMP levels elicit dose-dependent and specific stimulation of the already precocious chondrogenic differentiation that subridge mesoderm expiants undergo in organ culture in the absence of the AER (Kosher et al., 1979b). In addition, agents that elevate cyclic AMP levels promote chondrogenic differentiation of stage 19 limb mesenchymal cells subjected to high-density monolayer culture under con­ ditions in which chondrogenic differentiation does not normally occur (Ahrens et al., 1977), and these agents also stimulate chondrogenesis of cells from the nonchondrogenic proximal peripheral regions of the limb (Solursh et al., 1981). Furthermore, in the presence of cyclic AMP derivatives, subridge mesoderm expiants cultured in the presence of the AER fail to undergo the striking proximalto-distal outgrowth and contour changes characteristic of control expiants, and the cessation of AER-directed morphogenesis in the presence of these agents is accompanied by the precocious chondrogenic differentiation of the mesenchymal cells (Kosher and Savage, 1980). Agents that elevate cyclic AMP levels enable subridge mesenchymal cells to overcome the negative influence on cartilage differentiation and the positive influence on morphogenesis imposed upon them

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by the AER (Kosher and Savage, 1980). Thus, it has been suggested that when limb mesenchymal cells move from the influence of the AER, their cyclic AMP content increases triggering chondrogenic differentiation (Kosher and Savage, 1980). The increase in cyclic AMP content is thought to result from the cellular condensation and resultant intimate cell-cell interaction that occurs when sub­ ridge mesenchymal cells move from the influence of the AER (Kosher and Savage, 1980; see also Solursh et al, 1978). Support for this latter suggestion derives from the observation that a widespread cellular condensation process has not been detected preceding overt cartilage formation in subridge mesoderm expiants cultured in the presence of cyclic AMP derivatives (Kosher et al., 1979b; Kosher and Savage, 1980). This suggests that elevating the cyclic AMP content of the subridge mesenchymal cell precludes the necessity of cells passing through a condensation phase prior to overt cartilage formation. If this is so, it further suggests that in the absence of exogenous cyclic AMP, the intimate association between molecules on adjacent cell surfaces oc­ curring during cellular condensation may provide the elevated cyclic AMP levels required to trigger chondrogenic differentiation. C. The Role of Cellular Condensation in Limb Cartilage Differentiation As previously described, the first morphological change characterizing mes­ enchymal cells that have initiated chondrogenic differentiation in vivo, in highdensity monolayer culture, and in organ culture is a widespread cellular con­ densation process. A variety of observations indicate this cellular condensation is critically important in cartilage differentiation. For example, it appears that certain skeletal malformations such as those seen in brachypod mouse mutants can be attributed to impairment in the condensation process (Gruneberg and Lee, 1973; Elmer and Selleck, 1975; Duke and Elmer, 1977, 1978). Both in vivo (Gruneberg and Lee, 1973) and in vitro (Elmer and Selleck, 1975; Duke and Elmer, 1977, 1978) brachypod post-axial mesenchymal cells undergo delayed and reduced condensations and a correspondent subsequent delay and reduction in cartilage formation. In addition, a variety of studies indicate that chondro­ genesis of limb mesenchymal cells in monolayer culture is dependent upon conditions promoting the formation of cellular aggregates or condensations during the initial period of culture (see, for example, Umansky, 1968; Caplan, 1970; von der Mark and von der Mark, 1977; Newman, 1977; Ahrens et al., 1977; Hassell et al., 1978a; and Karasawa et al., 1979). For example, when precar­ tilaginous limb mesenchymal cells are cultured at densities below confluence, the cells fail to differentiate into cartilage even after 12 days in culture (Caplan, 1970; Levitt and Dorfman, 1972; von der Mark and von der Mark, 1977; New­ man, 1977). The cells in such subconfluent low-density cultures demonstrate only a low-level synthesis of sulfated glycosaminoglycans (GAG) (Caplan, 1970, 1972; Levitt and Dorfman, 1972) and also fail to synthesize cartilage-charac­ teristic type II collagen (von der Mark and von der Mark, 1977; Newman, 1977).

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Only when the cells are seeded at densities greater than confluence do they form cartilage nodules, show amplified sulfated GAG production (Caplan, 1970, 1972; Levitt and Dorfman, 1972), and initiate the synthesis of type II collagen (von der Mark and von der Mark, 1977; Newman, 1977). In other words, dissociated prechondrogenic limb mesenchymal cells differentiate into cartilage under con­ ditions promoting the formation of cellular aggregates or condensations and intimate cell-cell interactions. In contrast, chondrogenic differentiation does not occur under conditions precluding cellular condensation and intimate cell-cell interactions. As mentioned before, it has been suggested that during condensation an in­ teraction between molecules on adjacent cell surfaces occurs, resulting in increase in the cyclic AMP content of the cells which triggers chondrogenic differentiation (Kosher and Savage, 1980). It is of some interest that, during the cellular con­ densation process subridge mesenchymal cells undergo in organ culture, an interaction between cell surface galactosyltransferases and acceptors on adjacent cell surfaces occurs (Shur et al., 1982). It is of some interest to determine if the interaction between these surface molecules is causally related to chondrogenic differentiation. D. The Role of Hyaluronate in Limb Cartilage Differentiation Toole (1972) has observed that the amount of hyaluronate synthesized relative to chondroitin sulfate is higher at stage 23 in whole limb buds than in older limb buds in which overt cartilage formation has begun. Furthermore, hyaluronidase activity becomes detectable at about the time a metachromatic matrix appears in the proximal regions of the limb (Toole, 1972). Thus, Toole (1972) has suggested that synthesis of hyaluronate by limb mesenchymal cells is associated with inhibition of their differentiation, and that removal of hyaluronate may be necessary for organized chondrogenic differentiation. Consistent with this is a recent study demonstrating a gradation of hyaluronate accumulation along the proximodistal axis of the limb correlated with both distance of cells from the AER and their state of differentiation (Kosher et al., 1981). Hyaluronate is by far the predominant glycosaminoglycan synthesized by mesenchymal cells di­ rectly subjacent to the AER, and there is a progressive decline in hyaluronate accumulation by more proximal mesodermal cells that is correlated with the initiation of condensation (Kosher etal., 1981). Thus, it has been suggested that the AER may inhibit differentiation of subjacent mesenchymal cells by causing them to secrete a considerable quantity of hyaluronate (Kosher et al., 1981). Accumulating extracellular hyaluronate may maintain the physical separation of subridge mesenchymal cells and thus prevent the cellular condensation that is required to trigger differentiation. It may feed back upon the cells, inhibiting their synthesis of cartilage matrix components (Kosher et al., 1981). The physicochemical properties of hyaluronate make it an ideal candidate for physically separating cells and thus preventing extensive cell-cell interactions (see Toole,

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1976), and, in fact, Toole et al., (1972) have demonstrated that exogenous hyaluronate inhibits the aggregation of dissociated stage 26 limb bud cells in monolayer culture (see, however, Finch et al., 1978). Furthermore, it has been suggested that as subridge mesenchymal become located progressively farther from the AER and its inhibitory influence as a result of polarized proximal-todistal outgrowth, their synthesis of hyaluronate may progressively decline and preexisting extracellular hyaluronate may be removed by activation or increase in activity of hyaluronidase; these events may result in the initiation of the condensation process (Kosher et al., 1981). Consistent with this suggestion is the finding by Shambaugh and Elmer (1980) that limbs of brachypod mouse mutants, whose skeletal malformations are attributed to an impairment in the condensation process, delay the reduction of hyaluronate synthesis occurring in normal limbs on day 12.5 of gestation, and this delay correlates with the reduced cellular condensations in the mutant limb. In view of the key role hyaluronate may play in inhibiting cartilage differ­ entiation, it is of some interest that hyaluronate is synthesized by the AER, and, in fact, there is a selective and substantial increase in the amount of hyaluronate produced by the AER compared to limb and nonlimb ectodermal tissues that do not promote limb bud outgrowth (Kosher and»Savage, 1981). Thus, it has been suggested that excess hyaluronate produced by the AER may not only be involved in the unique outgrowth-promoting effect of the AER, but also may be involved in inhibiting differentiation by acting on the immediately subjacent mesenchymal cells, causing them to maintain their own high rate of hyaluronate synthesis or perhaps simply contributing to the hyaluronate-rich matrix surrounding the sub­ jacent mesenchymal cells (Kosher and Savage, 1981). E. The Regulation of Somite Chondrogenesis In contrast to limb chondrogenesis in which a heterologous tissue, the AER, exerts a negative effect on the cartilage differentiation of limb mesenchymal cells, the formation of vertebral cartilage by mesenchymal sclerotomal cells of the somite requires a positive differentiative influence from heterologous tissues (i.e., the embryonic notochord and spinal cord). The positive inductive influence of the notochord and spinal cord on somite chondrogenesis has been demonstrated both in vivo and in vitro in an organ culture system (see Hall, 1977, for review). A considerable amount of evidence has been obtained indicating that extra­ cellular matrix components (i.e., collagen and proteoglycans) produced by the embryonic notochord are involved in promoting somite chondrogenic differen­ tiation. During its interaction with somites in vivo, the embryonic notochord synthesizes and secretes proteoglycans (predominantly proteochondroitin 4- and 6-sulfates; Hay and Meier, 1974; Kosher and Lash, 1975) and collagen (pre­ dominantly type II collagen; Linsenmayer et al., 1973b; Miller and Mathews, 1974; von der Mark et al., 1976). These initially accumulate extracellularly in the perinotochordal sheath and in the cell-free region adjacent to the notochord

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and subsequently become distributed among somitic sclerotomal cells following migration of the sclerotomal cells into this cell-free region just prior to differ­ entiation of the cells into cartilage (Ruggeri, 1972). Notochords from which perinotochordal extracellular matrix components have been removed are impaired in their ability to support in vitro somite chondrogenesis (Kosher and Lash, 1975). Furthermore, exogenous proteoglycans and purified type I or type II collagen substrates greatly stimulate in vitro somite chondrogenesis in the absence of the notochord or other inducing tissues (Kosher et al., 1973; Kosher and Church, 1975; Lash and Vasan, 1978). It has been suggested that extracellular matrix components exert their regu­ latory effect on somite chondrogenic differentiation by interacting with com­ ponents of the somite cell surface, and this interaction might result in lowered intracellular cyclic AMP levels that may trigger chondrogenic differentiation (Kosher, 1976). A variety of agents that elevate intracellular cyclic AMP levels inhibit formation of the small amount of cartilage normally formed by somites in vitro in the absence of inducing tissues and inhibit the ability of somites to respond to the inductive influence of the notochord or collagen (Kosher, 1976). In addition, collagen-induced in vitro somite chondrogenesis is accompanied by reduction in the cyclic AMP content of the expiants (Kosher and Savage, 1979). Although cyclic AMP derivatives stimulate chondrogenic differentiation of limb mesenchymal cells, these same agents inhibit somite chondrogenesis. This observation is not as surprising as it may seem initially when one considers that precartilaginous limb and somite mesodermal cells not only differ considerably from one another (see Section IV) but also that the process of somite chondro­ genesis differs in several significant respects from the process of limb chondro­ genesis (see preceding and in Kosher et al., 1979b, Discussion). That the regulatory mechanisms controlling the process of limb chondrogenesis differ from those controlling somite chondrogenesis is emphasized by the observation that whereas somites of brachyury (T/T) mutant mouse embryos fail to differ­ entiate into cartilage in vitro even in the presence of normal (nonmutant) inducing tissues, limb buds of the same mutant embryos cultured under the same conditions as the mutant somites do produce cartilage (Bennett, 1958).

VI. MAINTENANCE OF THE CHONDROCYTE PHENOTYPE As previously described, when chondrogenic tissue from the proximal central core of stage 25 and older limb buds is transplanted into the peripheral myogenic region of the limb, the tissue maintains its phenotype and forms ectopic cartilage in the presence of influences from the limb periphery that normally elicit for­ mation of nonchondrogenic tissues (Searls and Janners, 1969). In addition, when intact slices of cartilage are subjected to organ culture the chondrocytic phenotype is maintained for an extended period of time (Benya and Nimni, 1979). However, when differentiated chondrocytes are liberated from the extracellular matrix that

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normally surrounds them, the phenotype of the isolated chondrocytes is quite susceptible to modulation, particularly in monolayer culture. Such modulation involves not only a change in the morphology of chondrocytes yielding cells with a fibroblastic appearance, but also involves a switch from synthesis of cartilage-characteristic type II collagen to synthesis of type I collagen (Abbott andHoltzer, 1966; Coon, 1966; May ne et al., 1976a; Muller etal., 1977). These observations indicate that the extracellular matrix normally surrounding chon­ drocytes plays a key role in maintaining their differentiated phenotype. Further evidence supporting this notion derives from the observation that exogenous cartilage proteoglycans stimulate synthesis of sulfated proteoglycans by differentiated chondrocytes in suspension culture (Nevo and Dorfman, 1972) and in monolayer culture (Schwartz and Dorfman, 1975; Huang, 1974). In addition, when chondrocytes in monolayer culture are treated with sufficient testicular hyaluronidase to remove all cell-associated proteoglycan, the cells undergo a transition to a fibroblastic morphology and switch from synthesis of type II to synthesis of type I collagen (Pennypacker and Goetinck, 1976b, 1979; see, however, Caplan, 1981). Other indications that maintenance of the chon­ drocytic phenotype is sensitive to the extracellular environment are studies dem­ onstrating that exogenous hyaluronate, which is only a minor constituent of mature cartilage matrix, exerts an inhibitory effect on proteoglycan synthesis and cartilage matrix deposition by mature chondrocytes in suspension culture (Wiebkin and Muir, 1973, 1977) and in monolayer culture (Solursh et al., 1974; Handley and Lowther, 1976). Another extracellular macromolecule that may have an effect on maintenance of the chondrocytic phenotype is fibronectin. Fibronectin is a high molecular weight glycoprotein that is a major constituent of the cell surface of cultured embryonic and adult fibroblasts; it has also been found on the surface of several other cell types (see Yamada and Olden, 1978, and Yamada et al., 1978, for recent reviews). Initial studies indicated that mature chondrocytes do not possess fibronectin at their surfaces or in their intercellular matrices (Linder et al., 1975; Dessau et al., 1978). During the initial aggregation or condensation phase the mesenchymal cells of 10-day-old mouse limbs or stage 24 chick limbs undergo in high density monolayer culture, they synthesize fibronectin and accumulate it on their surfaces (Lewis et al., 1978; Hassell et al., 1978b). However, later in the culture period when the cells have deposited a cartilage matrix, the syn­ thesis of fibronectin is no longer detectable and fibronectin is no longer detected on the surfaces of the cells (Lewis et al., 1978; Hassell et al., 1978b). Similarly, when isolated chondrocytes are subjected to monolayer culture, they initially accumulate fibronectin on their surfaces, but, as cartilage matrix deposition proceeds, fibronectin accumulation ceases (Dessau et al., 1978). Thus, it has been suggested that loss of fibronectin may be required for chondrogenesis to occur (Lewis et al., 1978) and for maintenance of the chondrocytic phenotype (Pennypacker et al., 1979; West et al., 1979). Consistent with these suggestions

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are reports indicating that in the presence of exogenous fibronectin, chondrocytes in monolayer culture assume a fibroblast-like morphology, undertake depressed synthesis of sulfated glycosaminoglycans, and initiate synthesis of type I collagen (Pennypacker et al., 1979; West et al., 1979). It is important to point out, however, that immunohistochemical studies demonstrate that fibronectin is not only present during the critical early stages (stages 22-24) of chondrogenic differentiation in the proximal central core of the limb but also is present as late as stage 27 in the differentiated cartilage rudiments when chondrocytes are surrounded by a metachromatic cartilage matrix containing type II collagen (Dessau et al, 1980; Kosher et al, 1982; Tomasek et al, 1982; Melnick et al, 1981). In addition, continued synthesis of fibronectin during the chondrogenesis elicited by subcutaneous implantation of demineralized bone matrix has been noted (Weiss and Reddi, 1980). Thus, the relationship of fibronectin to the acquisition and maintenance of the chondrocytic phenotype is unclear and ambiguous. Other less well defined factors have both positive and negative effects on the maintenance of the chondrocytic phenotype in monolayer culture. For example, a heat-labile high molecular weight component of embryo extract not only mod­ ulates the morphology of chondrocytes to a fibroblast-like appearance but also elicits a switch in the type of collagen synthesized from type II to type I (Coon and Cahn, 1966; Schiltz et al, 1973; Mayne et al, 1976b). In addition, dif­ ferentiated chondrocytes in monolayer culture release a heat- and trypsinsensitive factor with a molecular weight of 30,000-150,000 into the culture medium, stimulating the synthesis of sulfated glycosaminoglycans and collagen by chondrocytes (Solursh et al, 1973). Although the morphological and biochemical chondrocytic phenotype is mod­ ulated in monolayer culture, there is no evidence that modulated cells have dedifferentiated to the extent that they can redifferentiate into other specialized nonchondrogenic cell types. However, cells whose phenotype has been modu­ lated for several generations can readily reacquire their chondrocytic phenotype under appropriate conditions of culture (Coon, 1966). In addition, loss of the chondrocyte phenotype elicited by embryo extract, fibronectin, and testicular hyaluronidase can be reversed upon subculturing in media without these agents (Mayne et al, 1976b; Pennypacker et al, 1979; West et al, 1979; Pennypacker and Goetinck, 1976b; 1979).

References Abbott, J., and Holtzer, H. (1966). The loss of phenotypic traits by differentiated cells. III. The reversible behavior of chondrocytes in primary cultures. J. Cell Biol. 28, 473-488. Abrahamsohn, P. Α., Lash, J. W., Kosher, R. Α., and Minor, R. R. (1975). The ubiquitous oc­ currence of chondroitin sulfates in chick embryos. J. Exp. Zool. 194, 511-518. Ahrens, P. B., Solursh, M., and Reiter, R. S. (1977). Stage-related capacity for limb chondrogenesis in cell culture. Dev. Biol. 60, 69-82.

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Duke, J., and Elmer, W. A. (1978). Cell adhesion and chondrogenesis in brachypod mouse limb mesenchyme: fragment fusion studies. J. Embryol. Exp. Morphol. 48, 161-168. Ede, D. Α., Flint, O. P., Wilby, Ο. K., and Colquhoun, P. (1977). The development of precartilage condensations in limb bud mesenchyme in vivo and in vitro. In "Vertebrate Limb and Somite Morphogenesis" (D. A. Ede, J. R. Hinchliffe, and M. Balls, eds.), pp. 161-180. Cambridge Univ. Press, Cambridge. Elmer, W. Α., and Selleck, D. K. (1975). In vitro chondrogenesis of limb mesoderm from normal and brachypod mouse embryos. J. Embryol. Exp. Morphol. 33, 371-386. Eyre, D. R. (1980). Collagen: molecular diversity in the body's protein scaffold. Science, 207, 1315-1322. Fell, Η. B., and Canti, R. G. (1934). Experiments on the development in vitro of the avian kneejoint. Proc. Roy. Soc. London, Ser. Β 116, 316-349. Finch, R. A., Parker, C. L., and Walton, S. T. (1978). The lack of an inhibitory effect of hyaluronate on chondrogenesis in chick limb-bud mesoderm cells grown in culture. Cell Differ. 7, 283-293. Franco-Browder, S., DeRydt, J., and Dorfman, A. (1963). The identification of a sulfated muco­ polysaccharide in chick embryos, stages 11-23. Proc. Natl. Acad. Sci. U.S.A. 49, 643-647. Globus, M., and Vethamany-Globus, S. (1976). An in vitro analogue of early chick limb bud outgrowth. Differentiation, 6, 91-96. Godman, G. C , and Porter, K. R. (1960). Chondrogenesis studied with the electron microscope. J. Biophys. Biochem. Cytol. 8, 719-760. Goel, S. C. (1970). Electron microscope studies on developing cartilage. I. The membrane system related to the synthesis and secretion of extracellular materials. J. Embryol. Exp. Morphol. 23, 169-184. Goetinck, P. F., Pennypacker, J. P., and Royal, P. D. (1974). Proteochondroitin sulfate synthesis and chondrogenic expression. Exp. Cell Res. 87, 241-248. Gruneberg, H., and Lee, A. J. (1973). The anatomy and development of brachypodism in the mouse. J. Embryol. Exp. Morphol. 30, 119-141. Hall, Β. K. (1977). Chondrogenesis of the somitic mesoderm. Adv. Anat. Embryol. Cell Biol. 53(4), 7-50. Hamburger, F., and Hamilton, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88, 49-92. Handley, C. J., and Lowther, D. A. (1976). Inhibition of proteoglycan biosynthesis by hyaluronic acid in chondrocytes in cell culture. Biochim. Biophys. Acta, 444, 69-74. Hascall, V. C. (1977). Interaction of cartilage proteoglycans with hyaluronic acid. J. Supramol. Struct. 7, 101-120. Hascall, V. C , and Hascall, G. K. (1981). Proteoglycans. In "Cell Biology of Extracellular Matrix" (E. D. Hay, ed.), pp. 39-63. Plenum, New York. Hascall, V. C , and Heinegard, D. (1975). The structure of cartilage proteoglycans. In "Extracellular Matrix Influences on Gene Expression" (H. C. Slavkin and R. C. Greulich, eds.), pp. 423-434. Academic Press, New York. Hascall, V. C , Oegema, T. R., Brown, M., and Caplan, A. I. (1976). Isolation and characterization of proteoglycans from chick limb bud chondrocytes grown in vitro. J. Biol. Chem. 251, 3511-3519. Hassell, J. R., Pennypacker, J. P., and Lewis, C. A. (1978a). Chondrogenesis and cell proliferation in limb bud cell cultures treated with cytosine arabinoside and vitamin A. Exp. Cell Res. 112, 409-417. Hassell, J. R., Pennypacker, J. P., Yamada, Κ. M., and Pratt, R. M. (1978b). Changes in cell surface proteins during normal and vitamin Α-inhibited chondrogenesis in vitro. Ann. N.Y. Acad. Sci. 312, 406-409. Hay, E. D., and Meier, S. (1974). Glycosaminoglycan synthesis by embryonic inductors: neural tube, notochord, and lens. J. Cell Biol. 62, 889-898. Hilfer, S. R., Searls, R. L., and Fonte, V. (1973). An ultrastructural study of early myogenesis in the chick wing bud. Dev. Biol. 30, 374-391.

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