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Two distinct regulatory steps in cartilage differentiation
DEVELOPMENTAL
BIOLOGY
94, 311-325
(1982)
Two Distinct Regulatory Steps in Cartilage Differentiation’ MICHAEL SOL.URSH,* KAREN L. JENSEN,* CARL T. SINGLEY,*” AND REBECCA S. REITER* *Department
THOMAS
F. LINSENMAYER,~
of Zoology, University of Iowa, Iowa City, Iowa 52242; and tThe Developmental Biology Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Received March 2, 1982; accepted in revised form July 21, 1982
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 trseated 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 mat.rix 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, inclucling 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. INTRODUCTION
In general, the differentiation of particular cell types involves a sequence of distinct steps. Each step brings the progenitor cell closer to the final differentiated product. One way to identify and study individual steps or transitions is to isolate cells at different times during their development and compare their differentiative properties in a particular test system. This approach has been used to identify distinct steps in the conversion of limb mesenchyme cells into overtly differentiated chondrocytes (Solursh et al., 1978). The test system involves high-density cell cultures which permit the differentiat:ion of several cell types (Umansky, 1966; Caplan, 1970; Osdoby and Caplan, 1979), especially cartilage. In these cultures, initially aggregates of cells form in which there is more extensive cell multilayering (Ahrens let al., 1977). In cultures prepared from wings of certain stage chick embryos (stages 2025 of Hamburger and Hamilton (1951)), an Alcian bluestaining extracellular matrix accumulates in aggregates as cartilage differentiation occurs. However, in cultures 1 Supported by NIH Grant HD05505 and NSF Grant PCM 77. 01154 to MS. and NIH Grants EY02261 and AM03564 to T.F.L. ’ Present address: Depart:ment of Zoology, Ohio State University, Columbus, Ohio 43210.
prepared from both earlier (stage 19) and later stages (stage 26), the aggregates do not form cartilage matrix spontaneously, but can be stimulated to do so if the cultures are treated with db cyclic AMP (Solursh et al., 1981). Previous information concerning the properties of wing cells from stages 19, 24, and 26 is summarized below. At stage 19, the mesenchyme cells in the wing bud appear morphologically homogeneous (Singley and Solursh, 1981). While organ cultures prepared from wing buds of this stage do form cartilage (Solursh and Reiter, 1980), high-density cell cultures produce only aggregates, which fail to accumulate an Alcian blue-staining extracellular matrix (Ahrens et al., 1977). Apparently, dissociation and/or randomization of the cells at this early stage interferes with some step in chondrogenic differentiation. These cells appear unable to interact with other cells in a manner which is apparently required for chondrogenic differentiation in this culture system (Solursh and Reiter, 1980). By stage 24, a number of regional changes have occurred in the wing bud. Early skeletal primordia become recognizable by histology (Fell and Canti, 1934) as well as by their relatively higher uptake of 35SO; (Searls, 1965) and lower uptake of [3H]thymidine (Janners and Searls, 1970). However, overt chondrogenic differentia-
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tion is not yet detectable in situ. High-density cultures prepared from wings from stage 24 embryos form aggregates which spontaneously accumulate an Alcian blue-staining extracelltilar matrix (Caplan, 1970; Ahrens et al., 1977). By stage 26, chondrogenesis has begun in the proximal regions of the wing. This can be demonstrated by histology (Fell and Canti, 1934) and by biochemical (Linsenmayer et al., 1973) or immunological (Dessau et al., 1980) detection of cartilage collagen. The more distal regions of the limb are ,at progressively younger stages of development (Saunders, 1948). High-density cultures of wing buds from stage 26 embryos again produce aggregates which fail to accumulate spontaneously an Alcian blue-staining matrix. In addition, one finds small clusters of chondrocytes which apparently had already in situ (Solursh et al., 1981). differentiated Since the presence of an Alcian blue-staining matrix can only detect overtly differentiated cartilage cells, additional, more sensitive criteria for chondrogenesis are needed. This present study is concerned with a further analysis of the in vitro behavior of cells from these three stages. Transmission electron microscopy and immunohistochemistry (Von der Mark and Von der Mark, 1977) with a monoclonal antibody directed against type II collagen (Linsenmayer and Hendrix, 1980) are used to define further the cell phenotypes in cultures prepared from stages 19, 24, and 26. The results indicate that cells from stage 19 wings become blocked at a step prior to the production of detectible levels of type II collagen. On the other hand, stage 26 cells contain type II collagen, but resemble mesenchyme in other respects. The existence of these two phenotypes suggests that there are at least two (distinct regulatory steps in the expression of the cartilage phenotype. MATERIALS
AND
METHODS
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26 wings) for 10 min at 37°C and mechanically dissociated by pipetting. The enzyme solution (2 ml) was diluted with 3 ml of medium, sometimes containing 100 pg/ml of soybean trypsin inhibitor (Sigma Chemical Corp., St. Louis, MO.). The cells were collected by centrifugation and suspended in medium sometimes containing 100 pg/ml soybean trypsin inhibitor. Cell suspensions were passed through two layers of No. 20 Nitex screen, and the cell concentration was determined with a hemacytometer. The cells were collected by centrifugation and resuspended in culture medium consisting of Ham’s Fiz nutrient mixture and 10% fetal bovine serum (both from Grand Island Biological Co., Grand Island, N. Y .) and antibiotics. In some cases, chick cells were mixed with Japanese quail cells prepared from stage 24 wings as described above or from chick chondrocytes prepared either from limb bud mesenchyme by maintaining cells in suspension culture for 48 hr (Solursh et al., 1980) or from sterna prepared according to the method described by Cahn et al. (1967). In all cases, the final cell concentration was 2 X lo5 cells per 10 ~1. Ten microliters of cell suspension was placed on a 35 mm tissue culture dish or on an autoclaved borosilicate coverslip (Bellco) in a culture dish. After 1 hr at 37°C in culture 2 ml of culture medium was added. The medium was replaced daily. In some experiments, the cultures were treated in various ways. For db cyclic AMP treatment, cultures were maintained in medium containing 1 mM N6, 02’dibutyryl adenosine 3’:5’-monophosphoric acid (Sigma) from Day 1 to 3 of culture. Ascorbate treatment (50 pg/ml) was begun on Day 0 or Day 1 of culture; the effects were similar in either case. Cultures were usually maintained in an atmosphere of 5% CO2 and 95% air. For low oxygen, cultures were placed in a humidified jar gassed with a mixture of 5% C02, 25% air, and 70% N2, and kept at 37°C.
Cultures
Fixation
Micromass cultures were prepared as described previously (Ahrens et al., 1977) from wing buds of stage 19, 24, or 26 (Hamburger and Hamilton, 1951) White Leghorn chick embryos (Welp Hatchery, Bancroft, Iowa). Wings were incubated in trypsin and collagenase (0.05% each for stage 19 wings and 0.1% each for stage 24 or
Cultures were washed with Saline G and in some cases were fixed for 10 min in Kahle’s fixative as described previously (Ahrens et al., 1977). These cultures were stained with Alcian blue at pH 1 in order to visualize sulfated glycosaminoglycans in the extracellular matrix. Nodule numbers per culture were counted on tracings
and Histology
FIG. 1. Stage 19 wing bud mesenchyme control culture on Day 3. (a) Thick Epon section in the plane of culture illustrating aggregate and interaggregate morphology. Aggregates consist of a core of rounded, tightly packed cells surrounded by less tightly packed, round and crescentshaped cells. There is little extracellular space in the aggregate core. The darkly stained cells in this and subsequent micrographs appear to have been fixed in various stages of cell death. X140. (b) Electron micrograph showing the close cell contact typical of stage 19 aggregates. Aggregate cells possess few cell processes and form numerous gap junctions (not illustrated). X6000. (c) Electron micrograph of an interaggregate area. Cells in interaggregate areas are generally more elongate, flattened, and possess numerous cell processes. There is considerably more extracellular space in these areas as compared with aggregates. The extracellular material consists of numerous collagen fibrils as well as some proteoglycan-like material and 3-nm filaments. X6000.
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made by use of a microslide projector. Nodule diameters were measured directly from the cultures with the aid of an ocular micrometer in a microscope at 400X. Two measurements at right angles to each other were made on each of 10 different nodules in three different cultures. An analysis of variance with a priori contrast was performed to determine if there were significant differences between pairs of means. Some cultures were dehydrated, embedded in paraffin, serially sectioned at 7 pm, and stained according to the Feulgen method (Humason, 1972), and then with Alcian blue at pH 1. The numbers of quail and chick nuclei were counted under an oil immersion objective. For electron microscopy, washed cultures were fixed for 30 min in 3% glutaraldehyde in one-half strength Karnovsky’s buffer at pH 6.6 and postfixed, dehydrated, and embedded in Epon 812, all as described previously (Ahrens et al., 1979). For light microscopy, sections 0.25 pm thick were cut with glass, mounted on slides, and stained with Azure II and methylene blue. For transmission electron microscopy, sections, about 50 to 80 nm thick, were cut with a diamond knife with a Sorval MT-2B ultramicrotome, stained with methanolic uranyl acetate (Stempak and Ward, 1964) and lead citrate (Venable and Coggeshall, 1965), and examined with a Philips EM300 electron microscope. All sections were cut parallel to the culture dish. Immunofluorescence Cultures inoculated on coverslips were washed three times at 37°C with Saline G and then once with 0.02 M phosphate-buffered saline (pH 7.2) at room temperature. They were then fixed in 100% acetone for 10 min and air-dried at room temperature for 4 days. For antibody staining, all subsequent steps were done at room temperature. The cultures were rehydrated in phosphate-buffered saline for 10 min and then incubated in the various antibody-containing mouse ascites fluids for 30 min. These were l/1600 dilutions of ascites fluids containing either monoclonal antibody to type II collagen (Linsenmayer and Hendrix, 1980), type I collagen (Linsenmayer et al., 1979), or the control ascites fluid. The cultures were washed four times with phosphate-
FIG. 2. Stage 19 wing bud mesenchyme culture (Day 3) treated with db CAMP. (a) Thick Epon section in the plane of culture. Note that the aggregate cells are widely separated by extensive extracellular space as compared with the tightly packed cells of control cultures (Fig. la). The interaggregate areas contain more intercellular space as compared to controls. X140. (b) Electron micrograph of an aggregate core. The extensive extracellular spaces are filled with a granular and filamentous matrix having the appearance of aggregate proteoglycan (inset; Hascall, 1980). Very little collagen is seen in aggregates in db CAMP-treated cultures. X1500 (inset X90,000). (inset bar = 100 am).
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buffered saline and incubated with fluorescein-conjugated IgG fraction of rabbit anti-mouse IgG (heavy and light chains; Cappel Laboratories, Lot No. 13039), l/300 dilution in phosphate-buffered saline, for 20 min. To intensify the fluorescent signal, the cultures were then washed four times in phosphate-buffered saline and incubated for 20 min in fluorescein-conjugated IgG fraction of goat anti-rabbit IgG (heavy and light chains;
FIG. 3. Stage 19 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate area in a control culture. (b) A few cells in the aggregate shown in (a) exhibit extracellular fluorescence after being stained with antibody to type II collagen. Small clusters of stained cells, usually in aggregate centers, are observed throughout the culture. The intensity is usually faint, however, approaching background. (c) Phase-contrast micrograph showing an aggregate area in a culture which had been exposed to db CAMP from Day 1 to 3. (d) The area shown in c shows intense intra- and extracellular fluorescence after being stained with antibody to type II collagen. The fluorescence intensity is drastically increased, as is the size of the staining area, compared to control cultures (all x200).
FIG. 4. Stage 24 wing bud mesenchyme control culture at Day 2. (a) Thick Epon section in the plane of culture. The aggregates in these cultures blend into interaggregate areas and are less distinct than those of stage 19 cultures (Fig. la). Aggregates are characterized by the rounded shape of the core cells and the crescentic shape of the more peripheral cells. As in stage 19 cultures, aggregate cells on Day 2 of culture are tightly associated with little intervening space. X140. (b) Electron micrograph of an aggregate. Aggregate cells are rounded and have few cell processes. The ECM here is sparse and consists of collagen fibrils and few proteoglycan-like granules (not illustrated). x1500.
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Cappel Laboratories, Lot No. 13259) diluted l/300 in phosphate-buffered saline. After four more washes, the coverslips were mounted in glycerol and observed or photographed with a Leitz Planopak illuminator with an LZ filter cube. The specificity of the staining was verified on cultures of chick dermal fibroblasts or chick sternal chondrocytes for types I and II collagens, respectively. There was no detectable fluorescence with the control ascites fluid. RESULTS
Stage 19 Cultures Control cultures. Micromass cultures prepared from wing mesenchyme of stage 19 chick embryos form numerous aggregates but no Alcian blue-staining nodules (Ahrens et al., 1977). These are examined on Day 3 of culture, since nodules do not form after even longer periods. Viewed in cross section, the aggregates appear as areas of mesenchyme in which cells are more extensively multilayered than in adjacent areas (Solursh et al., 1978). Cut horizontally to the culture dish, the aggregates appear as regions containing tightly packed cells surrounded by more dispersed interaggregate cells (Fig. la). Based on transmission electron microscopy, cells within an aggregate appear closely applied and have little intercellular space (Fig. lb). On the other hand, the interaggregate cells are separated by more extracellular space in which one finds collagen fibrils, 3-nm filaments, and some 30-nm proteoglycan-like granules. Based on the absence of an Alcian blue-staining extracellular matrix and matrix components identifiable at the electron microscopic level, the aggregates have failed to differentiate into cartilage nodules. The type of collagen present in the cells can be used as a more qualitative criterion for the differentiation of cartilage cells than the accumulation of extracellular matrix. Observed from above in whole mounted cultures, type I collagen is detected throughout the stage 19 cultures (not shown). However, when cultures are stained with antibody to type II collagen, fluorescence is observed only in a few restricted areas. Small clusters of cells within aggreg,ates exhibit a faint fluorescence (Figs. 3a,b) suggesting the presence of type II collagen in a few cells. db CAMP-treated cultures. It has been reported earlier
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that db CAMP treatment of micromass cultures from wings of stage 19 embryos from Day 1 to 3 of culture stimulates some aggregates to develop into Alcian bluestaining nodules (Ahrens et al., 1977; Solursh et al., 1981). After such treatment, cells in both aggregate and interaggregate areas are separated by more extensive extracellular space (Fig. 2a). In aggregate areas, the extracellular matrix resembles that of cartilage matrix, containing proteoglycan aggregates. Furthermore, the cell aggregates now stain intensely with type II collagen antibody (Figs. 3c,d). On the other hand, the interaggregate areas do not stain with type II antibody. These results demonstrate that db cyclic AMP treatment greatly increases the proportion of cells associated with type II collagen. In addition, it drastically stimulates the accumulation of an extracellular cartilage matrix. A number of other treatments, including ascorbate and soybean trypsin inhibitor, had no detectable effect on stage 19 cultures, in contrast to cells from the later stages considered below. Stage 24 Cultures Control cultures. In cultures derived from stages 2025 (Ahrens et al., 1977; Solursh et al., 1981), aggregates spontaneously develop into Alcian blue-staining nodules. By 40 hr of culture, small areas in the center of aggregates become associated with an Alcian blue-staining matrix. These areas enlarge progressively with time. A similar pattern is observed when the cultures are stained with antibody to type II collagen (not shown). At the electron microscope level, after 48 hr of culture, aggregate cells appear rounded and have few cell processes (Fig. 4). Still at this time the extracellular matrix is sparse, consisting of collagen fibrils and a few proteoglycan-like granules. After 72 hr of culture, cartilage nodules accumulate extensive cartilage matrix (Fig. 5). The extracellular material is rich in proteoglycan-like granules, but contains little identifiable collagen. Together with the inflated ER cisternae, these features suggest that these chondrocytes are scorbutic (Meier and Solursh, 1978). By immunofluorescence, type II collagen is detected in discrete nodules and appears primarily intracellular (Figs. 7a,b). On the other hand, the entire culture stains with antibody against type I collagen (not shown). The
FIG. 5. Stage 24 wing bud mesenchyme control culture at Day 3. (a) Thick Epon section in the plane of culture. Distinct cartilage nodules (n) are present throughout the culture. Cartilage nodules display an extensive extracellular component surrounding relatively rounded cells. Internodular areas appear similar to interaggregate areas of Day 2 cultures (Fig. 4a). X140. (b) Electron micrograph of a cartilage nodule. Nodular cells are rounded, have inflated ER cisternae and possess only a few short cell processes. X1500. (c) The extracellular matrix is rich in proteoglycan-like granular material. Collagen fibrils (C) are sparse as compared with normal cartilage in situ (Meier and Solursh, 1978). X90,000. (d) Internodular cells are more flattened, elongate, and possess numerous cell processes. The ECM consists of collagen fibrils and sparse proteoglycan-like granular material (Fig. 5~). X6000.
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internodular areas still resemble the interaggregate areas of Day 2 cultures. The internodular cells appear flattened and are associated with collagen fibrils and some proteoglycan-like material (Fig. 5d). db CAMP-treated cultures. Treatment of micromass cultures from Day 1 to 3 with db CAMP causes the formation of extensive cartilage throughout the culture, based on Alcian blue staining, 35S autoradiography and staining with antibody directed against cartilage-specific proteoglycan (Solursh et al., 1981). As can be seen in Fig. 6, in treated cultures the nodules are less distinct and appear to anastomose. Cells in the interaggregate areas are in small clusters which appear to be undergoing chondrogenesis, as judged by the extensive staining with antibody against type II collagen (Figs. 7c,d). On the other hand, treatment with ascorbate or soybean trypsin inhibitor does not stimulate the interaggregate areas to form cartilage. Stage 26 Cultures Control cultures. Micromass cultures prepared from wings of stage 26 embryos form numerous aggregates; however, most of these aggregates fail to develop into Alcian blue-staining nodules even with an extended time of culture (Solursh et al., 1981). While the cells in these aggregates still appear mesenchymal in morphology (Fig. 8), they are more polymorphic and less tightly packed than those in cultures from stage 19 or 24 embryos (cf. Figs. 1 and 5). In further contrast to stage 19 and 24 cultures, the entire aggregate areas show intracellular staining for type II collagen (Fig. 10). Based on the three combined criteria, (mesenchyme morphology, absence of Alcian blue-staining matrix but the presence of type II collagen), the stage 26 aggregate cells are chondrocytes, but they are not overtly differentiated. Treated cultures. Several treatments have been found to stimulate the development of aggregates in stage 26 cultures into Alcian blue-staining nodules. However, by
FIG. 6. Stage 24 wing bud mesenchyme db CAMP treated culture at Day 3. (a) Thick Epon section in the plane of a micromass culture of stage 24 chick wing bud mesenchyme treated with db CAMP and fixed at the end of Day 3 of culture. The nodules (n) are less distinct than in Day 3 control cultures and appear to anastomose. Internodular areas (I) form aggregate-like clusters of cells which appear to be undergoing chondrogenesis. x140. (b) Low-magnification TEM micrograph of a nodule/internodule interfacial area like that indicated by the box in (a). The nodular matrix is more dense than that of untreated controls, and there is considerable cartilage-like matrix in the internodular areas. More fibrillar collagen is present in the matrix of these nascent nodules as compared with the aggregates of control cultures. It is noteworthy that cells within mature nodules do not display a typical chondrocyte morphology, but are generally flattened in the plane of culture and often possess large cell processes. X1500.
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late stage 26 many of these treatments become less effective. db CAMP treatlment, for example, stimulates nodule formation, as reported previously (Solursh et al., 1981). However, this treatment becomes ineffective with advancing stage. Based on transmission electron microscope examination, db CAMP-treated cultures contain nodules resembling those seen in stage 24 cultures, except that the cells are highly polymorphic and the extracellular matrix is less dense (Fig. 9). Furthermore, nodules in these treated cultures stain with antibody to type II collagen in a manner indistinguishable from untreated stage 26 cultures (see Fig. 10). Several other treatments besides db CAMP also promote the formation of Alcian blue-staining nodules by stage 26 cultures. The addition of ascorbic acid increases nodule number (Table l; Fig. 11) in stage 26 cultures but has no such effect on stage 19 or stage 24 cultures. Since its effect is synergistic with that produced by db CAMP (Table 1; Fig. ll), these agents must act by distinct mechanisms. Maintenance of cultures in an atmosphere of 5% O2 also dramatically increases the formation of Alcian blue-staining nodules (Table 1). One expected effect of ascorbate treatment is to increase the accumulation. of collagen. As can be seen in Fig. lOd, ascorbate-treated stage 26 cultures stain intensely with antibody to type II collagen and a considerable proportion of the staining appears to be extracellular. A similar effect of ascorbic acid is seen in stage 24 cultures. Transmission electron microscopic observation of such treated cultures demonstrates the presence of numerous collagen fibrils in the extracellular spaces and the reduced inflation of the ER cisternae in treated cells (Fig. 12). Behavior of Cultures of Mixtures 26 Cells
of Stage 24 and Stage
The results presentecl above demonstrate that wing mesenchyme from stage 24 embryos can undergo chondrogenic expression spontaneously while those from stage 26 embryos fail to express the chondrogenic phenotype overtly. The dissociation of stage 26 wings into a suspension of single cells could remove a required factor. A possible mechanism for the failure of expression by stage 26 cells is that there is some factor which is required for chondrogenic expression and which is synthesized in adequate am.ounts by stage 24 cells but not by stage 26 cells. If this hypothesis is correct, the addition of small numbers of stage 24 cells to cultures of stage 26 mesenchyme is expected to improve nodule formation. The results shown in Table 2 are consistent with this hypothesis. The addition of progressively greater proportions of stage 23 cells increases nodule number. The addition of even one stage 23 cell out of
FIG. 7. Stage 24 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate (large arrow) and interaggregate area (small arrow) in a control culture. (b) Cells within the aggregate exhibit intracellular fluorescence after being stained with antibody to type II collagen. Only background fluorescence is detected in the internodular area. Nodules throughout the culture fluoresce and are separated by nonfluorescing internodular regions. (c) Phase-contrast micrograph of an aggregate and interaggregate area in a culture which has been exposed to db CAMP from Day 1 to 3. (d) Both the aggregate and interaggregate areas stain and the whole culture shows generally increased fluorescence compared to control cultures (all x200). Ascorbate-treated cultures (not shown) show a similar distribution of fluorescence throughout the culture, but the intensity is drastically increased. In addition, there is increased extracellular staining.
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VOLUME 94, 1982 TABLE 1 THE EFFECT OF VARIOUS TREATMENTS ON NODULE FORMATION BY STAGE 26 CULTURES
Treatment” Control db AMP Ascorbate Ascorbate Low 02
+ db CAMP
Number of Alcian bluestaining nodules/culture* 0 8f6 23 f 6 74 f 4
2+4 10 f 4 9t4 70 + 13
4*5 8fl 34 f 15 92 f 5 59 f 23
“All treatments are from Day 1 to 3 of culture except for the low 02, which was begun on Day 0. * Values are means + SD of four cultures. Three representative trials are shown.
three stage 26 cells produces a significant increase in nodule number. Examination of cultures prepared from stage 24 quail wings and stage 26 chick wings, in which quail and chick cells can be distinguished (LeDouarin, 1973), shows the expected proportion and distribution of cells from the two stages, as reported previously (Solursh and Reiter, 1980). The similar diameters of nodules regardless of the proportion of stage 26 cells, other than the 3:l mixture of stage 26 and stage 23 cells, suggest that stage 26 cells are participating in nodule formation (Table 2). Sections through chimeric cultures prepared from a 1:3 mixture of stage 24 chick cells and stage 26 quail cells clearly demonstrate that stage 26 cells are surrounded by Alcian blue staining matrix (Fig. 13a). In contrast, in a 1:3 mixture of quail limb or sternal cartilage and stage 26 cells, an Alcian blue-staining matrix is not associated with the stage 26 cells (Fig. 13b). It is often associated with the cartilage cells, however. These results suggest that stage 24 cells can promote the overt expression of chondrogenesis by stage 26 cells and support the hypothesis that there is a temporally regulated, mesenchyme-derived influence which promotes chondrogenic expression. DISCUSSION
By use of micromass cultures as a test system, the chondrogenic capacities of limb mesenchyme cells can be demonstrated to undergo distinct changes with deFIG. 8. Stage 26 wing bud control culture at Day 3. (a) Thick Epon section in plane of culture. The aggregates in stage 26 cultures are less distinct than those of stages 19 or 24. Interaggregate areas display considerable extracellular space which, however, contains little visible matrix material. X140. (b) Electron micrograph of an aggregate. The cells here are more polymorphic and are less tightly packed as compared with aggregates of stage 19 or 24 cultures. Myoblasts are often observed within aggregates of stage 26 mesenchyme (arrow). X1500.
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Expression
TABLE 2 THE FORMATION OF CARTILAGE NODULES BY MIXTURES OFCELLSFROMSTAGE~~ AND~~WINGS Ratio of stage 26:stage 23derived cells
Number of Alcian blue-staining nodules”
1:o 2O:l 3:l 1:l 1:3 0:l
0.25 f 0.5 Ok0 31 +- 9 46 f 31 141 ? 34 188 + 25
Mean nodule diameter*
4.97 6.00 6.67 6.38
f + f +
0.27’ 0.28 0.33 0.25
n Results are means f SD of four cultures and are typical of two trials. * Results are expressed as means + SE. Each unit = 0.018 mm. c Only this value is significantly different from the 0:l cultures (P 5 0.0006).
After dissociation and randomization, stage 19-derived wing mesenchyme can only form aggregates of closely associated cells. These cells do not undergo chondrogenesis as defined both by ultrastructural features and by immunofluorescence for type II collagen. Because these cells interfere with chondrogenesis when they are mixed with cultures from stage 24 wing mesenchyme, it was suggested that the stage 19-derived cells are unable to interact with wing mesenchyme from stage 24 embryos in a manner which is required for chondrogenesis (Solursh and Reiter, 1980). The aggregated cells are, however, potentially chondrogenic since treatment with db cyclic AMP causes the accumulation of cartilage matrix, demonstrated by Alcian blue staining and ultrastructural features, and the appearance of type II collagen, demonstrated by immunofluorescence. While the mechanism of action of db cyclic AMP on these cells is not known, it has been hypothesized that it bypasses required cell-cell interaction (Solursh et al., 1978). In fact, particular cell-cell interactions might lead to increased endogenous levels of cyclic AMP (Solursh et al., 1979) which in turn might promote chondrogenesis. Cells derived from wings of stage 24 embryos in highdensity cultures form aggregates which spontaneously differentiate into cartilage nodules, as defined by histochemical (Caplan, 1970; Ahrens et al., 1977) and biovelopment.
FIG. 9. Stage 26 wing bud culture at Day 3 treated with db CAMP. (a) Thick Epon section in plane of culture. The entire culture displays a greater amount of extracellular space as compared with Day 3 controls (Fig. Ba). X140. (b) Electron micrograph of a cartilage nodule. The cells of this nodule are highly polymorphic and possess numerous cell processes as compared with the nodular cells of stage 24 cultures (Fig. 5b). In addition, the extracellular component of the nodule is less extensive, and the matrix is not as dense. The cells here continue to display a highly inflated ER. X1500.
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FIG. 10. Stage 26 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate area in a control culture. (b) The cells within the aggregate area shown in (a) exhibit a diffuse intracellular fluorescence in aggregate areas but not in the interaggregate areas after being stained with antibody to type II collagen. The aggregates show fluorescence even though they do not appear to have formed nodules, based on Alcian blue staining. Cultures treated with db CAMP from Day 1 to 3 contain many Alcian blue-staining nodules, but the fluorescence after staining with antibody to type II is only slightly brighter than that shown in Fig. lob. (cl Phase-contrast micrograph of an aggregate area of an ascorbate-treated culture. (d) The area shown in c exhibits intense extracellular fluorescence. The internodular areas do not stain with antibody to type II collagen. The nodules also stain with Alcian blue in these cultures. Cultures treated with both ascorbate and db CAMP stain nearly as intensely as that shown in Fig. 10d (all X200).
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chemical (Von der Mark and Von der Mark, 1979) criteria. Because the nodules enlarge by assimilating adjacent mesenchyme, it has been suggested that the differentiating cartilage has an autoinductive influence on cartilage differentiation (Solursh, 1980). The assimilative process can be greatly accelerated by treatment with db cyclic AMP, as determined by a number of criteria (Solursh et al., 1981), including ultrastructure and immunofluorescence for type II collagen. Cultures prepared from wings of stage 26 embryos form numerous aggregates; these contain cells which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells ultrastructurally. However, based on the presence of immunologically detectable type II collagen, these aggregated cells are chondrocytes which have not undergone overt differentiation. This intermediate state in differentiation has been called the protodifferentiated state (Rutter et al., 1968). The existence of a protodifferentiated state suggests that there might be two transitions during chondrogenesis: one involving the initial acquisition of the chondrogenic capacity and another involving overt expression. In the high-density culture system used here, several environmental agents are found to promote expression of chondrogenesis. Caplan (1970) noted that treatment with 3-acetylpyridine promotes nodule formation in cultures from stage 26 limbs. The mechanism is not known. Treatment with db cyclic AMP also causes many of the aggregates to accumulate an extracellular matrix which stains with Alcian blue and which ultrastructurally resembles that produced by stage 24-derived cultures. Cyclic AMP analogues have been shown to promote the accumulation of proteoglycan by other chondrocytes (Miller et al., 1979). The lack of any detectable change in type II collagen immunofluorescence after this treatment might be due to the scorbutic culture conditions used (Meier and Solursh, 1979). Treatment with ascorbic acid also causes an increase in the number of Alcian blue staining nodules formed in stage 26-derived cultures and the accumulation of an ultrastructurally normal cartilage matrix, resembling that found in situ (Meier and Solursh, 1979). db Cyclic AMP and ascorbate have synergistic effects on nodule number and therefore, are likely to act by different, but complementary, mechanisms. Both of these mechanisms might be related to effects on secretory processes. The action of low oxygen is most likely to be related to the effects of ascorbate, since hypoxia can enhance collagen formation by fibroblasts by activating prolyl hydroxylase (Levine and Bates, 1976). Effects of both ascorbate (Hall, 1981) and hypoxia (Thorogood, 1979) on chondrogenesis have been reported. It would be interesting if these effects were on the process of overt expression.
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The ability of small numbers of stage 24-derived cells to promote chondrogenic expression by stage 26 cultures is particularly noteworthy. The participation of stage 26-derived cells in chonldrogenesis in mixed cultures is suggested by the observations that nodule size is the same in stage 24 cultures and in cultures containing a
a
b
FIG. 12. Stage 26 wing bud culture at Day 3 treated with ascorbate. Electron micrograph of a nodule. The cells here are more polymorphic than nodule cells of stage 24 controls, but somewhat less so than stage 26 control or db CAMP-treated cultures. A striking feature of ascorbate-treated cultures is the presence of numerous collagen fibrils in the ECM. Note also that the ER of these cells is less inflated than that of control cells. X1500.
FIG. 11. Whole mounts of micromass cultures prepared from stage 26 wings and fixed and stained with Alcian blue after 72 hr of culture. (a) Control culture. No nodules are present. (b) Culture treated with db CAMP from Day 1 to 3. A few nodules are seen. (c) Culture treated with 50 @g/ml ascorbate. (d) Culture treated with both db CAMP and ascorbate. While both db CAMP and ascorbate added alone increased the number of nodules formed, their effect is synergistic when they are present together (X10).
1:l mixture of stage 24 and stage 26 cells and that stage 26 cells are associated with Alcian blue-staining matrix in chimeric nodules. The effect of adding one out of three stage 24 cells to stage 26 cells contrasts with the effect of mixing similar proportions of stage 19 and stage 24 cells. In this case, chondrogenesis was not expressed (Solursh and Reiter, 1980). More important, adding one out of three differentiated chondrocytes to stage 26 cells
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FIG. 13. Cross sections of chimeric wing bud cultures at Day 3 stained by the Alcian blue and Feulgen method. (a) This culture was prepared from a 1:3 mixture of stage 24 chick and stage 26 quail wing bud cells, respectively. Note that the nodule contains numerous stage 26 quail cells, all surrounded by Alcian blue-staining matrix (X1350). (b) This culture was prepared from a 1:3 mixture of quail sternal cartilage and stage 26 chick wing bud cells. Note the presence of quail cartilage (arrow). The stage 26 cells have failed to produce an Alcian blue staining matrix (X1330).
did not promote expression. These later results suggest that an influence which is actively produced by stage 24 cells, but not by chondrocytes, can promote chondrogenic expression. The observation that nodules are smaller in cultures containing a 3:l mixture of stage 26 cells and stage 24 cells than in the stage 24 cultures also supports this suggestion, since the chondrogenic stimulus may not be propagated as the result of chondrogenie expression. It is possible that the mesenchymederived influence was produced in situ at a time prior to stage 26 but could not be replaced by stage 26 mesenchyme cells after cell dissociation. Whether this influence is a component of the mesenchyme extracellular matrix or cell surface is presently being investigated. The protodifferentiated cartilage phenotype seen in aggregates formed in cultures from stage 26 wings is a phenotype found in the prechondrogenic limbs. Micromass cultures prepared from the proximal half of stage 23 or 24 wings behave similarly to those prepared from whole stage 26 wings (Swalla et al., 1983), producing aggregates of protodifferentiated cells. Addition of as few as one out of ten distal wing cells to proximal cultures stimulates nodule formation. These observations are also consistent with a role for a mesenchyme-derived influence in initial chondrogenic expression.
The behavior of different stage wing mesenchyme in the micromass culture system suggests that there is a progressive change in the properties of subsets of limb mesenchyme cells. At relatively early stages some cells can form aggregates, which can differentiate into cartilage after treatment with db cyclic AMP. Otherwise, cells from this early stage become blocked at a predifferentiated stage, prior to the accumulation of detectible levels of type II collagen. Later in situ, these cells can provide a chondrogenic stimulus as well as respond to the signal. However, at a later stage, they can no longer provide the signal, but they can still express chondrogenesis in response to a permissive environment. The response involves the overt expression of the chondrogenie phenotype by protodifferentiated cartilage cells. The demonstration of cells having these three phenotypes suggests the existence of at least two regulatory steps in cartilage differentiation. REFERENCES AHRENS, P. B., SOLURSH, M., and REITER, R. S. (1977). Stage-related capacity for limb chondrogenesis in cell culture. Deu. Biol. 60, 6982. AHRENS, P. B., SOLURSH, M., REITER, R. S., and SINGLEY, C. T. (1979). Position-related capacity for differentiation of limb mesenchyme in cell culture. Deu. Biol. 69, 436-450. CAHN, R. D., COON, H. G., and CAHN, M. B. (1967). Cell culture and cloning techniques. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 493-530. Crowell, New York. CAPLAN, A. (1970). Effects of the nicotinamide-sensitive teratogen 3acetylpyridine on chick limb bud cells in culture. Erp. Cell. Res. 62, 341-355. DESSAU, W., VON DER MARK, H., VON DER MARK, K., and FISHER, S. (1980). Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol. 57, 51-60. FELL, H. B., and CANTI, R. G. (1934). Experiments on the development in vitro of the avian knee joint. Proc. Roy. Sot. Ser. B 116, 316349. HALL, B. K. (1981). Modulation of chondrocyte activity in vitro in response to ascorbic acid. Acta Anat. 109, 51-63. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,4992.
HASCALL, G. K. (1980). Cartilage-proteoglycans: Comparison of sectioned and spread whole molecules. J. Ultrastruct. Res. 70, 369-375. HUMASON, G. L. (1972). “Animal Tissue Techniques,” 3rd ed. pp. 317, 320, 332. Freeman, San Francisco. JANNERS, M. Y., and SEARLS, R. L. (1970). Changes in the rate of cellular proliferation during the differentiation of cartilage and muscle in the mesenchyme of the embryonic chick wing. Deu. Biol. 23, 136-165. LEDOUARIN, N. (1973). A biological cell labeling technique and its use in experimental embryology. Deu. Biol. 30, 217-222. LEVINE, C. I., and BATES, C. J. (1976). The effect of hypoxia on collagen synthesis in cultured 3T6 fibroblasts and its relationship to the mode of action of ascorbate. Biochim. Biophys. Acta 444, 446452. LINSENMAYER, T. F., and HENDRIX, M. J. C. (1980). Monoclonal antibodies to connective tissue macromolecules: type II collagen. Biochem.
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SOLURSH ET AL. LINSENMAYER, T. F., HENDRIX., M. J. C., and LITTLE, C. D. (1979). Production and characteriz.ation of a monoclonal antibody to chicken type I collagen. Proc. Nut. Acad. Sci. USA 76, 3703-3707. LINSENMAYER, T. F., TOOLE, B. P., and TRELSTAD, R. L. (1973). Temporal and spatial transitions in collagen types during embryonic chick limb bud development. Deu. Biol. 35, 232-239. MEIER, S., and SOLURSH, M. (1978). Ultrastructural analysis of the effects of ascorbic acid on secretion and assembly of extracellular matrix by cultured chick embryo chondrocytes. J. Ultrastruct. Res.
65, 48-59. MILLER, R. P., HUSAIN, M., and LOHIN, S. (1979). Long acting CAMP analogues enhance sulfate incorporation into matrix proteoglycans and suppress cell division of fetal rat chondrocytes in monolayer culture. J. Cell Physiol. 100, 63-76. OSDOBY, P., and CAPLAN, A. I. (1979). Osteogenesis in cultures of limb mesenchymal cells. Deu. Biol. 73, 84-102. RUTTER, W. J., CLARK, W. R., KEMP, J. D., BRADSHAW, W. S., SANDERS, T. G., and BALL, W. D. (1968). Multiphasic regulation in cytodifferentiation. In “Epithelial Mesenchymal Interactions” (R. Fleischmajer and R. E. Billingham, eds.), pp. 114-131. Williams & Wilkins, Baltimore. SAUNDERS, J. W. (1948). The proximo-distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J. Exp. Zool.
108,363-403. SEARLS, R. L. (1965). An autoradiographic study of the uptake of S35 sulfate during the differentiation of limb bud cartilage. Deu. Biol.
11,155-168. SINGLEY, C. T., and SOLURSH, M. (1981). The spatial distribution of hyaluronic acid and mesenchymal condensation in the embryonic chick wing. Deu. Biol. 84, 10~2-120. SOLURSH, M. (1980). Histogemc Mechanisms in in vitro limb Chondrogenesis. In “Current Research Trends in Prenatal Craniofacial Development” (R. M. Pratt and R. L. Christiansen, eds.), pp. 315327. Elsevier North-Holland, New York.
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SOLURSH, M., AHRENS, P. B., and REITER, R. S. (1978). A tissue culture analysis of the steps in limb chondrogenesis. In Vitro 14, 51-61. SOLURSH, M., HARDINGHAM, T. E., HASCALL, V. C., and KIMURA, J. H. (1980). Separate effects of hyaluronic acid on proteolgycan synthesis and deposition of pericellular matrix by cultured chick embryo limb chondrocytes. Deu. Biol. 75, 121-129. SOLURSH, M., and REITER, R. S. (1980). Evidence of histogenic interactions during in vitro limb chondrogenesis. Deu. Biol. 78, 141150. SOLURSH, M., REITER, R. S., AHRENS, P. B., and VERTEL, B. M. (1981). Stage and position-related changes in chondrogenic response of chick embryonic wing mesenchyme to treatment with dibutyryl cyclic AMP. Deu. Biol. 83, 9-19. SOLURSH, M., REITER, R. S., AHRENS, P. B., and PRATT, R. M. (1979). Increase in levels of cyclic AMP during avian limb chondrogenesis in uitro. Differentiation 15, 1833186. STEMPAK, J. C., and WARD, R. T. (1964). An improved staining method for electron microscopy. J. Cell. Biol. 22, 697-701. SWALLA, B. J., OWENS, E. M., LINSENMAYER, T. F., and SOLURSH, M. (1983). Two distinct classes of prechondrogenic cell types in the embryonic limb bud. Submitted for publication. THOROGOOD, P. (1979). In vitro studies on skeletogenic potential of membrane bone periosteal cells. J. Embryol. Exp. Morphol. 54, 185-
207. UMANSKY, R. (1966). The effect of cell population density on the developmental fate of reaggregating mouse limb bud mesenchyme. Dew. Biol. 13, 31-56. VENABLE, J. H., and COGGESHALL, R. (1965). A simplified lead citrate stain for use in electron microscopy. J. Cell. Biol. 25, 407-408. VON DER MARK, K., and VON DER MARK, H. (1977). Immunological and biochemical studies of collagen type transition during in vitro chondrogenesis of chick limb mesodermal cells. J. Cell Biol. 73,736-
747.
BIOLOGY
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(1982)
Two Distinct Regulatory Steps in Cartilage Differentiation’ MICHAEL SOL.URSH,* KAREN L. JENSEN,* CARL T. SINGLEY,*” AND REBECCA S. REITER* *Department
THOMAS
F. LINSENMAYER,~
of Zoology, University of Iowa, Iowa City, Iowa 52242; and tThe Developmental Biology Laboratory, Massachusetts General Hospital and Harvard Medical School, Boston, Massachusetts 02114 Received March 2, 1982; accepted in revised form July 21, 1982
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 trseated 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 mat.rix 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, inclucling 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. INTRODUCTION
In general, the differentiation of particular cell types involves a sequence of distinct steps. Each step brings the progenitor cell closer to the final differentiated product. One way to identify and study individual steps or transitions is to isolate cells at different times during their development and compare their differentiative properties in a particular test system. This approach has been used to identify distinct steps in the conversion of limb mesenchyme cells into overtly differentiated chondrocytes (Solursh et al., 1978). The test system involves high-density cell cultures which permit the differentiat:ion of several cell types (Umansky, 1966; Caplan, 1970; Osdoby and Caplan, 1979), especially cartilage. In these cultures, initially aggregates of cells form in which there is more extensive cell multilayering (Ahrens let al., 1977). In cultures prepared from wings of certain stage chick embryos (stages 2025 of Hamburger and Hamilton (1951)), an Alcian bluestaining extracellular matrix accumulates in aggregates as cartilage differentiation occurs. However, in cultures 1 Supported by NIH Grant HD05505 and NSF Grant PCM 77. 01154 to MS. and NIH Grants EY02261 and AM03564 to T.F.L. ’ Present address: Depart:ment of Zoology, Ohio State University, Columbus, Ohio 43210.
prepared from both earlier (stage 19) and later stages (stage 26), the aggregates do not form cartilage matrix spontaneously, but can be stimulated to do so if the cultures are treated with db cyclic AMP (Solursh et al., 1981). Previous information concerning the properties of wing cells from stages 19, 24, and 26 is summarized below. At stage 19, the mesenchyme cells in the wing bud appear morphologically homogeneous (Singley and Solursh, 1981). While organ cultures prepared from wing buds of this stage do form cartilage (Solursh and Reiter, 1980), high-density cell cultures produce only aggregates, which fail to accumulate an Alcian blue-staining extracellular matrix (Ahrens et al., 1977). Apparently, dissociation and/or randomization of the cells at this early stage interferes with some step in chondrogenic differentiation. These cells appear unable to interact with other cells in a manner which is apparently required for chondrogenic differentiation in this culture system (Solursh and Reiter, 1980). By stage 24, a number of regional changes have occurred in the wing bud. Early skeletal primordia become recognizable by histology (Fell and Canti, 1934) as well as by their relatively higher uptake of 35SO; (Searls, 1965) and lower uptake of [3H]thymidine (Janners and Searls, 1970). However, overt chondrogenic differentia-
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tion is not yet detectable in situ. High-density cultures prepared from wings from stage 24 embryos form aggregates which spontaneously accumulate an Alcian blue-staining extracelltilar matrix (Caplan, 1970; Ahrens et al., 1977). By stage 26, chondrogenesis has begun in the proximal regions of the wing. This can be demonstrated by histology (Fell and Canti, 1934) and by biochemical (Linsenmayer et al., 1973) or immunological (Dessau et al., 1980) detection of cartilage collagen. The more distal regions of the limb are ,at progressively younger stages of development (Saunders, 1948). High-density cultures of wing buds from stage 26 embryos again produce aggregates which fail to accumulate spontaneously an Alcian blue-staining matrix. In addition, one finds small clusters of chondrocytes which apparently had already in situ (Solursh et al., 1981). differentiated Since the presence of an Alcian blue-staining matrix can only detect overtly differentiated cartilage cells, additional, more sensitive criteria for chondrogenesis are needed. This present study is concerned with a further analysis of the in vitro behavior of cells from these three stages. Transmission electron microscopy and immunohistochemistry (Von der Mark and Von der Mark, 1977) with a monoclonal antibody directed against type II collagen (Linsenmayer and Hendrix, 1980) are used to define further the cell phenotypes in cultures prepared from stages 19, 24, and 26. The results indicate that cells from stage 19 wings become blocked at a step prior to the production of detectible levels of type II collagen. On the other hand, stage 26 cells contain type II collagen, but resemble mesenchyme in other respects. The existence of these two phenotypes suggests that there are at least two (distinct regulatory steps in the expression of the cartilage phenotype. MATERIALS
AND
METHODS
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26 wings) for 10 min at 37°C and mechanically dissociated by pipetting. The enzyme solution (2 ml) was diluted with 3 ml of medium, sometimes containing 100 pg/ml of soybean trypsin inhibitor (Sigma Chemical Corp., St. Louis, MO.). The cells were collected by centrifugation and suspended in medium sometimes containing 100 pg/ml soybean trypsin inhibitor. Cell suspensions were passed through two layers of No. 20 Nitex screen, and the cell concentration was determined with a hemacytometer. The cells were collected by centrifugation and resuspended in culture medium consisting of Ham’s Fiz nutrient mixture and 10% fetal bovine serum (both from Grand Island Biological Co., Grand Island, N. Y .) and antibiotics. In some cases, chick cells were mixed with Japanese quail cells prepared from stage 24 wings as described above or from chick chondrocytes prepared either from limb bud mesenchyme by maintaining cells in suspension culture for 48 hr (Solursh et al., 1980) or from sterna prepared according to the method described by Cahn et al. (1967). In all cases, the final cell concentration was 2 X lo5 cells per 10 ~1. Ten microliters of cell suspension was placed on a 35 mm tissue culture dish or on an autoclaved borosilicate coverslip (Bellco) in a culture dish. After 1 hr at 37°C in culture 2 ml of culture medium was added. The medium was replaced daily. In some experiments, the cultures were treated in various ways. For db cyclic AMP treatment, cultures were maintained in medium containing 1 mM N6, 02’dibutyryl adenosine 3’:5’-monophosphoric acid (Sigma) from Day 1 to 3 of culture. Ascorbate treatment (50 pg/ml) was begun on Day 0 or Day 1 of culture; the effects were similar in either case. Cultures were usually maintained in an atmosphere of 5% CO2 and 95% air. For low oxygen, cultures were placed in a humidified jar gassed with a mixture of 5% C02, 25% air, and 70% N2, and kept at 37°C.
Cultures
Fixation
Micromass cultures were prepared as described previously (Ahrens et al., 1977) from wing buds of stage 19, 24, or 26 (Hamburger and Hamilton, 1951) White Leghorn chick embryos (Welp Hatchery, Bancroft, Iowa). Wings were incubated in trypsin and collagenase (0.05% each for stage 19 wings and 0.1% each for stage 24 or
Cultures were washed with Saline G and in some cases were fixed for 10 min in Kahle’s fixative as described previously (Ahrens et al., 1977). These cultures were stained with Alcian blue at pH 1 in order to visualize sulfated glycosaminoglycans in the extracellular matrix. Nodule numbers per culture were counted on tracings
and Histology
FIG. 1. Stage 19 wing bud mesenchyme control culture on Day 3. (a) Thick Epon section in the plane of culture illustrating aggregate and interaggregate morphology. Aggregates consist of a core of rounded, tightly packed cells surrounded by less tightly packed, round and crescentshaped cells. There is little extracellular space in the aggregate core. The darkly stained cells in this and subsequent micrographs appear to have been fixed in various stages of cell death. X140. (b) Electron micrograph showing the close cell contact typical of stage 19 aggregates. Aggregate cells possess few cell processes and form numerous gap junctions (not illustrated). X6000. (c) Electron micrograph of an interaggregate area. Cells in interaggregate areas are generally more elongate, flattened, and possess numerous cell processes. There is considerably more extracellular space in these areas as compared with aggregates. The extracellular material consists of numerous collagen fibrils as well as some proteoglycan-like material and 3-nm filaments. X6000.
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made by use of a microslide projector. Nodule diameters were measured directly from the cultures with the aid of an ocular micrometer in a microscope at 400X. Two measurements at right angles to each other were made on each of 10 different nodules in three different cultures. An analysis of variance with a priori contrast was performed to determine if there were significant differences between pairs of means. Some cultures were dehydrated, embedded in paraffin, serially sectioned at 7 pm, and stained according to the Feulgen method (Humason, 1972), and then with Alcian blue at pH 1. The numbers of quail and chick nuclei were counted under an oil immersion objective. For electron microscopy, washed cultures were fixed for 30 min in 3% glutaraldehyde in one-half strength Karnovsky’s buffer at pH 6.6 and postfixed, dehydrated, and embedded in Epon 812, all as described previously (Ahrens et al., 1979). For light microscopy, sections 0.25 pm thick were cut with glass, mounted on slides, and stained with Azure II and methylene blue. For transmission electron microscopy, sections, about 50 to 80 nm thick, were cut with a diamond knife with a Sorval MT-2B ultramicrotome, stained with methanolic uranyl acetate (Stempak and Ward, 1964) and lead citrate (Venable and Coggeshall, 1965), and examined with a Philips EM300 electron microscope. All sections were cut parallel to the culture dish. Immunofluorescence Cultures inoculated on coverslips were washed three times at 37°C with Saline G and then once with 0.02 M phosphate-buffered saline (pH 7.2) at room temperature. They were then fixed in 100% acetone for 10 min and air-dried at room temperature for 4 days. For antibody staining, all subsequent steps were done at room temperature. The cultures were rehydrated in phosphate-buffered saline for 10 min and then incubated in the various antibody-containing mouse ascites fluids for 30 min. These were l/1600 dilutions of ascites fluids containing either monoclonal antibody to type II collagen (Linsenmayer and Hendrix, 1980), type I collagen (Linsenmayer et al., 1979), or the control ascites fluid. The cultures were washed four times with phosphate-
FIG. 2. Stage 19 wing bud mesenchyme culture (Day 3) treated with db CAMP. (a) Thick Epon section in the plane of culture. Note that the aggregate cells are widely separated by extensive extracellular space as compared with the tightly packed cells of control cultures (Fig. la). The interaggregate areas contain more intercellular space as compared to controls. X140. (b) Electron micrograph of an aggregate core. The extensive extracellular spaces are filled with a granular and filamentous matrix having the appearance of aggregate proteoglycan (inset; Hascall, 1980). Very little collagen is seen in aggregates in db CAMP-treated cultures. X1500 (inset X90,000). (inset bar = 100 am).
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buffered saline and incubated with fluorescein-conjugated IgG fraction of rabbit anti-mouse IgG (heavy and light chains; Cappel Laboratories, Lot No. 13039), l/300 dilution in phosphate-buffered saline, for 20 min. To intensify the fluorescent signal, the cultures were then washed four times in phosphate-buffered saline and incubated for 20 min in fluorescein-conjugated IgG fraction of goat anti-rabbit IgG (heavy and light chains;
FIG. 3. Stage 19 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate area in a control culture. (b) A few cells in the aggregate shown in (a) exhibit extracellular fluorescence after being stained with antibody to type II collagen. Small clusters of stained cells, usually in aggregate centers, are observed throughout the culture. The intensity is usually faint, however, approaching background. (c) Phase-contrast micrograph showing an aggregate area in a culture which had been exposed to db CAMP from Day 1 to 3. (d) The area shown in c shows intense intra- and extracellular fluorescence after being stained with antibody to type II collagen. The fluorescence intensity is drastically increased, as is the size of the staining area, compared to control cultures (all x200).
FIG. 4. Stage 24 wing bud mesenchyme control culture at Day 2. (a) Thick Epon section in the plane of culture. The aggregates in these cultures blend into interaggregate areas and are less distinct than those of stage 19 cultures (Fig. la). Aggregates are characterized by the rounded shape of the core cells and the crescentic shape of the more peripheral cells. As in stage 19 cultures, aggregate cells on Day 2 of culture are tightly associated with little intervening space. X140. (b) Electron micrograph of an aggregate. Aggregate cells are rounded and have few cell processes. The ECM here is sparse and consists of collagen fibrils and few proteoglycan-like granules (not illustrated). x1500.
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Cappel Laboratories, Lot No. 13259) diluted l/300 in phosphate-buffered saline. After four more washes, the coverslips were mounted in glycerol and observed or photographed with a Leitz Planopak illuminator with an LZ filter cube. The specificity of the staining was verified on cultures of chick dermal fibroblasts or chick sternal chondrocytes for types I and II collagens, respectively. There was no detectable fluorescence with the control ascites fluid. RESULTS
Stage 19 Cultures Control cultures. Micromass cultures prepared from wing mesenchyme of stage 19 chick embryos form numerous aggregates but no Alcian blue-staining nodules (Ahrens et al., 1977). These are examined on Day 3 of culture, since nodules do not form after even longer periods. Viewed in cross section, the aggregates appear as areas of mesenchyme in which cells are more extensively multilayered than in adjacent areas (Solursh et al., 1978). Cut horizontally to the culture dish, the aggregates appear as regions containing tightly packed cells surrounded by more dispersed interaggregate cells (Fig. la). Based on transmission electron microscopy, cells within an aggregate appear closely applied and have little intercellular space (Fig. lb). On the other hand, the interaggregate cells are separated by more extracellular space in which one finds collagen fibrils, 3-nm filaments, and some 30-nm proteoglycan-like granules. Based on the absence of an Alcian blue-staining extracellular matrix and matrix components identifiable at the electron microscopic level, the aggregates have failed to differentiate into cartilage nodules. The type of collagen present in the cells can be used as a more qualitative criterion for the differentiation of cartilage cells than the accumulation of extracellular matrix. Observed from above in whole mounted cultures, type I collagen is detected throughout the stage 19 cultures (not shown). However, when cultures are stained with antibody to type II collagen, fluorescence is observed only in a few restricted areas. Small clusters of cells within aggreg,ates exhibit a faint fluorescence (Figs. 3a,b) suggesting the presence of type II collagen in a few cells. db CAMP-treated cultures. It has been reported earlier
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that db CAMP treatment of micromass cultures from wings of stage 19 embryos from Day 1 to 3 of culture stimulates some aggregates to develop into Alcian bluestaining nodules (Ahrens et al., 1977; Solursh et al., 1981). After such treatment, cells in both aggregate and interaggregate areas are separated by more extensive extracellular space (Fig. 2a). In aggregate areas, the extracellular matrix resembles that of cartilage matrix, containing proteoglycan aggregates. Furthermore, the cell aggregates now stain intensely with type II collagen antibody (Figs. 3c,d). On the other hand, the interaggregate areas do not stain with type II antibody. These results demonstrate that db cyclic AMP treatment greatly increases the proportion of cells associated with type II collagen. In addition, it drastically stimulates the accumulation of an extracellular cartilage matrix. A number of other treatments, including ascorbate and soybean trypsin inhibitor, had no detectable effect on stage 19 cultures, in contrast to cells from the later stages considered below. Stage 24 Cultures Control cultures. In cultures derived from stages 2025 (Ahrens et al., 1977; Solursh et al., 1981), aggregates spontaneously develop into Alcian blue-staining nodules. By 40 hr of culture, small areas in the center of aggregates become associated with an Alcian blue-staining matrix. These areas enlarge progressively with time. A similar pattern is observed when the cultures are stained with antibody to type II collagen (not shown). At the electron microscope level, after 48 hr of culture, aggregate cells appear rounded and have few cell processes (Fig. 4). Still at this time the extracellular matrix is sparse, consisting of collagen fibrils and a few proteoglycan-like granules. After 72 hr of culture, cartilage nodules accumulate extensive cartilage matrix (Fig. 5). The extracellular material is rich in proteoglycan-like granules, but contains little identifiable collagen. Together with the inflated ER cisternae, these features suggest that these chondrocytes are scorbutic (Meier and Solursh, 1978). By immunofluorescence, type II collagen is detected in discrete nodules and appears primarily intracellular (Figs. 7a,b). On the other hand, the entire culture stains with antibody against type I collagen (not shown). The
FIG. 5. Stage 24 wing bud mesenchyme control culture at Day 3. (a) Thick Epon section in the plane of culture. Distinct cartilage nodules (n) are present throughout the culture. Cartilage nodules display an extensive extracellular component surrounding relatively rounded cells. Internodular areas appear similar to interaggregate areas of Day 2 cultures (Fig. 4a). X140. (b) Electron micrograph of a cartilage nodule. Nodular cells are rounded, have inflated ER cisternae and possess only a few short cell processes. X1500. (c) The extracellular matrix is rich in proteoglycan-like granular material. Collagen fibrils (C) are sparse as compared with normal cartilage in situ (Meier and Solursh, 1978). X90,000. (d) Internodular cells are more flattened, elongate, and possess numerous cell processes. The ECM consists of collagen fibrils and sparse proteoglycan-like granular material (Fig. 5~). X6000.
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internodular areas still resemble the interaggregate areas of Day 2 cultures. The internodular cells appear flattened and are associated with collagen fibrils and some proteoglycan-like material (Fig. 5d). db CAMP-treated cultures. Treatment of micromass cultures from Day 1 to 3 with db CAMP causes the formation of extensive cartilage throughout the culture, based on Alcian blue staining, 35S autoradiography and staining with antibody directed against cartilage-specific proteoglycan (Solursh et al., 1981). As can be seen in Fig. 6, in treated cultures the nodules are less distinct and appear to anastomose. Cells in the interaggregate areas are in small clusters which appear to be undergoing chondrogenesis, as judged by the extensive staining with antibody against type II collagen (Figs. 7c,d). On the other hand, treatment with ascorbate or soybean trypsin inhibitor does not stimulate the interaggregate areas to form cartilage. Stage 26 Cultures Control cultures. Micromass cultures prepared from wings of stage 26 embryos form numerous aggregates; however, most of these aggregates fail to develop into Alcian blue-staining nodules even with an extended time of culture (Solursh et al., 1981). While the cells in these aggregates still appear mesenchymal in morphology (Fig. 8), they are more polymorphic and less tightly packed than those in cultures from stage 19 or 24 embryos (cf. Figs. 1 and 5). In further contrast to stage 19 and 24 cultures, the entire aggregate areas show intracellular staining for type II collagen (Fig. 10). Based on the three combined criteria, (mesenchyme morphology, absence of Alcian blue-staining matrix but the presence of type II collagen), the stage 26 aggregate cells are chondrocytes, but they are not overtly differentiated. Treated cultures. Several treatments have been found to stimulate the development of aggregates in stage 26 cultures into Alcian blue-staining nodules. However, by
FIG. 6. Stage 24 wing bud mesenchyme db CAMP treated culture at Day 3. (a) Thick Epon section in the plane of a micromass culture of stage 24 chick wing bud mesenchyme treated with db CAMP and fixed at the end of Day 3 of culture. The nodules (n) are less distinct than in Day 3 control cultures and appear to anastomose. Internodular areas (I) form aggregate-like clusters of cells which appear to be undergoing chondrogenesis. x140. (b) Low-magnification TEM micrograph of a nodule/internodule interfacial area like that indicated by the box in (a). The nodular matrix is more dense than that of untreated controls, and there is considerable cartilage-like matrix in the internodular areas. More fibrillar collagen is present in the matrix of these nascent nodules as compared with the aggregates of control cultures. It is noteworthy that cells within mature nodules do not display a typical chondrocyte morphology, but are generally flattened in the plane of culture and often possess large cell processes. X1500.
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late stage 26 many of these treatments become less effective. db CAMP treatlment, for example, stimulates nodule formation, as reported previously (Solursh et al., 1981). However, this treatment becomes ineffective with advancing stage. Based on transmission electron microscope examination, db CAMP-treated cultures contain nodules resembling those seen in stage 24 cultures, except that the cells are highly polymorphic and the extracellular matrix is less dense (Fig. 9). Furthermore, nodules in these treated cultures stain with antibody to type II collagen in a manner indistinguishable from untreated stage 26 cultures (see Fig. 10). Several other treatments besides db CAMP also promote the formation of Alcian blue-staining nodules by stage 26 cultures. The addition of ascorbic acid increases nodule number (Table l; Fig. 11) in stage 26 cultures but has no such effect on stage 19 or stage 24 cultures. Since its effect is synergistic with that produced by db CAMP (Table 1; Fig. ll), these agents must act by distinct mechanisms. Maintenance of cultures in an atmosphere of 5% O2 also dramatically increases the formation of Alcian blue-staining nodules (Table 1). One expected effect of ascorbate treatment is to increase the accumulation. of collagen. As can be seen in Fig. lOd, ascorbate-treated stage 26 cultures stain intensely with antibody to type II collagen and a considerable proportion of the staining appears to be extracellular. A similar effect of ascorbic acid is seen in stage 24 cultures. Transmission electron microscopic observation of such treated cultures demonstrates the presence of numerous collagen fibrils in the extracellular spaces and the reduced inflation of the ER cisternae in treated cells (Fig. 12). Behavior of Cultures of Mixtures 26 Cells
of Stage 24 and Stage
The results presentecl above demonstrate that wing mesenchyme from stage 24 embryos can undergo chondrogenic expression spontaneously while those from stage 26 embryos fail to express the chondrogenic phenotype overtly. The dissociation of stage 26 wings into a suspension of single cells could remove a required factor. A possible mechanism for the failure of expression by stage 26 cells is that there is some factor which is required for chondrogenic expression and which is synthesized in adequate am.ounts by stage 24 cells but not by stage 26 cells. If this hypothesis is correct, the addition of small numbers of stage 24 cells to cultures of stage 26 mesenchyme is expected to improve nodule formation. The results shown in Table 2 are consistent with this hypothesis. The addition of progressively greater proportions of stage 23 cells increases nodule number. The addition of even one stage 23 cell out of
FIG. 7. Stage 24 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate (large arrow) and interaggregate area (small arrow) in a control culture. (b) Cells within the aggregate exhibit intracellular fluorescence after being stained with antibody to type II collagen. Only background fluorescence is detected in the internodular area. Nodules throughout the culture fluoresce and are separated by nonfluorescing internodular regions. (c) Phase-contrast micrograph of an aggregate and interaggregate area in a culture which has been exposed to db CAMP from Day 1 to 3. (d) Both the aggregate and interaggregate areas stain and the whole culture shows generally increased fluorescence compared to control cultures (all x200). Ascorbate-treated cultures (not shown) show a similar distribution of fluorescence throughout the culture, but the intensity is drastically increased. In addition, there is increased extracellular staining.
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Treatment” Control db AMP Ascorbate Ascorbate Low 02
+ db CAMP
Number of Alcian bluestaining nodules/culture* 0 8f6 23 f 6 74 f 4
2+4 10 f 4 9t4 70 + 13
4*5 8fl 34 f 15 92 f 5 59 f 23
“All treatments are from Day 1 to 3 of culture except for the low 02, which was begun on Day 0. * Values are means + SD of four cultures. Three representative trials are shown.
three stage 26 cells produces a significant increase in nodule number. Examination of cultures prepared from stage 24 quail wings and stage 26 chick wings, in which quail and chick cells can be distinguished (LeDouarin, 1973), shows the expected proportion and distribution of cells from the two stages, as reported previously (Solursh and Reiter, 1980). The similar diameters of nodules regardless of the proportion of stage 26 cells, other than the 3:l mixture of stage 26 and stage 23 cells, suggest that stage 26 cells are participating in nodule formation (Table 2). Sections through chimeric cultures prepared from a 1:3 mixture of stage 24 chick cells and stage 26 quail cells clearly demonstrate that stage 26 cells are surrounded by Alcian blue staining matrix (Fig. 13a). In contrast, in a 1:3 mixture of quail limb or sternal cartilage and stage 26 cells, an Alcian blue-staining matrix is not associated with the stage 26 cells (Fig. 13b). It is often associated with the cartilage cells, however. These results suggest that stage 24 cells can promote the overt expression of chondrogenesis by stage 26 cells and support the hypothesis that there is a temporally regulated, mesenchyme-derived influence which promotes chondrogenic expression. DISCUSSION
By use of micromass cultures as a test system, the chondrogenic capacities of limb mesenchyme cells can be demonstrated to undergo distinct changes with deFIG. 8. Stage 26 wing bud control culture at Day 3. (a) Thick Epon section in plane of culture. The aggregates in stage 26 cultures are less distinct than those of stages 19 or 24. Interaggregate areas display considerable extracellular space which, however, contains little visible matrix material. X140. (b) Electron micrograph of an aggregate. The cells here are more polymorphic and are less tightly packed as compared with aggregates of stage 19 or 24 cultures. Myoblasts are often observed within aggregates of stage 26 mesenchyme (arrow). X1500.
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TABLE 2 THE FORMATION OF CARTILAGE NODULES BY MIXTURES OFCELLSFROMSTAGE~~ AND~~WINGS Ratio of stage 26:stage 23derived cells
Number of Alcian blue-staining nodules”
1:o 2O:l 3:l 1:l 1:3 0:l
0.25 f 0.5 Ok0 31 +- 9 46 f 31 141 ? 34 188 + 25
Mean nodule diameter*
4.97 6.00 6.67 6.38
f + f +
0.27’ 0.28 0.33 0.25
n Results are means f SD of four cultures and are typical of two trials. * Results are expressed as means + SE. Each unit = 0.018 mm. c Only this value is significantly different from the 0:l cultures (P 5 0.0006).
After dissociation and randomization, stage 19-derived wing mesenchyme can only form aggregates of closely associated cells. These cells do not undergo chondrogenesis as defined both by ultrastructural features and by immunofluorescence for type II collagen. Because these cells interfere with chondrogenesis when they are mixed with cultures from stage 24 wing mesenchyme, it was suggested that the stage 19-derived cells are unable to interact with wing mesenchyme from stage 24 embryos in a manner which is required for chondrogenesis (Solursh and Reiter, 1980). The aggregated cells are, however, potentially chondrogenic since treatment with db cyclic AMP causes the accumulation of cartilage matrix, demonstrated by Alcian blue staining and ultrastructural features, and the appearance of type II collagen, demonstrated by immunofluorescence. While the mechanism of action of db cyclic AMP on these cells is not known, it has been hypothesized that it bypasses required cell-cell interaction (Solursh et al., 1978). In fact, particular cell-cell interactions might lead to increased endogenous levels of cyclic AMP (Solursh et al., 1979) which in turn might promote chondrogenesis. Cells derived from wings of stage 24 embryos in highdensity cultures form aggregates which spontaneously differentiate into cartilage nodules, as defined by histochemical (Caplan, 1970; Ahrens et al., 1977) and biovelopment.
FIG. 9. Stage 26 wing bud culture at Day 3 treated with db CAMP. (a) Thick Epon section in plane of culture. The entire culture displays a greater amount of extracellular space as compared with Day 3 controls (Fig. Ba). X140. (b) Electron micrograph of a cartilage nodule. The cells of this nodule are highly polymorphic and possess numerous cell processes as compared with the nodular cells of stage 24 cultures (Fig. 5b). In addition, the extracellular component of the nodule is less extensive, and the matrix is not as dense. The cells here continue to display a highly inflated ER. X1500.
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FIG. 10. Stage 26 cultures on Day 3 viewed from above. (a) Phasecontrast micrograph showing an aggregate area in a control culture. (b) The cells within the aggregate area shown in (a) exhibit a diffuse intracellular fluorescence in aggregate areas but not in the interaggregate areas after being stained with antibody to type II collagen. The aggregates show fluorescence even though they do not appear to have formed nodules, based on Alcian blue staining. Cultures treated with db CAMP from Day 1 to 3 contain many Alcian blue-staining nodules, but the fluorescence after staining with antibody to type II is only slightly brighter than that shown in Fig. lob. (cl Phase-contrast micrograph of an aggregate area of an ascorbate-treated culture. (d) The area shown in c exhibits intense extracellular fluorescence. The internodular areas do not stain with antibody to type II collagen. The nodules also stain with Alcian blue in these cultures. Cultures treated with both ascorbate and db CAMP stain nearly as intensely as that shown in Fig. 10d (all X200).
VOLUME 94, 1982
chemical (Von der Mark and Von der Mark, 1979) criteria. Because the nodules enlarge by assimilating adjacent mesenchyme, it has been suggested that the differentiating cartilage has an autoinductive influence on cartilage differentiation (Solursh, 1980). The assimilative process can be greatly accelerated by treatment with db cyclic AMP, as determined by a number of criteria (Solursh et al., 1981), including ultrastructure and immunofluorescence for type II collagen. Cultures prepared from wings of stage 26 embryos form numerous aggregates; these contain cells which fail to accumulate an Alcian blue-staining matrix and which resemble mesenchyme cells ultrastructurally. However, based on the presence of immunologically detectable type II collagen, these aggregated cells are chondrocytes which have not undergone overt differentiation. This intermediate state in differentiation has been called the protodifferentiated state (Rutter et al., 1968). The existence of a protodifferentiated state suggests that there might be two transitions during chondrogenesis: one involving the initial acquisition of the chondrogenic capacity and another involving overt expression. In the high-density culture system used here, several environmental agents are found to promote expression of chondrogenesis. Caplan (1970) noted that treatment with 3-acetylpyridine promotes nodule formation in cultures from stage 26 limbs. The mechanism is not known. Treatment with db cyclic AMP also causes many of the aggregates to accumulate an extracellular matrix which stains with Alcian blue and which ultrastructurally resembles that produced by stage 24-derived cultures. Cyclic AMP analogues have been shown to promote the accumulation of proteoglycan by other chondrocytes (Miller et al., 1979). The lack of any detectable change in type II collagen immunofluorescence after this treatment might be due to the scorbutic culture conditions used (Meier and Solursh, 1979). Treatment with ascorbic acid also causes an increase in the number of Alcian blue staining nodules formed in stage 26-derived cultures and the accumulation of an ultrastructurally normal cartilage matrix, resembling that found in situ (Meier and Solursh, 1979). db Cyclic AMP and ascorbate have synergistic effects on nodule number and therefore, are likely to act by different, but complementary, mechanisms. Both of these mechanisms might be related to effects on secretory processes. The action of low oxygen is most likely to be related to the effects of ascorbate, since hypoxia can enhance collagen formation by fibroblasts by activating prolyl hydroxylase (Levine and Bates, 1976). Effects of both ascorbate (Hall, 1981) and hypoxia (Thorogood, 1979) on chondrogenesis have been reported. It would be interesting if these effects were on the process of overt expression.
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The ability of small numbers of stage 24-derived cells to promote chondrogenic expression by stage 26 cultures is particularly noteworthy. The participation of stage 26-derived cells in chonldrogenesis in mixed cultures is suggested by the observations that nodule size is the same in stage 24 cultures and in cultures containing a
a
b
FIG. 12. Stage 26 wing bud culture at Day 3 treated with ascorbate. Electron micrograph of a nodule. The cells here are more polymorphic than nodule cells of stage 24 controls, but somewhat less so than stage 26 control or db CAMP-treated cultures. A striking feature of ascorbate-treated cultures is the presence of numerous collagen fibrils in the ECM. Note also that the ER of these cells is less inflated than that of control cells. X1500.
FIG. 11. Whole mounts of micromass cultures prepared from stage 26 wings and fixed and stained with Alcian blue after 72 hr of culture. (a) Control culture. No nodules are present. (b) Culture treated with db CAMP from Day 1 to 3. A few nodules are seen. (c) Culture treated with 50 @g/ml ascorbate. (d) Culture treated with both db CAMP and ascorbate. While both db CAMP and ascorbate added alone increased the number of nodules formed, their effect is synergistic when they are present together (X10).
1:l mixture of stage 24 and stage 26 cells and that stage 26 cells are associated with Alcian blue-staining matrix in chimeric nodules. The effect of adding one out of three stage 24 cells to stage 26 cells contrasts with the effect of mixing similar proportions of stage 19 and stage 24 cells. In this case, chondrogenesis was not expressed (Solursh and Reiter, 1980). More important, adding one out of three differentiated chondrocytes to stage 26 cells
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b
FIG. 13. Cross sections of chimeric wing bud cultures at Day 3 stained by the Alcian blue and Feulgen method. (a) This culture was prepared from a 1:3 mixture of stage 24 chick and stage 26 quail wing bud cells, respectively. Note that the nodule contains numerous stage 26 quail cells, all surrounded by Alcian blue-staining matrix (X1350). (b) This culture was prepared from a 1:3 mixture of quail sternal cartilage and stage 26 chick wing bud cells. Note the presence of quail cartilage (arrow). The stage 26 cells have failed to produce an Alcian blue staining matrix (X1330).
did not promote expression. These later results suggest that an influence which is actively produced by stage 24 cells, but not by chondrocytes, can promote chondrogenic expression. The observation that nodules are smaller in cultures containing a 3:l mixture of stage 26 cells and stage 24 cells than in the stage 24 cultures also supports this suggestion, since the chondrogenic stimulus may not be propagated as the result of chondrogenie expression. It is possible that the mesenchymederived influence was produced in situ at a time prior to stage 26 but could not be replaced by stage 26 mesenchyme cells after cell dissociation. Whether this influence is a component of the mesenchyme extracellular matrix or cell surface is presently being investigated. The protodifferentiated cartilage phenotype seen in aggregates formed in cultures from stage 26 wings is a phenotype found in the prechondrogenic limbs. Micromass cultures prepared from the proximal half of stage 23 or 24 wings behave similarly to those prepared from whole stage 26 wings (Swalla et al., 1983), producing aggregates of protodifferentiated cells. Addition of as few as one out of ten distal wing cells to proximal cultures stimulates nodule formation. These observations are also consistent with a role for a mesenchyme-derived influence in initial chondrogenic expression.
The behavior of different stage wing mesenchyme in the micromass culture system suggests that there is a progressive change in the properties of subsets of limb mesenchyme cells. At relatively early stages some cells can form aggregates, which can differentiate into cartilage after treatment with db cyclic AMP. Otherwise, cells from this early stage become blocked at a predifferentiated stage, prior to the accumulation of detectible levels of type II collagen. Later in situ, these cells can provide a chondrogenic stimulus as well as respond to the signal. However, at a later stage, they can no longer provide the signal, but they can still express chondrogenesis in response to a permissive environment. The response involves the overt expression of the chondrogenie phenotype by protodifferentiated cartilage cells. The demonstration of cells having these three phenotypes suggests the existence of at least two regulatory steps in cartilage differentiation. REFERENCES AHRENS, P. B., SOLURSH, M., and REITER, R. S. (1977). Stage-related capacity for limb chondrogenesis in cell culture. Deu. Biol. 60, 6982. AHRENS, P. B., SOLURSH, M., REITER, R. S., and SINGLEY, C. T. (1979). Position-related capacity for differentiation of limb mesenchyme in cell culture. Deu. Biol. 69, 436-450. CAHN, R. D., COON, H. G., and CAHN, M. B. (1967). Cell culture and cloning techniques. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 493-530. Crowell, New York. CAPLAN, A. (1970). Effects of the nicotinamide-sensitive teratogen 3acetylpyridine on chick limb bud cells in culture. Erp. Cell. Res. 62, 341-355. DESSAU, W., VON DER MARK, H., VON DER MARK, K., and FISHER, S. (1980). Changes in the patterns of collagens and fibronectin during limb-bud chondrogenesis. J. Embryol. Exp. Morphol. 57, 51-60. FELL, H. B., and CANTI, R. G. (1934). Experiments on the development in vitro of the avian knee joint. Proc. Roy. Sot. Ser. B 116, 316349. HALL, B. K. (1981). Modulation of chondrocyte activity in vitro in response to ascorbic acid. Acta Anat. 109, 51-63. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morphol. 88,4992.
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