Inhibition of limb chondrogenesis by fibronectin

Differential ion

Differentiation (1984) 26:42-48

T,:

Springer-Verlag 19x4

Inhibition of limb chondrogenesis by fibronectin Billie J. Swalla and Michael Solursh Department of Zoology. University of Iowa, Iowa City, Iowa 52242, USA

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

Introduction Fibronectin is a high molecular weight glycoprotein, associated with the cell surface, that aids in the attachment of cells to different substrates, including collagen [see 31 for review]. Recent findings suggest that in some systems exogenous fibronectin can influence differentiation. For instance, fibronectin added to neural crest cells in culture promotes the differentiation of catecholamine-containing cells [14, 221. Exogenous fibronectin also inhibits the fusion of L, myoblasts [20]. The addition of fibronectin to sternal chondrocyte cultures causes the cells to assume a fibroblastic appearance, while cartilage-specific proteoglycan and type I1 collagen synthesis is decreased [19, 301. This effect is enhanced if chondrocytes are cultured on a collagen substrate rather than on a plastic tissue culture dish [18]. If fibronectin is removed from the serum present in the culture medium, the chondrocytes round up and 35S incorporation into proteoglycan is increased. Nevertheless, fibronectin is found on the surface of sternal chondrocytes in monolayer culture [9] and on chondroblasts’ surfaces prior to matrixinduced endochondral bone formation [29]. Later, fibronectin is no longer detected by indirect immunofluorescence as intracellular type I1 collagen appears and cartilage-specific proteoglycan begins to reaccumulate in the pericellular matrix [9].

This evidence led to a search for a role of fibronectin in chondrogenic differentiation from limb mesenchyme during embryonic development. Based on indirect immunofluorescence, mouse forelimb [23], chick forelimb [12, 281, and chick hindlimb [8, 151 all contain a uniform distribution of fibronectin until prechondrogenic condensations are seen. Then fibronectin appears to be more concentrated in the condensations than in the muscle-forming areas [7]. Type I [8, 231 and type I11 [23] collagens are also found in these condensations prior to cartilage development. As cartilage differentiation proceeds, an apparent decrease in fibronectin concentration occurs with a concomitant increase in type I1 collagen in both the mouse [23] and chick [8]. Although the mesenchymal distal tip of the limb bud is associated with a high concentration of fibronectin until stage 28 or 29 [12, 281, there is less immunologically detectable fibronectin in the developmentally more advanced proximal region as overt cartilage differentiation occurs. However, pretreatment with hyaluronidase [12, 15, 231 enhances the immunological staining of fibronectin in the cartilage matrix, suggesting that glycosaminoglycansmask the fibronectin present. Because fibronectin is normally found distributed throughout the embryonic limb and is seen on the freshly dissociated chondrocyte surface in culture, there has been speculation about whether fibronectin is involved in the normal differentiation of cartilage from mesenchymal tissue in vivo [8, 161. However, no study has yet been reported in which fibronectin is shown to affect the differentiation of limb mesenchyme into cartilage. This study shows that fibronectin decreases the chondrogenic expression of cells obtained from the proximal portion of stage 23-24 chick wing buds in micromass culture. Swalla et al. [27j described a population of cells in the proximal region of the chick wing bud that in culture contains low levels of immunologically detectable type I1 collagen, but that fails to make cartilage nodules which stain with alcian blue, pH 1. The present report shows that these cells can form cartilage nodules in low serum or serum-free media. Addition of fibronectin to the low serum medium inhibits chondrogenic expression. Methods Stage 23 or 24 embryos [lo] were obtained from fertile White Leghorn chicken eggs (Welp Hatchery, Bancroft, Iowa). Wing buds were pinched off with fine watchmaker’s forceps and in some cases were separated into proximal and distal portions [27]. The distal piece consisted of the

Fig. 1 6 . Proximal cultures grown in medium supplemented with 10% fetal calf serum (a, b), 5% fetal calf serum (c, d), or 5% fetal calf serum and 50 pg/ml fibronectin (e, f). Cultures in a, c, and e were stained with alcian blue to demonstrate cartilage nodules. Note that nodules are absent in 10% fetal calf serum while numerous nodules are present in 5% serum. The addition of fibronectin to 5% fetal calf serum-supplemented medium inhibits chondrogenesis. The cultures in b, d, and f are the same cultures as those in a, c, and e, respectively, restained with hematoxylin to demonstrate that the formation of prechondrogenic aggregates is similar in all three media. ( x 13)

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apical ectodermal ridge (AER) and underlying mesenchyme. The proximal part contained the ectoderm and mesoderm from the edge of the AER to the flank, including any precartilage condensations that had formed. Proximal and distal limb pieces were dissociated as described in detail elsewhere [3]. In brief, dissected parts were incubated 10 min in 0.1% trypsin (Difco, 1 :200), 0.1% collagenase (Worthington Cop.), and 10% chick serum (Grand Island Biological Co.) in calcium- and magnesium-free saline G at 37" C. Following a brief mechanical dissociation, the cells were pelleted and resuspended in fresh medium. After passing the suspension through a two-ply No. 20 Nitex screen, the number of cells was counted in a hemacytometer. The cells were again pelleted and resuspended in medium containing 10% fetal calf serum at a concentration of 2 x lo5 cells/lO p1 of suspension. One 10 pI sample of cells was added to each 35 mm tissue culture dish. The cells were left to attach for one hour in a 37" C humidified incubator before each dish was flooded with the appropriate medium. Ham F,, nutrient medium (Grand Island Biological Co.) with antibiotics was the base medium LO which either fetal calf serum (Grand Island Biological Co.) in different concentrations or 1 mg/ml fetuin (Type HI, Sigma) was added. In some cases, fibronectin (human plasma fibronectin, Collaborative Research, Inc., or Bethesda Research Laboratories) was also added to the medium. Each day the medium was withdrawn and replaced with fresh medium. Cell fibronectin from chick embryo fibroblasts was generously provided by Dr. Kenneth Yamada. For some experiments, proximal and distal cells were suspended in a collagen gel solution containing 1 mg/ml of type I collagen, Ham F,, nutrient medium, and 10% fetal bovine serum with or without 50 pg/ml of fibronectin. Then, 10 pl samples, each containing 5 x lo4 cells, were plated out and fed 10 min later with the appropriate medium. The collagen gel was prepared from rat tails according to the method described by Solursh et al. [26]. After three days of incubation, cultures were rinsed in saline G, fixed for 30 min in Kahle's fixative, rinsed again in distilled water, and stained with alcian blue, pH 1 [I31 overnight. This allowed the number of cartilage nodules to be counted and recorded. Any aggregate which had accumulated an extensive alcian blue staining matrix was counted as a nodule, regardless of size. In some cases, cultures were then stained with hematoxylin in order to visualize the cell aggregates and culture area.

Results When a stage 23 or 24 chick wing bud is separated into proximal and distal parts before dissociation, proximal micromass cultures make few, if any, alcian blue staining nodules (Fig. 1 a) while distal micromass cultures make a con-

Fig. 2s-c. Distal cultures stained with alcian blue to demonstrate cartilage formation. The nature and extent of alcian blue matrix makes nodule counts diflicult to determine. Cultures are to be comparcd qualitativcly. a This was grown in medium supplemented with 10% fclal calf serum, b with 5% fetal calf serum, and c with 5'X fetal calr serum and 50 pg/ml fibronectin. Chondrogenesis by distal cultures appears relatively unaffected by exogenous fibronectin. Of numerous trials, this was the most extrcme effect that was observed. ( x 13)

a

b

c

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Table 1. Effect of serum variations on nodule and aggregate formation by stage 23 proximal chick wing bud cultures'

Table 3. Effect of varying fibronectin concentration on nodule formation by proximal cultures in serum-free medium"

Serum in F,, medium

Nodules/ culture XfSD

Aggregates/ culture XfSD

Fibronectin concentration (Crglml)

Nodules/culture

10% fetal calf serumb Heat-inactivated 10% fetal calf serumc 10% newborn calf serum' 5% fetal calf serum 5% fetal calf serum + 50 pg/ml plasma fibronectin

4+4 174f8

265 f34

0 1 5 10 50

230 42 144f41 29f8

a

168f23 173f21 9+1

384 f21 355f41

*

5+5

0.3 -f 0.6

' Results are x + S D for triplicate plates in one trial. While the actual number of nodules varied from one experiment to the next, increased concentration of fibronectin always resulted in a decrease in nodule number in all four separate trials. The cultures were treated for the entire 72 h culture period

Results are from triplicate plates in one experiment Treatments had a similar effect in four separate trials Treatments had a similar effect in two separate trials

Table 4. Effect of time and duration of treatment with fibronectin' Table 2. Effect of time and duration of treatment of proximal cultures with 10% fetal calf serum. Treatment 10% serum Fetuin

5% serum

Nodules/cultureb day 0-3 day 0-3 day 0-1 day 1-2 day &3 day &I day 1-2

If1 311 +23 95 f52 21+16 47 f34 20f16

5f8

Days of treatment

Nodules/culture

Control Day 0 - day 3 Day 0 - day 2 Day I - day 3 Day 0 - day I Day 2 - day 3

299 f26 97*44 229 5 49 253 f63 310+54 346f144

a

Results are 8 f S D for triplicate cultures in one trial out of three repeated trials. Cultures were treated with 10 pg/ml plasma fibronectin in serum-free medium

' Cells were inoculated from a single cell suspension made in F,, medium containing 10% fetal calf serum. At the time of adding medium or of daily feeding, some cultures received F,, medium supplemented with 5% fetal calf serum or fetuin at the times and for the durations indicated Results are means f S D of triplicate cultures and are typical of three trials

fluent sheet of cartilage in Ham F,, medium supplemented with 10% fetal calf serum (Fig. 2a). If the fetal calf serum is heat-inactivated at 60" C for 45 min, lowered to a concentration of less than 7%, or replaced with newborn calf serum, the proximal micromass cultures make alcian blue staining cartilage nodules (Table 1, Fig. 1c). However, the extent of the cartilage formed in the distal cultures is decreased by lowering the concentration of fetal calf serum in the medium (Fig. 2). As shown in Table 2, removal of 10% serum by replacing the medium with either serum-free, fetuin-containing medium, or medium supplemented with 5% serum during the first day of culture is more effective in promoting nodule formation than on the second day of culture. However, at any time the presence of 10% fetal calf serum decreases nodule formation and a continuous exposure for three days is most inhibitory. When purified human plasma fibronectin is present in the low serum media, there are very few cartilage nodules in the proximal cultures (Fig. 1e, Table 1). In contrast, the distal cultures are not obviously affected by the addition of fibronectin and do not differ in appearance from the unsupplemented controls (Fig. 2c). In serum-free medium (F12stock plus antibiotics and 1 mg/ml fetuin), distal cultures accumulate less alcian blue staining matrix (not shown) than in low serum concentrations. The proximal

cells make large numbers of small, distinct nodules (for nodule counts, see Tables 3 and 4). Because the distal cultures grow so poorly in this serum-free medium, they are dificult to study. Proximal cultures, however, make distinct nodules which are easily counted. Fibronectin was added to serum-free medium to avoid any contamination by fibronectin present in the serum. The effect of exogenous fibronectin is concentration-dependent. At high concentrations (50 pg/ml) there are few nodules (Table 3). Also, the fibronectin was most effective when the cultures were treated for the entire culture period (day 0-3). Treating for only one day had no appreciable effect on the number of nodules formed, and treating for two days (day 1-3 or day 0-2) reduced the number of nodules only slightly (Table 4). In addition, there appears to be no specific sensitive period. Some cultures were also treated with cell fibronectin which was no more effective in preventing chondrogenesis than plasma fibronectin. Proximal and distal cells were cultured at a subconfluent cell density, suspended in a collagen gel with or without the addition of 50 pg/ml fibronectin in the gel and culture medium. In this test system 10% fetal calf serum is present. Nevertheless, proximal cells (Fig. 3a) make some cartilage nodules, but many fewer than distal cells form (Fig. 3c). Most important, exogenous fibronectin still inhibits chondrogenesis by the non-confluent proximal cells (Fig. 3 b). Again, chondrogenesis by distal cells is not affected by the presence of added fibronectin (Fig. 3 d). Therefore, a differential response to fibronectin by proximal and distal cells is seen even when the cells are suspended at lower density in a collagen gel.

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Fig. 3a-d. Proximal (a, b) and distal (c, d) cells grown in collagen gel and stained with hematoxylin to see groups of cartilage cells formed within the gel. These masses also stained intensely with alcian blue. a Proximal cells make some nodules if cultured in the gel. b Addition of 50 pg/ml fibronectin inhibits nodule formation. c Distal cultures form many nodules in a collagen gel. d Distal

cultures with fibronectin look the same as thc untreated cultures (c). ( x 14)

Discussion Chick wing bud cells from the proximal part of stage 23 or 24 limb buds do not make nodules in F,, medium supplemented with 10% fetal calf serum, although they do make low levels of cartilage-specific cell products [27]. These cells have been called 'protodifferentiated' and have similar properties to stage 26 cells in micromass culture [25]. This paper shows that proximal cells can express their chondrogenic capacity if cultured in medium containing a lower serum concentration. There appears to be a factor in the serum that inhibits chondrogenesis by proximal cells. The inhibitory factor is likely to be a protein because it is inactivated by heat. The nature of this inhibitor and mechanism of action are not yet known. The factor does not appear to be fibronectin since removal of traces of fibronectin from the serum by absorption on gelatin does not

decrease the inhibitory activity (results not shown). It is presently being purified in this laboratory. It is likely to be a cell attachment factor since Cairns [5] noted that explants from the proximal part of the limb bud spread faster than those from the distal region of the limb bud. Other cell attachment factors are present in serum and some might even utilize the same cell surface receptor as fibronectin [l 11. It is possible in the present study that the serum factor and fibronectin have a similar mechanism of action. This suggestion is supported by the observation that high serum or fibronectin inhibits chondrogenesis only with prolonged treatment. It is significant that the developmentally younger distal cells are not sensitive to this inhibitor of differentiation of proximal cells. Distal cells are spontaneously chondrogenic even in the presence of 10% fetal calf serum. However, if distal cells are cultured in low serum medium, chon-

47

drogenesis is drastically decreased. Both the attachment and survival of the cells appear to be decreased, resulting in a lower density culture that stains faintly with alcian blue. However, chondrogenesis is more extensive in a low serum medium than in serum-free medium. It seems likely that there is another component in the serum which facilitates the growth and attachment of distal cells. These results emphasize the differences between proximal and distal cells. Because proximal cells are isolated from the developmentally older part of the stage 24 chick limb, the population contains some nonchondrogenic cell types such as myoblasts [21]. Abbott and Holtzer [i] showed that myoblasts can inhibit differentiation of chondroblasts in culture, a phenomenon referred to as ‘interference’ [6]. However, when quail breast myoblasts are mixed with chick distal wing cells in a ratio of 1 :5 respectively, the cultures still make an extensive cartilage matrix (N.C. Zanetti and M. Solursh, unpublished work). Furthermore, cells from the isolated prechondrogenic core region dissected from the proximal region of the limb bud exhibit the ‘proximal’ phenotype [24]. The qualitative differences in chondrogenic capacity of proximal and distal cells in culture are therefore more likely due to differences in properties of the cells, rather than the proportion of different cell types in the two limb regions. On the other hand, differences in the proportion of different cell types probably produce some of the quantitative differences in the extent of chondrogenesis between proximal and distal cultures. Most important, proximal and distal cells show different susceptibility to treatment of the cultures with exogenous fibronectin. Distal cells are not noticeably affected by the treatment either in micromass cultures or in hydrated collagen gels. On the contrary, proximal cells do not form cartilage nodules in the presence of exogenous fibronectin in either test system. The mechanism of this inhibition has not been characterized yet. One possible mechanism could involve cell attachment and flattening. Abbott and Holtzer [2] considered the role of cell shape in the maintenance of differentiated chondroblasts in culture. Benya and Shaffer [4] have since confirmed this correlation between cell shape and chondrogenic expression. Pennypacker [18] showed that adding fibronectin to sternal chondrocytes caused the cells to flatten. It may be that cell shape regulates the expression of the cartilage cell phenotype and that fibronectin inhibits expression by causing chondrocytes to assume a flattened morphology. Cell shape may also be important in the initial differentiation of cartilage from mesenchyme cells. Solursh et al. [26] demonstrated that single limb mesenchyme cells can become chondrogenic if they are maintained in a round configuration. In addition, treating proximal cultures for 24 hours with cytochalasin D causes the cells to round up and become chondrogenic by day 3 of culture [32]. It is interesting that this effect of cytochalasin D is also seen in proximal cultures treated with fibronectin [32]. Fibronectin may promote cell flattening and in turn inhibit chondrogenesis by proximal cells. The results indicate that chondrogenesis by proximal cultures is particularly sensitive to a serum component and to fibronectin. On the other hand, distal cells have a very different or no response to serum concentration and exogenous fibronectin. It may be that the differences in these two cell phenotypes can be traced to differences in cell surface properties. This suggestion is supported by the obser-

vation that serum-dependent cell aggregation occurs faster in proximal cell suspensions than in distal cell suspensions [lq.The proximal cells could have different or more accessible fibronectin binding sites than the distal cells. A maturation-related or position-related change in receptors for prevalent matrix components like fibronectin could have a central role in regulating the onset of chondrogenesis during normal development. Acknowledgments. The authors would like to thank Chris Goerdt, Karen Jensen, Rebecca Reiter, and Nina Zanetti for generously sharing their results and ideas. Special thanks also to Dr. K.M. Yamada for his gift of cell fibronectin. This work was supported by NIH grant HD05505.

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Received June 1983 / Accepted in revised form October 1983