Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid

Differentiation

Differentiation (1988) 39: 123-130

Ontogeny and Neoplasia 0 Springer-Verlag 1988

Selective stimulation of in vitro limb-bud chondrogenesis by retinoic acid Douglas F. Paulsen’”, Robert M. Langille

’, Virginia Dress’,

and Michael Solursh’

Department of Anatomy, Morehouse School of Medicine, Atlanta, GA 30310, USA Department of Biology, University of Iowa, Iowa City, IA 52242, USA

Abstract. Embryonic exposure to pharmacologic doses of vitamin A analogs (retinoids) is a well-known cause of limbskeletal deletions, limb truncation and other skeletal malformations. The exclusively inhibitory effect of retinoic acid (RA) on chondrogenesis in standard serum-containing cultures of limb-bud mesenchymal cells is equally well known and has provided a means to explore the cellular basis for RA-mediated skeletal teratogenesis. Recent studies showing that lower RA concentrations can cause skeletal duplication when applied directly to the anterior border of a developing limb, suggest that RA may have a role in normal limb development as a diffusible morphogen capable of regulating skeletal pattern. While RA treatment causes both, skeletal deletions and duplications are clearly different (if not opposing) effects, the latter of which is difficult to reconcile with RA’s heretofore exclusively inhibitory effect on in vitro chondrogenesis. In the present study, RA’s effects on chondrogenesis and myogenesis were examined in serum-free cultures of chick limb-bud mesenchymal cells and compared with its effects on similar cultures grown in serum-containing medium. When added to serum-free medium, concentrations of RA known to cause skeletal duplication in vivo dramatically enhanced in vitro chondrogenesis (to over 200% of control values) as judged by both Alcian-blue staining and [35S]sulfate incorporation, while having little effect on myogenesis. Higher concentrations inhibited both chondrogenesis and myogenesis. The results indicate that at physiological concentrations, RA can selectively modulate chondrogenic expression and suggest that at higher concentrations, RA’s inhibitory effects are less specific. The system described provides the first cell-culture model for exploring the cellular basis for RA’s concentration-dependent capacity to both enhance and inhibit phenotypic expression.

Introduction

The ability of exogenous RA to cause limb truncation, limbskeletal deletions, and craniofacial anomalies in developing vertebrate embryos is well established [15-17, 44, 491. Numerous in vitro studies have been carried out using standard serum-containing culture media in an effort to determine the cellular basis for RA’s teratogenic effects [8, 10, 19, 20, 24, 31, 36, 50, 511. Under these conditions, RA con-

* To whom offprint requests should be sent

sistently inhibits chondrogenesis from mesenchyme [6, 91 and inhibits phenotypic expression by mature chondrocytes [12, 411. These findings have established RA treatment of limb mesenchymal cell cultures as a standard model for exploring the cellular and molecular basis of skeletal teratogenesis [9, 10, 13, 141. Recent studies have focused increased attention on the effects of RA on skeletal development by indicating that endogenous RA in developing chick limbs [45] and in regenerating amphibian limbs [30] may provide important positional cues to differentiating cells regarding their proper developmental fates, and thus influence normal skeletal patterning [40]. The RA in developing chick limbs occurs in higher concentrations posteriorly than anteriorly [45]. These results are consistent with the suggestion that RA acts as a normal limb-skeletal morphogen, exerting concentrationdependent effects on mesenchymal cells lying at different points along an anteroposterior RA gradient [40]. Exogenous RA, when applied to the anterior border of a limb bud such that the anterior RA concentration becomes equivalent to that normally found posteriorly, induces a mirror-image duplication of the limb skeleton [42, 47, 481. In so doing, RA mimics the effects of implants of the zone of polarizing activity (ZPA) [47], a posterior limb subregion associated with potent morphogenic activity [26, 381, but whose mechanism of action is unknown. Similar treatment with higher RA concentrations causes limb truncation reminiscent of RA’s teratogenic effects [48]. The ability of lower RA concentrations to stimulate the formation of additional skeletal elements and higher RA concentrations to inhibit skeletogenesis has also been reported in studies of amphibian limb regeneration. Here, low RA concentrations induce the duplication of stump skeletal elements in outgrowths from amputated limbs, whereas higher RA concentrations can inhibit regeneration entirely [27]. In mouse limb primordia grown in serum-free organ culture, high RA concentrations reduce, and low RA concentrations increase, the total cartilage mass [21]. Whether RA’s dose-related opposing effects on skeletogenesis operate through similar or completely different cellular mechanisms is presently unknown. Analysis of the mechanism of action of RA in vivo, especially at the cellular level, is complicated by the multitude of developmental processes occurring simultaneously in the limb and in other organ systems. All these processes may influence, and/or be influenced by, the composition of the blood in the common embryonic circulation. It would be useful therefore to have a cell-culture system in which

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the diverse cellular effects of RA can be explored in relative isolation. Previous studies of the exclusively inhibitory effects of retinoids on chondrogenesis in serum-containing cell culture have been useful in exploring the cellular basis of RA-mediated teratogenesis. However, the presence of unknown amounts of retinoids in the serum supplements used in such studies prohibits knowledge of the precise retinoid concentrations encountered by the cultured cells. This complicates analyses of the effects of RA in the minute (nanomolar) concentrations capable of causing limb-skeletal duplication [47], and now known to be present in normal embryonic limbs [45]. In this report, we describe the use of a recently developed serum-free medium [35] to examine the effects of nanomolar concentrations of all-trans-RA on chondrogenesis and myogenesis in microtiter cultures of stage 23-24 chick limb mesenchyme. The results show that, depending on its concentration, RA can either stimulate or inhibit chondrogenic expression in serum-free cell culture. This model will be useful in future studies of the cellular basis of skeletal patterning and the relationship between cytodifferentiation and morphogenesis. Methods Media and additives. The defined medium (DM) used in these studies was composed of a mixture of 60% Ham's F-12 nutrient mixture (Gibco) and 40% Dulbecco's modified Eagle's medium (DMEM ; high-glucose type, Gibco) supplemented with 5 pg/ml insulin (Collab. Res.), 5 pg/ml chicken transferrin (Conalbumin, Sigma), 50 pg/ml L-ascorbic acid, 100 n M hydrocortisone (Sigma) and antibiotics. The serum-containing medium (SCM) was composed of the same F-l2/DMEM mixture supplemented with 10% fetal calf serum, 50 pg/ml L-ascorbic acid and antibiotics [35]. A primary stock solution of 1 mg all-trans retinoic acid (RA; Sigma) per ml in 100% ethanol was prepared and stored in a light-tight container at -20" C. A secondary stock containing 1 pg RA/ml of the appropriate medium (and 0.1% ethanol) was then prepared and diluted with medium to obtain sufficient media with the appropriate amounts of RA (1, 5, 10, 25, 50 and 75 ngiml) for the 4-day incubation period. The prepared media were stored in light-protected 50 ml conical centrifuge tubes at 4" C during the incubation period. Media containing ethanol at a concentration equal to that in the 75 ng RA/ml media experiments (0.0075% v/v) were used as vehicle-only controls. There was was no significant difference between the amount of chondrogenesis in ethanol-free and vehicle-only controls. Microtiter cultures. Single-cell suspensions of stage 23-24 chick [7] limb mesenchyme were obtained and seeded into 96-well tissue-culture-treated microtiter plates (Corning) at a density of 3.5 x lo5 cells per well in 150 pl D M or SCM as previously described [35]. The cells were allowed to attach for 1.5-2 h before aspirating the attachment medium and replacing it with 250 pl medium containing the appropriate amount of all-trans RA or vehicle. Cultures were allowed to grow for 4 days, with complete medium changes every 12 h. Histology. Cultures destined for morphological analysis were fixed by the method of Carlson et al. [5] for 30 min at 0'4" C, and washed with distilled water. Cartilage was

demonstrated by staining with Alcian blue at pH 1.0 [23], and myoblasts were demonstrated by an indirect immunoperoxidase technique using the anti-sarcomere myosin monoclonal antibody, MF-20 [I] as described elsewhere 1351. Quantitation of chondrogenesis Alcian-blue extraction. The amount of cartilage matrix produced in these cultures was estimated according to methods published in detail elsewhere [35]. Briefly, after 4 days, the cultures were fixed and stained as described above. Unbound stain was removed with two washes of 0.1 N HC1. The final rinse with distilled water was aspirated to near dryness and replaced with 100 pl 4 M guanidine HCl, pH 5.8 (GuHCl) per well. The plates were then incubated at 4" C overnight to extract the specifically bound dye [9]. After warming to room temperature, the gross absorbance values for triplicate cultures were read on a microtiter plate reader using a 600-nm filter. The fluid was removed and each well washed twice with water. The final wash was again aspirated to near dryness and replaced with 100 pl fresh GuHC1. The plates were read again to obtain blank values that were subtracted from the respective gross values to give the net absorbance of the extracted dye, which was taken as a measure of the amount of cartilage matrix present. Nodule size and number. The relative numbers of cartilage nodules within the cultures was ascertained for each RA treatment in both types of media and compared. Alcianblue-stained cultures were examined under a dissecting microscope fitted with a 10 square by 10 square ocular grid, which measured 2.56 mmZ of the culture dish surface. All of the nodules within this grid were counted and the number adjusted to noduleslmm'. The results of at least three cultures for each treatment were used to derive the average number of nodules/mm2. In addition, the widest diameters of ten random nodules was recorded, with the aid of a linear ocular grid, from each of three representative cultures for each treatment, to provide a relative measurement of nodular area. r3'Ss/ Sulfate incorporation. The amounts of [35S]sulfate incorporated into macromolecules in the medium, a GuHCl extract of the cell layer, and in the extracted cell layer residue were analyzed in triplicate cultures grown as described above. Carrier-free [35S]sulfate (specific activity 9.7 mCi/ mM) dissolved in 1 M H,S04 (Amersham) was diluted in unsupplemented FZ2/DMEM containing an equimolar amount of NaOH to a final concentration of 0.2pCi/pl. Six hours before terminating the cultures on day 4, 10 pl (2 pCi) [35S]S04 solution was added to each culture well. Blank samples for each treatment schedule were prepared by growing companion cultures and adding the [35S]S04 just prior to collecting the samples. The medium and three washes of each well in 0.02 A4 phosphate buffered saline, pH 7.4 (PBS) were pooled separately and frozen at - 20" C. The medium fraction from each culture grown in DM received 30 pl fetal bovine serum (equivalent to that in this fraction of SCM cultures) prior to freezing. After delivering 200 p1 GuHCl to each well, the plate was sealed with Parafilm and kept overnight at 4" C. The GuHCl extract and two PBS washes for each well were pooled separately in

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1.5-ml microfuge tubes. A third PBS wash was delivered to each well and a small policeman fashioned from Tygon tubing was used to free the extracted cell-layer from the well. The third wash and the extracted cell-layer residue were transferred by pipet to the appropriate microfuge tube along with a fourth PBS wash of the well. The microfuge tubes were then spun for 5 min at high speed in a fixed-angle microfuge at 4" C. The supernatant was transferred to separate tubes and the pellet washed with a final wash of 100 p1 PBS. The final washes were transferred to the appropriate tubes containing the pooled GuHCl extracts and washes. The tubes containing the extracted cell-layer pellets and those containing the GuHCl extracts were frozen at - 20" C. The tubes containing the three samples obtained from each well were thawed, 10 p1 1% NaCl in distilled water was added to each, and the contents of each tube were added to three volumes of 95% ethanol and allowed to stand overnight at - 20" C. The resulting precipitates were collected by centrifugation and washed twice with a saturated solution of MgSO, in 70% ethanol. The final washes were carefully aspirated and each pellet was resuspended in 2 ml 10% trichloroacetic acid (TCA) containing 0.1 M MgSO, (TCA/MgSO,) and allowed to stand overnight at 4" C. Each pellet was then washed twice with 2 ml TCA/MgSO, and dissolved in 1 ml 0.2 A4 Tris, pH 8. Each sample then received 100 p1 10 mg/ml pronase in 0.2 M Tris and was incubated for 5 h at 37" C. Next, each sample received 1 ml 0.2 M Tris and 100 pl 20 mg/ml carrier chondroitin sulfate in water. After vortexing, each sample received 1 ml 1% cetylpyridinium chloride (CPC) in distilled water, was mixed again and allowed to stand at room temperature for 10 min. The precipitates formed were resuspended and collected by vacuum on separate 25-mm Millipore HA filters. The filters bearing the precipitates were washed twice with 5 m l 0.3% CPC and a final wash of ice-cold distilled water and air dried on a wire rack. Each dried filter was cut into 16 pieces and placed in a 7-ml glass scintillation vial. Each vial received 5 ml Beckman Ready Gel scintillation fluid and was capped, mixed vigorously, and allowed to equilibrate overnight before counting on a Beckman LS 5801 liquid scintillation counter. Blank values were all below 100 counts per minute (cpm) and were subtracted from the corresponding gross cpm values obtained for the samples. Quantitation of myogenesis. The level of myogenesis in these cultures was measured by a modification of methods developed for ELISA assays and is described in detail elsewhere [35]. Cultures destined for these studies were fixed in acidified alcohol at 4" C overnight and then postfixed for 2 h in 10% buffered formalin to inactivate the endogenous phosphatases. After washing twice with PBS, the wells were blocked with 5% normal rabbit serum (NRS) in 0.01 M PBS (pH 9), then incubated first with 50% MF-20 monoclonal antibody hybridoma supernatant in 0.02 M PBS (primary antibody) and then with alkaline phosphatase-conjugated rabbit-antibodies to mouse IgG (Zymed; secondary antibody). The wells were washed with 0.02 M PBS containing 0.02% Tween 20 between and after the antibody incubations. The final wash was replaced with 100 pl PNPP substrate solution (p-nitrophenyl-phosphate in 0.75 M aminomethyl-propanediol, pH 10.3). After a 40-min incubation at room temperature, absorbance per well was measured on a microtiter plate reader using a 405-nm filter. The

values obtained for blank wells (receiving no primary antibody) were subtracted from those obtained for corresponding experimental wells to give a net absorbance value [35]. D N A assay. Values for total DNA per culture well were obtained using a modification of the assay first described by Brunk et al. [3]. This assay, which has been described elsewhere [35], involved the use of chicken liver DNA as the standard and Hoechst 33258 (Polysciences) as the fluor. Results

The effects of RA on DNA accumulation, chondrogenesis and myogenesis in 4-day cultures of limb-bud mesenchymal cells from stage 23-24 chick embryos were examined. As shown previously [35], total DNA/culture varied with the presence or absence of serum in the culture medium, but was not significantly affected by RA treatment, except at concentrations higher than those employed in the present study (Fig. 1 E, J and [35]). Effects on chondrogenesis and myogenesis differed depending upon both the type of medium used and the RA concentration to which the cells were exposed. Effects on chondrogenesis Alcian-blue absorbance. In cultures grown in DM, RA treatment both enhanced and inhibited chondrogenesis, depending on the amount of RA in the medium. At concentrations of 1, 5 and 10 ng/ml (3.3, 16.5 and 33.3 n M respectively), RA dramatically enhanced the accumulation of Alcianblue-stainable cartilage matrix materials. Peak enhancement (222% of the control value) occurred at 5 ng RA/ml (Fig. 1A). This is the first report of RA-mediated stimulation of chondrogenesis in cell culture. At 25 ng RA/ml D M (82.5 nM), chondrogenic expression returned to near control levels. Above this concentration, RA caused a dosedependent inhibition of chondrogenesis (Fig. 1A and [35]). In cultures grown in SCM, RA caused the expected dosedependent inhibition of matrix accumulation at all concentrations tested (Fig. 3 F). Nodule numbers and morphology. RA-induced differences in the values for the total amount of Alcian-blue-stainable matrix accumulation (Fig. 1A, C) could reflect differences in the number, size and/or staining intensity of the cartilage nodules. The photomicrographs in Fig. 2 suggest that RA's greatest effect is on the amount of extracellular matrix per cartilage nodule (staining intensity). Measurement of nodule diameters confirmed this observation, indicating that while larger nodules typically form in SCM than in DM (Fig. 1C, H and [35]), RA treatment does not significantly affect nodule size in either medium when compared to respective untreated controls (Fig. 1C, H). As shown previously [35] the number of nodules per culture did not differ significantly between control cultures grown in SCM vs. DM. An RA-mediated increase in the number of cartilage nodules per culture could explain the increase in total matrix accumulation that occurs at low RA concentrations in DM (Fig. 1A). RA treatment however causes a dosedependent decrease in the number of nodules formed in both types of medium (Fig. 1B, G). In DM, increasing RA concentrations caused a gradual reduction in the number of nodules per culture, while in SCM there was a precipitous

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Fig. 1A-J. Effects of retinoic acid (RA) on 4-day cultures of stage 23-24 chick wing-bud mesenchymal cells grown in defined medium (DM, A-E) and in serum-containing medium (SCM, F-J). Effects on chondrogenesis (A-C, F-H): Note the enhancement of cartilagematrix accumulation in DM (A) at RA concentrations of 1-10 ng/ml and inhibition in DM above 25 ng/ml and in SCM (F) at all RA concentrations tested. In both DM and SCM, RA treatment decreased nodule numbers (B, G) and had little effect on nodule size (C, H). Effects on myogenesis (D, I): In DM (D), note the lack of a significant effect on myogenesis at 1-5 ng RA/ml and inhibition above 10 ng RA/ml. In SCM (I) RA inhibited myogenesis at all concentrations tested. Effects on DNA accumulation (C, F): Note the lack of a significant effect of RA on DNA accumulation in either DM (E) or SCM (J). Each data point represents the mean value obtained from at least three separate cultures. Error bars represent standard deviation; *, broad pale Alcian-blue-positive areas with indistinct borders

3 27

Fig. 2A-F. Morphology of 4-day cultures grown in defined medium (DM, A-C) and in serum-containing medium (SCM, D-F). Control cultures (A, D) received ethanol vehicle (0.0075% v/v). Cultures shown in B and E received 5 ng RA/ml and those shown in C and F received 25 ng RA/ml. Note the increased staining intensity of the cartilage nodules (arrows) in B as compared to A. Note the decreased numbers and staining intensity of the MF-20-positive myoblasts (arrowheads) and cartilage nodules (arrows) in C and F, bar, 0.5 mm

Table 1. Effects of retinoic acid (RA) on [35S]sulfate incorporation during the final 6 h of the 4-day culture period by stage 23-24 chick limb-bud mesenchyme cells grown in defined medium (DM) or in serum-containing medium (SCM) Culture conditions

[35S]CPMper culture (% of total CPM per culture)a Total

Cell layer

Medium

Residue

DM Control l o n g RA/ml DM 75 ng RA/ml DM

4777.85 410.3 13276.1k1308.0b 1367.65 68.0b

292.5+ 87.4 689.5k 55.6b 98.9 i 2.Sb

GuHCl extract (6.1 k2.0) (5.3k0.9) (7.2i0.1)

SCM Control 13661.7k 372.7 1190.1 k214.6 (8.7kl.6) l o n g RA/ml SCM 7273.3k 605.7b 798.2k 33.0b (11.0&1.3) 75 ng RA/ml SCM 3916.7k 370.Yb 535.8+ 64.2b (13.7f0.Y)b a

3.236.6 5 349.7 (67.7 52.5) 10763.3 1060.7b (81.0 f2.0) 953.0i 58.3b (69.7k0.8)

1248.7 i199.6 (26.2 i 4 . 2 ) 1823.2k437.1 (13.7 +2.4)b 315.7k 6.7b (23.1 k0.7)

10157.4+ 704.2 (74.4k5.1) 5284.8k 5Y3.7b (72.6k2.1) 2467.5-t 239.7b (63.0i1.7)b

2314.2k495.3 (16.Yk3.6) 1190.3k 34.6b (16.4+1.0) Y13.4k 94.4b (23.3k0.8)b

+

Values are mean standard deviation for triplicate cultures Significantly different from corresponding control (P<0.05); two-tailed T-test

drop between 5 and 10ng/ml from nodule numbers near control levels to near zero (Fig. 1G). Thus, the enhancement of chondrogenesis by RA concentrations between 1 and 25 ng/ml in DM appears to be due mainly to an effect on the accumulation of Alcian-blue-stainable cartilage matrix components (proteoglycans [9]) ; a result confirmed by analyzing [35S]sulfate incorporation as described below. RA’s inhibitory effects appear to involve decreases in both staining intensity and nodule number. Nodule size appears to be unaffected, except possibly at RA concentrations above 50ng/ml in DM, where the number of organized, compact nodules decreases and broad areas of very pale Alcian-blue staining occur (Fig. 2 C). Incorporation of (35S]sulfate. Other studies have shown that while the majority of the proteoglycans secreted by these cells in vitro are found in the GuHC1-extractable extracellular matrix, a considerable proportion are released into the medium [43]. It is thus possible that the RA-mediated stimulation of chondrogenesis indicated by the Al-

cian blue studies simply reflects an effect on the proportion of these macromolecules in the extracellular matrix versus the medium, with little or no effect on overall synthetic rate or total accumulation. However, [3sS]sulfate incorporation during the last 6 h of the culture period in cultures grown in SCM and DM argues against this possibility. Incorporation of [35S]sulfate was analyzed in three fractions for each culture; the medium, a GuHCl extract of the cell layer, and the extracted cell-layer residue. Since previous studies show that the counts remaining in the GuHC1-extracted cell-layer residue require detergent to release them, they are considered to represent intracellular sulfate, while the counts in the GuHCl extract represent sulfate incorporated in the extracellular matrix [ll]. Table 1 shows that the pattern of RA’s effect on [35S]incorporation is quite similar to that on the accumulation of extractable Alcianblue-stainable matrix. Thus, for cultures grown in DM, 10 ng RA/ml stimulates and 75 ng RA/ml inhibits [35S]incorporation both in terms of total counts and in the various fractions. The stimulatory RA concentration (10 ng/ml) did

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increase the proportion of the total [35S]incorporated in the matrix (GuHC1 extract) and decrease that in the medium. However, this effect was small in relation to its effect on total incorporation. In cultures grown in SCM, both RA concentrations inhibited total [ ”S]incorporation and did so in a dose-dependent fashion. In SCM, the proportion of the total counts incorporated by the various fractions was unaltered at 10 ng RA/ml, but at 75 ng RA/ml there was a decrease in the amount incorporated in the GuHCl matrix extract and a concomitant increase in the medium and extracted cell-layer residue. RA’s effects at 75 ng RA/ ml in SCM corroborate previous reports of the effects of higher RA concentrations on chondrocyte cultures. In these studies, RA inhibited matrix synthesis [lo, 411 and reversibly stimulated the loss of proteoglycans from the matrix to the medium [4, 121.

Effects on myogenesis The effects of RA on the differentiation of muscle, the other major limb-mesenchymal derivative, were tested in companion cultures. Figure 1D shows that in DM, RA concentrations of 1 and 5 ng/ml did not significantly affect myogenesis. This indicates that the RA-mediated stimulation of condrogenesis is specific and does not simply reflect a general requirement for an essential nutrient otherwise lacking in DM. At l o n g RA/ml DM, there was a slight inhibition of myogenesis that reached a maximum at 25ng RA/ml DM and then levelled off. The profile of RA’s effect on myogenesis in SCM resembles that in DM, except that in SCM, the onset of both the decline and levelling off occurred at lower RA concentrations than in DM (Fig. 1 D, I). The inhibition of myogenesis involved a decrease both in the number of MF-20-positive cells and in the anti-myosin immunoperoxidase staining intensity (suggesting decreased myosin) in the myoblasts that remained MF-20 positive (Fig. 2C, E, F). Discussion It has been difficult to reconcile evidence of RA’s opposing effects on limb-skeletal development in vivo [46-481 and on limb-cartilage mass in serum-free organ culture [21], with evidence that its effects on chondrogenesis in cell culture are solely inhibitory [13]. The results of the present study represent the first report of a serum-free cell culture system in which a single retinoid (RA), depending on its concentration, has been shown to exert opposing effects on chondrogenic expression. The unknown amounts of RA and other retinoids in serum supplements (RA levels in normal human serum range from 2.8-6.6 ng/ml [32]) may account for the differences observed in the effects of RA in serum-containing and defined media. Retinoic acid’s greatest stimulation of chondrogenic expression occurred in the defined medium at 5 ng/ml, where its effects on myogenesis were negligible. Inhibition of chondrogenesis occurred at RA concentrations that also inhibited myogenesis. The results clearly show that exogenous RA, in the concentration range known to stimulate the formation of excess skeletal elements (1-50 n M or 0.3-15 ng/ml) [47] and now known to occur normally in chick limb buds (17-54 n M or 5.1-16.3 ng/ml) [45], dramatically and selectively enhances chondrogenic expression in serum-free cell cultures of chick limb mesenchyme. At these

physiological RA concentrations [45], the degree of chondrogenic enhancement is dose-related (Fig. 1A). The results are thus consistent with RA’s putative role as a positionalinformation-bearing morphogen that is able to influence limb-skeletal pattern by affecting the size and shape of skeletal elements forming at different points in an anteroposterior RA gradient [40,47]. However, nodule size and number were not significantly increased by the RA concentrations that enhanced chondrogenic expression (Fig. 1A-C), and enhancement of chondrogenic expression alone seems an unlikely explanation for the ability of RA treatment to elicit ectopic cartilage formation and pattern duplication in vivo. An additional RA-mediated effect on the number of available chondrogenic precursors or on their distribution (possibly due to changes in limb shape mediated through RA’s effects on the apical ectodermal ridge [22,46]) would appear to be required. Although no significant effect of RA on DNA accumulation was detected in the present study, it is possible that the inclusion of mesenchyme from the entire limb masked cell-type- or region-specific effects of RA on proliferation. In the in vivo experiments carried out by Tickle et al. [47, 481, when RA was delivered locally at appropriate concentrations, multiple, well-formed skeletal cartilages appeared, forming a mirror-image duplication of the normal skeleton without significantly increasing the total skeletal mass [47]. This suggests that RA initiates a complex patterning mechanism that does not necessarily involve direct RA-mediated recruitment of additional chondrogenic cells. Together, the in vivo and in vitro effects of nanomolar concentrations of exogenous RA suggests that RA’s ability to modulate chondrogenic expression is an important component of its putative role in skeletal patterning, but is probably only one of many factors involved in a complex mechanism orchestrated by RA. As previously stated, RA concentrations below 10 ng/ml stimulated chondrogenesis, but had no significant effect on myogenesis, suggesting a degree of specificity at the lower concentrations. Furthermore, the onset of RA-mediated inhibition of myogenesis occurs at lower concentrations (10 ng/ml) than does that of chondrogenesis (25 ng/ml; see Fig. 1A, D). These findings support a view that intrinsic factors, such as the level of expression of the various intracellular RA-binding proteins and receptors, may modulate the response of cells of different types and in different limb regions to the same level of RA [51]. The inhibition of both chondrogenesis and myogenesis at RA concentrations above 25 ng/ml suggests that at higher concentrations, RA’s effects on limb mesenchyme are less specific. However, due to regional differences in myoblast distribution in the developing limb [33], some RA-mediated effects on myogenesis may have also been masked in these whole-limb culturcs. The inability of a previous study to show RA-mediated inhibition of limb myogenesis [34] may reflect differences in culture conditions and/or the sensitivity of the analyses. Previous studies of RA’s dose-dependent opposing effects on amphibian limb regeneration suggested that RA’s effects at low concentrations are specific and mediated by cellular RA-binding proteins (cRABPs), whereas at higher concentrations, RA in excess of that which can be bound by cRABP has different, less specific and perhaps toxic effects [301 as is seen in other systems [25, 391. Specific cRABPs are known to exist in the cytoplasm of limb-bud mesenchymal cells [I 8, 281. However, toxicity, as indicated

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by a significant decrease in total DNA in comparison to controls, is seen under these conditions only at RA concentrations above 100 ng/ml[35]. Furthermore, a study by Maden and Summerbell [28] indicates that at RA concentrations that induce skeletal duplication in chick limb buds, only about 4% of the cRABP present in the limb is occupied by RA. Thus, RA’s inhibitory effects in this system cannot be directly attributed to toxicity, and clarification of the cellular basis for RA’s dose-related, opposing effects on patterning and phenotypic expression must await further study. It is clear that information regarding age- and regionrelated effects of RA on in vitro chondrogenesis and myogenesis by limb mesenchymal cells will be needed to clarify RA’s effects in this system at the cellular level. On a morepositive note, the recent discovery of two genes that encode distinct nuclear RA receptors [2, 371 and evidence that RA can activate expression of homeobox gene clusters [29] provide hope that significant advances in our knowledge of RA’s effects at the cellular and molecular levels are imminent. In summary, the results show for the first time that physiological concentrations of RA selectively enhance limb chondrogenesis in serum-free cultures and suggest that RA’s role in limb-skeletal patterning involves modulation of chondrogenic expression. They further provide an experimental model whereby analyses of RA’s stimulatory and inhibitory effects on phenotypic expression may be carried to the molecular level in serum-free culture. Here, concentrations of RA and other media additives can be more rigorously controlled. This will allow RA’s effects on gene expression and its relationship to cRABP and nuclear RA receptor levels, autocrine growth factors, protooncogenes and other cellular regulatory mechanisms to be examined in isolation from unknown entities in serum-supplemented media and away from the multitude of environmental contingencies associated with the intact embryo. Such studies will be useful in clarifying the relationship between cytodifferentiation and morphogenesis. Acknowledgements. This work was supported by grants RR08248, HD05505, and HD18577 from the NIH, USA and from the NSERC, Canada. MF-20 hybridoma supernatant was obtained from the Developmental Studies Hybridoma Bank, University of Iowa (supported by NIH contract NOlHD62915). We appreciate the technical assistance and advice of Karen Jensen, Rebecca Reiter, Lei Peng, Dwight Moulton and Jim Perry.

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Accepted in revised form October 10, 1988