Cartilage proteoglycan core protein gene expression during limb cartilage differentiation

DEVELOPMENTAL

BIOLOGY

118.112-117

(1986)

Cartilage Proteoglycan Core Protein Gene Expression during Limb Cartilage Differentiation ROBERTA. KOSHER,* STEVENW. GAY,*JOYCE R. KAMANITZ,* WILLIAM M. KULYK,* BARBARAJ. RODGERS,*S. SAI,~ T. TANAKA,t AND MARVIN L. TANzERt Departments of *Anatomy and tBiochemistry,

of Connecticut Health Center, Farmington, Connecticut 06032

University

Received April 4, 1986; accepted in revised

fm May

13, 1986

Changes in the steady-state cytoplasmic levels of mRNA for the core protein of the major sulfated proteoglycan of cartilage were examined during the course of limb chondrogenesis in vitro using cloned cDNA probes. Cytoplasmic core protein mRNA begins to accumulate at the onset of overt chondrogenesis in micromass culture coincident with the crucial condensation phase of the process, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix. Following condensation, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix by the cells. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during chondrogenesis in micromass culture. Cyclic AMP, an agent implicated in the regulation of chondrogenesis elicits a concomitant twoto fourfold increase in both cartilage core protein and type II collagen mRNA levels by limb mesenchymal cells. Core protein gene expression is more sensitive to CAMP than type II collagen gene expression. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes. 0 1986 Academic

Press. Inc.

INTRODUCTION

The process of cartilage differentiation in the developing vertebrate limb involves qualitative changes in the pattern of gene expression. As prechondrogenic limb mesenchymal cells differentiate into chondrocytes, they initiate the synthesis of a large cartilage-specific sulfated proteoglycan, cartilage-characteristic type II collagen, and several minor collagen species including type IX collagen, and cease synthesizing type I collagen and possibly fibronectin. The program of transcriptional and post-transcriptional molecular events that control this switch in specific gene activities is unknown. An essential step in understanding the molecular regulation of cartilage-specific gene activity is describing changes in the accumulation of relevant mRNA molecules during the progression of chondrogenesis, and determining how the production of these mRNAs is influenced by factors involved in the regulation of chondrogenesis. Recently, cloned cDNA probes complementary to mRNA for the protein core of the major sulfated proteoglycan of cartilage have been characterized (Sai et aL, 1986). These probes direct the synthesis of an immunologically identifiable core protein fragment in the expression

vector,

pUC9

(Sai

et cd,

1986). The cDNAs

hybridize to a single 8-9 kb sternal cartilage mRNA species, and analysis of their base sequence gives a single 0012-1606/86 $3.00 Copyright All rights

0 1986 by Academic Press, Inc. of reproduction in any form reserved.

112

open reading frame with a deduced amino acid sequence consistent with the amino acid composition and sequence (Perin et al., 1984) of cartilage proteoglycan core protein (Sai et al, 1986). In the present study, we have used these probes to examine changes in the steady-state cytoplasmic levels of cartilage proteoglycan core protein mRNA during the course of limb cartilage differentiation in vitro. Changes in core protein mRNA levels have been compared with the accumulation of mRNA for type II collagen, the other major constituent of hyaline cartilage matrix, and the effect of CAMP, a molecule implicated in the regulation of chondrogenesis, on core protein and type II collagen mRNA accumulation has been examined. Our results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during limb cartilage differentiation, although there are quantitative differences in the extent of expression of the two genes. MATERIALS

AND

METHODS

Cell and organ culture. Distal wing bud tips (subridge regions) were cut away from stage 25 (Hamburger and Hamilton, 1951) embryos of White Leghorn chicks as previously described, the size of the excised subridge regions being 0.3-0.4 mm from the distal apex of the tissue to the proximal cut edge (Kosher et aZ., 1979a).

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ET AL.

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Cartilage Proteoglgcan Core Protein Gene Expression

The ectoderm was removed from the distal subridge mesoderm, and micromass cultures were prepared from dissociated subridge mesenchymal cells as previously described (Gay and Kosher, 1984). In addition, intact subridge mesoderm explants were cultured on nutrient agar containing FlO medium supplemented with 10% fetal calf serum in the presence and absence of 1.0 mM dibutyryl-cyclic AMP (dbcAMP) as previously described (Kosher et ak, 1979a; Kosher and Savage, 1980). H@ridixatim probes. The cloned chick cartilage proteoglycan core protein cDNA plasmids utilized were ST1 and ST-2, similar 1200-base pair (bp) inserts into pUC9 (Sai et ak, 1986). The cloned chick type 11 collagen cDNA plasmid used was pCAR2, a 680-bp cDNA insert into pBR322 (Vuorio et aZ., 1982) which was provided by Dr. William Upholt (University of Connecticut Health Center). Probes were labeled with a-32P-dCTP (3000 Ci/ mmole, Amersham) by the standard nick translation procedure of Maniatis et al. (1982) to a specific activity of l-2 X lo* cpm/pg. The nick-translated probes were separated from unincorporated nucleotide by Sephadex G-50 spun-column chromatography (Maniatis et al, 1982). Hybridization anal&. Cytoplasmic mRNA levels were measured as previously described (Kosher et ah, 1986) by the cytoplasmic dot hybridization procedure of White and Bancroft (1982), except that 10 mM vanadylribonucleoside complex was included during preparation of cytoplasmic extracts and the nuclear pellets were utilized for determination of total DNA content (Brunk et aL, 1979). Aliquots of the cytoplasmic extracts were diluted with 15X SSC (standard saline citrate, 0.15 M NaCY0.015 M sodium citrate) and spotted onto nitrocellulose using the Schleicher and Schuell microsample filtration manifold. The nitrocellulose dot blots were baked at 80°C in a vacuum oven for 90 min, then prehybridized overnight at 42°C in a solution containing 50% formamide, 4X SSC, 50 mMsodium phosphate buffer (pH 6.5), 0.02% bovine serum albumin, 0.02% Ficoll400, 0.02% polyvinylpyrrolidone, 0.1% SDS, and 100 pg/ml of denatured salmon sperm DNA. Hybridization was then carried out for 48 hr at 55°C in a solution identical to the above except for the addition of 0.1 g/ml of dextran sulfate and 1.2 X 10’ cpm of the appropriate heat-denatured cloned cDNA plasmid. The nitrocellulose dot blots were washed four times for 5 min each at 55°C in 2X SSC, 0.1% SDS; four times for 15 min each at 55°C in 0.1X SSC, 0.1% SDS; and, exposed to Kodak X-Omat XAR-5 film for various lengths of time at -70°C with a DuPont Cronex lighting plus intensifying screen. The levels of hybridizable RNA sequences were quantified by scanning the dots of the resultant autoradiograms with a SL-TRFF soft laser scanning densitometer (Biomed Instruments, Fullerton, Calif.), interfaced to

an Apple IIe computer programmed to provide automatic peak integration. Several film exposures were scanned to be sure the densities of the dots were in the linear response range of the film. RESULTS

Cytoplasmic levels of cartilage proteoglycan core protein mRNA were initially examined during the course of the chondrogenic differentiation of the distal subridge mesenchymal cells of stage 25 wing buds in micromass culture. Stage 25 subridge mesoderm consists of a relatively homogenous population of undifferentiated chondrogenic progenitor cells, which uniformly progress through the phases of chondrogenesis in micromass culture and form a virtually uniform sheet of cartilage with little, if any, nonchondrogenic tissue detectable (Gay and Kosher, 1984). In such cultures, widespread prechondrogenie aggregates of cells are detected by the end of the first day of culture, after which there is a progressive and uniform accumulation of Alcian blue-positive cartilage matrix by the cells (Gay and Kosher, 1984). Cytoplasmic RNA sequences that hybridize to the cloned cartilage proteoglycan core protein cDNA probes are not detectable at 3 hr following the initiation of culture, which is prior to overt morphological indications of differentiation by the mesenchymal cells (Fig. 1). By A

HOURS

3

IN CULTURE

20

48

72

C

96

HOURS

3

IN CULTURE

20

48

72

96

4:

600

3

24

HOURS 0

48

72

96 HOURS

IN CULTURE

IN CULTURE

D

FIG. 1. (A) and (C). Autoradiographs demonstrating the hybridization of sap-labeled cartilage proteoglycan core protein cDNA plasmids, ST1 (A) and ST-2 (C) to RNA sequences in the cytoplasm of subridge limb mesenchymal cells at various times during the course of their chondrogenic differentiation in micromass culture. (B) and (D). The levels of core protein mRNA detectable with probes ST-1 (B) and ST2 (D) at each period of culture were determined by scanning densitometry as described under Materials and Methods. The amount of cytoplasmic core protein mRNA/pg DNA at each time point is presented as an amount relative to that at 96 hr of culture, which was arbitrarily set to 1000.

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DEVELOPMENTAL BIOLOGY

TABLE 1 CYTOPLASMIC CARTnAGE PROTEOGLYCAN CORE PROTEIN mRNA AND TYPE II COLLAGEN mRNA ACCUMULATION DURING THE COURSE OF THE CHONDROGENIC DIFFERENTIATION OF SUBRIDGE LIMB MESENCHYMAL CELLS IN MICROMASS CULTURE [mRNA]/pg Core protein Hours in culture 3 20 48 72 96

DNA Type II collagen

ST-1

ST-2

pCAR2

ND”

ND 100

13 100

100 210 540 1282

235 641

1449

378 1271 2222

Note. At each timepoint, aliquots of cytoplasmic extracts prepared from the same group of cells were hybridized with ST-l, ST-2, and pCAR2. The levels of mRNA were quantified by scanning densitometry. The amount of mRNA/yg DNA at each timepoint is expressed as an amount relative to that at 20 hr of culture, which was arbitrarily set at 100. The normalized mRNA levels detected with the various cDNA probes cannot be directly compared. a Not detectable.

20 hr, which corresponds to the onset of overt chondrogenesis as characterized by the formation of extensive prechondrogenic aggregates of cells throughout the culture, substantial amounts of hybridizable core protein RNA sequences are detectable in the cytoplasm of the cells (Fig. 1). During the subsequent 3 days of culture, there is a continuous and progressive increase in the accumulation of cytoplasmic core protein mRNA which parallels the progressive accumulation of cartilage matrix by the cells. A virtually identical pattern of accumulation of cytoplasmic core protein mRNA is observed using either core protein cDNA plasmid ST-1 or ST-2 (Fig. 1 and Table 1). No hybridization to plasmid pUC9 is detectable at any time during the culture period, indicating that none of the hybridization detectable with the probes is due to the binding of plasmid DNA sequences. The initiation of detectable cytoplasmic core protein mRNA accumulation at the onset of chondrogenesis (at 20 hr) coincides with a dramatic increase in the accumulation of hybridizable type II collagen mRNA sequences (Table 1). Furthermore, the subsequent progressive increase in cytoplasmic core protein mRNA is accompanied by a progressive increase in cytoplasmic type II collagen mRNA. These results suggest that the proteoglycan core protein and type II collagen genes may be coordinately regulated during cartilage differentiation. It is noteworthy, however, that the relative rate of accumulation of cytoplasmic type II collagen mRNA is about twice that of core protein mRNA during chondrogenesis in

VOLUME 118, 1986

micromass culture (Table 1). The amount of type II collagen mRNA per cell increases, on the average, about 14-fold between 20 and 96 hr of culture, whereas core protein mRNA increases only 6-fold during the same period. It should also be noted that, in contrast to core protein mRNA, low levels of type II collagen mRNA are detectable prior to the onset of overt differentiation (Table 1; see also Kravis and Upholt, 1985; Kosher et aL, 1986). To determine if the core protein and type II collagen genes respond in a coordinate fashion to factors involved in regulating chondrogenesis, we examined the effect of dbcAMP, an agent previously shown to stimulate in vitro chondrogenesis (Kosher et CZL,197913,Kosher and Savage, 1980; Solursh et aL, 1981) on mRNA accumulation. As shown in Figs. 2 and 3, dbcAMP elicits a concomitant DAYS

A

IN CULTURE 1

2

3

CAMP CONTROL

1000 CAMP 2 0 m 3 2 z

oz E

600 -

600-

z iij

6

400/

E E! 0 0 -

*O"-

/ I 1

B

DAYS

CT I 2

I 3

IN CULTURE

FIG. 2. (A). Autoradiograph demonstrating the hybridization of 3zPlabeled cartilage proteoglycan core protein cDNA plasmid, ST-l to RNA sequences in the cytoplasm of subridge mesoderm explants cultured in the presence (CAMP) and absence (control) of 1.0 mMdbcAMP. (B). The levels of mRNA in the presence (CAMP) and absence (CT) of dbcAMP were quantitated by scanning densitometry. The amount of mRNA/pg DNA at each point is expressed as an amount relative to that present in Day 1 control explants, which was artibrarily set to 100.

KOSHER ET AL. DAYS

A

Cartilage Proteoglycan Core Protein Gene Expression

IN CULTURE

CAMP CONTROL

CAMP

CT

lOOO-

2000

/-:

-

1

DAYS

2

3

IN CULTURE

FIG. 3. (A) Autoradiograph demonstrating the hybridization of =Plabeled type II collagen cDNA plasmid, pCAR2 to RNA sequences in the cytoplasm of subridge mesoderm explants cultured in the presence (CAMP) and absence (control) of 1.0 mM dbcAMP. (B) The levels of mRNA in the presence of (CAMP) and absence (CT) of dbcAMP were quantified by scanning densitometry. The amount of mRNA/rg DNA at each point is expressed as an amount relative to that present in Day 1 control explants, which was arbitrarily set to 100.

increase in cytoplasmic core protein and type II collagen mRNA sequences in subridge mesoderm explants. There is, however, a greater increase in accumulation of core protein mRNA in response to dbcAMP than type II collagen mRNA. Cytoplasmic core protein mRNA increases about fourfold in response to dbcAMP, whereas type II collagen mRNA increases twofold (Figs. 2 and 3). DISCUSSION

The results of the present study indicate that cytoplasmic cartilage proteoglycan core protein mRNA begins to accumulate at the onset of chondrogenesis coincident with the crucial condensation or aggregation phase of the process in vitro, in which prechondrogenic mesenchymal cells become closely juxtaposed prior to

115

depositing a cartilage matrix. The initiation of core protein mRNA accumulation coincides with a dramatic increase in cytoplasmic type II collagen mRNA. These temporal correlations suggest that regulatory events occurring during the crucial condensation process may be responsible for the concurrent changes in core protein and type II collagen gene activity. Among the interrelated events that have been implicated in triggering chondrogenesis during condensation are cell-cell and/ or cell-matrix interactions (Kosher, 1983; Solrush, 1983); cytoskeletal-linked change in cell shape (Archer et CZZ., 1982; Zanetti and Solursh, 1984); and, prostaglandinmediated elevations in cellular CAMP levels (Kosher and Walker, 1983; Biddulph et ah, 1984; Chepenik et d., 1984; Kosher and Gay, 1985; Gay and Kosher, 1985). Following the concurrent changes in mRNA accumulation at condensation in vitro, there is a concomitant progressive increase in cytoplasmic core protein and type II collagen mRNA accumulation which parallels the progressive accumulation of cartilage matrix. These results suggest that the cartilage proteoglycan core protein and type II collagen genes are coordinately regulated during the course of limb cartilage differentiation. It is noteworthy, however, that there are quantitative differences in the extent of expression of the two genes during chondrogenesis. The relative rate of accumulation of cytoplasmic type II collagen mRNA is greater than twice that of core protein mRNA during the progression of chondrogenesis in micromass culture. This latter observation could reflect differential changes in the transcription rates of the two genes during chondrogenesis, and/or differences in the stability of the two mRNAs during the process. Distinguishing between these and other possibilities will require further study. Further support for the notion that the genes for the two major constituents of hyaline cartilage matrix are coordinately regulated during the course of chondrogenesis derives from our observation that dbcAMP elicits a concomitant two- to fourfold increase in both proteoglycan core protein and type II collagen mRNA levels. Core protein gene expression, however, is more sensitive to CAMP than type II collagen gene expression, since cytoplasmic core protein mRNA levels rise fourfold in response to CAMP, while type II collagen mRNA levels increase twofold. Further experimentation will be necessary for understanding the molecular basis of the differential stimulatory effect of CAMP. In any event, the striking effect dbcAMP has on cartilage-specific gene expression provides further support for a key role for CAMP in the regulation of chondrogenesis (Kosher et aZ.,1979b; Kosher and Savage, 1980; Solursh et cd., 1981). In the present study we did not detect cartilage proteoglycan core protein mRNA sequences in the cytoplasm of limb mesenchymal cells prior to onset of chon-

116

DEVELOPMENTAL BIOLOGY

drogenesis in vitro (i.e., at 3 hr of micromass culture). Previously, we were unable to detect hybridizable cartilage core protein mRNA sequences in the cytoplasm of the prechondrogenic mesenchymal cells comprising stage 18/19 wing buds and the distal subridge region of stage 25 wing buds (Sai et aL, 1986). These observations suggest that the core protein of the sulfated proteoglycan molecules being synthesized by prechondrogenic mesenchymal cells is a different gene product than the core protein of cartilage chondroitin sulfate proteoglycan. Immunological and genetic evidence for this conclusion has been presented by others (Goetinck, 1982, 1983). It is noteworthy, however, that in the present study, as well as in previous studies (Kravis and Upholt, 1985; Kosher et aL, 1986), low levels of type II collagen mRNA have been detected in prechondrogenic mesenchymal cells prior to the onset of overt cartilage differentiation and the accumulation of detectable amounts of type II collagen. The type II collagen mRNA detectable in prechondrogenic cells is not due to the cross-hybridization of type II collagen cDNA probes to type I collagen mRNA (Kravis and Upholt, 1985; Kosher et CAL, 1986). We have previously suggested that the low level expression of the type II collagen gene by chondrogenic progenitor cells prior to the onset of overt differentiation may represent a molecular manifestation of the state of determination of the cells and may serve as a molecular marker to elucidate when and where during embryogenesis that determination of a specific limb cell type occurs (Kosher et al, 1986). The results of the present study and previous investigations indicate that the qualitative changes in gene expression that occur during limb chondrogenesis are controlled at multiple levels. The dramatic coordinate changes in cartilage proteoglycan core protein and type II collagen mRNA accumulation that occur at the onset of chondrogenesis are very likely controlled at the transcriptional level. Since cytoplasmic cartilage core protein mRNA is not detectable in prechondrogenic mesenchyma1 cells, it is quite possible that the onset of chondrogenesis involves initiation of the transcription of the cartilage core protein gene. It is, of course, difficult to conclusively eliminate the possibility that prechondrogenie cells contain a small number of core protein transcripts that were not detected in our studies, although the cytoplasmic dot hybridization procedure utilized is an extremely sensitive assay reportedly capable of detecting as few as five copies of a specific RNA per cell (White et al., 1986). In any case, the onset of overt chondrogenesis very likely involves greatly accelerated transcription of the type II collagen gene, rather than the initiation of its transcription, since low levels of type II collagen mRNA are present in the cytoplasm of prechondrogenic mesenchymal cells at the earliest stages

VOLUME 118, 1986

of limb development, well before the accumulation of detectable levels of type II collagen (Kravis and Upholt, 1985; Kosher et d, 1986). This latter observation suggests the possibility that post-transcriptional regulatory controls may limit the translation of type II collagen mRNA prior to the overt differentiation of the cells. Moreover, our previous studies have indicated that type I collagen gene expression during limb chondrogenesis is regulated, at least in part, at the translational level (Kosher et al, 1986). Substantial amounts of mRNAs for the al(I) and a2(1) chains of type I collagen are present in the cytoplasm of well-differentiated chondrocytes which have ceased synthesizing detectable amounts of type I collagen (Kosher et al, 1976; see also Kravis and Upholt, 1985; Focht and Adams, 1984; Saxe et aL, 1985). The differentiation of limb mesenchymal cells into chondrocytes thus provides an excellent system for investigating the various ways in which extracellular influences and intracellular regulatory factors influence tissue specific gene expression. We thank Dr. William Upholt for helpful discussions, as well as for supplying probes. This work was supported by grants from the National Institutes of Health, and the University of Connecticut Research Foundation. REFERENCES ARCHER, C. W., ROONEY, P., and WOLPERT, L. (1982). Cell shape and cartilage differentiation of early chick limb bud cells in culture. Cell L@i$er. 11,245-251. BIDDULPH, D. M., SAWYER, L. M., and SMALES, W. P. (1984). Chondrogenesis of chick limb mesenchyme in vitro. Effects of prostaglandins on cyclic AMP. Exp. Cell Res. 153,270-274. BRLJNK, C. F., JONES, K. C., and JAMES, T. W. (1979). Assay for nanogram quantities of DNA in cellular homogenates. And &o&em 92,49’7500. CHEPENIK, K. P., Ho, W. C., WAITE, B. M., and PARKER, C. L. (1984). Arachidonate metabolism during chondrogenesis in vitro. Calcif: Tissue Int. 36,175-181. FOCHT, R. J., and ADAMS, S. L. (1984). Tissue specificity of type I collagen gene expression is determined at both transcriptional and posttranscriptional levels. MoL Cell. Biol. 4,1843-1852. GAY, S. W., and KOSHER, R. A. (1984). Uniform cartilage differentiation in micromass cultures prepared from a relatively homogeneous population of chondrogenic progenitor cells of the chick limb bud: Effect of prostaglandins. J. Ezp. ZooL 232,317-326. GAY, S. W., and KOSHER, R. A. (1985). Prostaglandin synthesis during the course of limb cartilage differentiation in vitro. J. Embrgol. Exp. Morphol. 89,367-382. GOETINCK, P. F. (1982). Proteoglycans in developing embryonic cartilage. In “The Glycoconjugates” (M. I. Horowitz, ed.), Vol. 3, pp. 197-229. Academic Press, New York. GOETINCK, P. F. (1983). Mutations affecting limb cartilage. In “Cartilage” (B. K. Hall, ed.), Vol. 3,165-189. Academic Press, New York. HAMBURGER, V., and HAMILTON, H. L. (1951). A series of normal stages in the development of the chick embryo. J. Morph01 88,49-92. KOSHER, R. A. (1983). The chondroblast and the chondrocyte. In “Cartilage” (B. K. Hall, ed.), Vol. 1, pp. 59-85. Academic Press, New York. KOSHER, R. A., and GAY, S. W. (1985). The effect of prostaglandins on

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Cartilage Proteoglycan Core Protein

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SAI, S., TANAKA, T., KOSHER,R. A., and TANZER,M. L. (1986). Cloning and sequence analysis of a partial cDNA for chicken cartilage proteoglycan core protein. Proc. NatL Acad. Sk. USA 83,5081-5085. SAXE, S. A., LUKENS,L. N., and PAWLOWSKI,P. J. (1985). Changes in the nuclear and cytoplasmic levels of type I and type II collagen RNAs during growth of chondrocytes in 5-bromo-2’-deoxyuridine. J. BioL Chem 260,3812-3819.

SOLURSH,M. (1983). Cell-cell interaction in chondrogenesis. In “Cartilage” (B. K. Hall, ed.), Vol. 2,121-141. Academic Press, New York. 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. Dev. Biol 83,9-19. VUORIO,E., SANDELL,L., KRAVIS,D., SHEFFIELD,V. C.,VUORIO,T., DORFMAN, A., and UPHOLT,W. (1982). Construction and partial characterization of two recombinant cDNA clones for procollagen from chicken cartilage. Nucleic Acid-s Res. 10, 1175-1192. WHITE, B. A., and BANCROFT,F. C. (1982). Cytoplasmic dot hybridization. Simple analysis of relative mRNA levels in multiple small cell or tissue samples. J. Biol. Chem. 257.8569-8572. WHITE, B. A., LUFKIN, T., PRESTON,G. M., and BANCROFT,C. (1986). RNA dot and blot hybridization: Selected procedures for endocrine and neuroendocrine studies. In “Methods in Enzymology” (P. M. Conn, ed.), Vol. 124, pp. 269-278. Academic Press, Orlando, Fla., in press. ZANETTI,N. C., and SOLURSH,M. (1984). Induction of chondrogenesis in limb mesenchymal cultures by disruption of the actin cytoskeleton. J. Cell Biol. 99, 115-123.