Biochemical and ultrastructural studies of collagen and proteochondroitin sulfate in normal and nanomelic cartilage

DEVELOPMENTAL

BIOLOGY

50,

Biochemical Proteochondroitin JOHN Department

of Animal

35-47

(19761

and Ultrastructural Studies of Collagen and Sulfate in Normal and Nanomelic Cartilage P. PENNYPACKER' Genetics,

University

Accepted

AND PAUL of Connecticut,

December

F. GOETINCK Storrs,

Connecticut

06268

5, 1975

Biochemical and ultrastructural analysis of the sternal cartilage of chick embryos homozygous for the autosomal recessive gene nanomelia suggest that the mutant cells are functional chondrocytes in all respects except in proteochondroitin sulfate synthesis. Proteochondroitin sulfate synthesized by normal and mutant sterna in vitro was chromatographed on 1% agarose. Two distinct fractions of proteochondroitin sulfate were resolved from both normal and mutant cartilage. In normal cartilage, the major fraction represents approximately 90% of the total material, and in the mutant, this fraction is reduced to lo%, while the second fraction remains unchanged. It is suggested that at the onset of chondrogenesis in the mutant, an augmentation in the syntheis of the major fraction does not occur. Collagen synthesis in the mutant cartilage was analyzed by hydroxyproline determination, carboxymethylcellulose chromatography, and amino acid analysis to determine the percentage hydroxylation of lysine residues. By these procedures, collagen synthesis in the mutant was found to be both quantitatively and qualitatively similar to normal. Ultrastructural studies on the mutant sterna revealed that while the mutant chondrocytes were normal in appearance, the amount of extracellular matrix was decreased. In conjunction with this decrease, there is a severe reduction in the number of proteochondroitin sulfate matrix granules. No differences were observed in the collagen Iibrils. INTRODUCTION

thymidine analog, BrdUrd, and a high moEmbryonic chick cartilage has been lecular weight fraction of embryo extract shown to synthesize two species of proteo- (H-fraction) affect the synthesis of both chondroitin sulfate (PCS; Palmoski and macromolecules in chondrocyte cultures Goetinck, 1972; Levitt and Dorfman, 1973). (Palmoski and Goetinck, 1972; Schiltz et al., 1973; Levitt and Dorfman, 1973). The Recent studies have demonstrated that both species are also synthesized by stage parallel changes involving these two chon18-19 limb mesenchyme and that chondro- drogenic events during normal developgenesis involves the preferential increase ment and during treatment with BrdUrd in synthesis of one of these two species and H-fraction suggest that their regulation might be coordinated. (Goetinck et al., 1974). Concurrent with The elaboration of cartilage macromolethe elevated synthesis of PCS, there is also cules into extracellular matrix involves a transition from the synthesis of [al(I)l,a2 the interaction of PCS and hyaluronic acid collagen to [al(II)l, collagen (Linsento form huge complexes (Hascall and Heimeyer et aZ., 1973; Levitt and Dorfman, negard, 1974a,b; Heinegard and Hascall, 1973). In the developing limb of the embry19741, and the assembly of collagen moleonic chick, these two chondrogenic events cules into distinct fibrils. There is also begin at stage 24 and continue until the thought to be interaction between PCS onset of ossification in the cartilaginous rudiment (Linsenmeyer et al., 1973). The and collagen, and it is suggested that this interaction might influence fibril formaI Present address: Laboratory of Developmental tion (Mathews, 1965; Wood, 1960; Mathews Biology and Anomalies, National Institute of Dental and Decker, 1968; Toole and Lowther, Research, National Institutes of Health, Bethesda, Maryland 20014. 1968). Specifically, it has been hypothe35 Copyright All nghts

0 1976 by Academic Press, Inc. of reproduction in any form reserved.

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DEVELOPMENTAL BIOLOGY

sized that the presence of large amounts of PCS prevent the assembly of large diameter collagen fibrils in cartilage. Chick embryos, homozygous for the autosomal recessive gene, nanomelia, develop an extreme form of micromelia (Landauer, 1960). The defect is confined to the cartilage of the mutant and results in a reduction in PCS concentration to 10% of normal levels (Mathews, 1967; Fraser and Goetinck, 1971). It has been reported that the mutation specifically affects the synthesis of the cartilage-related species of PCS (Palmoski and Goetinck, 1972). If the synthesis of PCS and collagen is by some means coordinated, then collagen synthesis in the mutant cartilage also might be affected. Although a previous report (Mathews, 1967) indicated that the hydroxyproline concentration in the mutant cartilage is equivalent to normal, it has not been established if the transition from the synthesis of [a1(I)lpa2 to [al(H)], collagen had occurred in the mutant. It is also possible that the reduction in PCS might influence matrix organization, particularly with respect to collagen fibril formation. If the relationship between fibril size and PCS content is correct, one might expect larger diameter fibrils, possibly with a distinct periodicity. In the present study, we have examined PCS and collagen synthesis in the sternal cartilage of normal and nanomelic chick embryos. In addition, ultrastructural studies on the mutant cartilage were carried out to examine the effects of the mutation on matrix organization, as well as the overall cartilage fine structure. The results indicate that PCS metabolism of nanomelic cartilage is similar to that of prechondrogenic mesenchyme of normal embryos whereas collagen synthesis and fibril formation is unaffected by the defect involving PCS synthesis in the mutant. METHODS

Sterna were dissected from 14-day normal and mutant chick embryos that re-

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sulted from the crossing of known heterozygotes for the gene, nanomelia. Phenotypically normal embryos served as controls. For analysis of PCS, the sterna were carefully stripped of their perichondria, and incubated at 37°C in 50% Ham’s F-10 (Grand Island Biological Co., GIBCO), with two times the vitamin and four times the amino acid concentration, 38% Hanks’ balanced salt solution (HBSS), 0.5% (w/v) bovine serum albumin (Sigma Chemical Co.), and 10% fetal calf serum (GIBCO). Radioactive NaZs5S0, (New England Nuclear, NEN) was added at a concentration of 50 @X/ml. Four sterna were placed in screw-cap vials containing 2.5 ml of medium. The vials were first equilibrated in an atmosphere of 5% CO,:95% air, and then sealed and maintained at 37°C with constant shaking. The incubation period was 4 hr. The labeled sterna were rinsed in cold HBSS, diced with a razor blade, and extracted for 3 days in 4 M guanidine-HCl. The extract was clarified by centrifugation and then dialyzed against 0.5 M NaCl. Analysis of this extract by 1% agarose chromatography was carried out as previously described (Palmoski and Goetinck, 1972). For the analysis of collagen, 14-day normal and mutant sterna were cultured as described above in Dulbecco-modified Eagle’s medium supplemented with 10% fetal calf serum, 50 pg/ml p-aminopropionitrile, and 100 pg/ml ascorbic acid. The sterna were labeled with 50 @i/ml of [2,3“H(N)&proline (sp act 33.8 CUmmole, NEN) or [2-“Hlglycine (sp act 114 mCi/mg; NEN) for a period of 24 hr. Extraction, purification, and carboxymethylcellulose (CM32, Reeve Angel) chromatography of the labeled collagen was carried out on a 1.6 x 7.0 cm jacketed column as described by Linsenmeyer et al. (1973). Carrier collagen was prepared from lathyritic chicks as described by Kang et al. (1966). The degree of hydroxylation of lysine in cul chains was also analyzed. For this pur-

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pose, normal and mutant 14-day embryonic sterna were incubated in Eagle’s minimal essential medium supplemented with 10% fetal calf serum, 100 Fg/ml ascorbic acid, and 50 Fg/ml fi-amino-propionitrile as described above. [4,5-“H(N)lL-lysine (sp act 30 Ci/mmole; NEN) was added at a concentration of 25 &i/ml and the sterna were incubated for 24 hr. The labeled collagen was chromatographed on a carboxymethylcellulose (CMC) column and the fraction in the al peak pooled. The pooled material was dialyzed against 0.1% acetic acid and lyophilized. The lyophilized material was redissolved in 6 N HCl and hydrolyzed under nitrogen for 16 hr in sealed tubes at 120°C. Amino acid analysis of the acid hydrolyzate of the (Y chains was carried out using a split effluent stream analyzer so that radioactively labeled lysine and hydroxylysine could be measured. All radioactivity was counted by liquid scintillation spectrometry using a counting fluid prepared by mixing 2 vol of a toluene solution of 2,5-diphenyloxazole (4 g/liter) and 1,4-di[2-(5-phenyloxazolyl)]benzene (0.1 g/liter) and 1 vol of Triton X-100. To determine the percentage of lysine residues oxidized to hydroxylysine using [4,5-:‘Hllysine, the number of hydroxylysine dpm was multiplied by 413 to take into account the loss of one tritium atom during the reaction (Miller, 1972). The precentage hydroxylation was then determined from this corrected value. For electron microscopy, normal and mutant 14-day embryonic sterna were

FIG.

extracted

Normal

and Nanomelic

Cartilage

37

fixed in cold 2% glutaraldehyde (Tousimis Research Corp.) buffered with 0.1 M phosphate buffer, pH 7.3. The sterna were then rinsed in cold phosphate buffer and postfixed in phosphate buffered 1% osmium tetroxide (Stevens Metallurgical Corp.) for 1V2 hr. The sterna were dehydrated in a graded series of alcohols and propylene oxide, and embedded in Epon 812. Silver to gold sections were cut on a LKB Ultratome III, stained with 4% uranyl acetate and Reynolds’ lead citrate, and examined on a Phillip’s 300 electron microscope. RESULTS

Proteochondroitin Sulfate Synthesis in Normal and Mutant Sternal Cartilage The intact PCS macromolecule synthesized by chick cartilage was initially analyzed. For this purpose, normal and nanomelic sterna were cultured in the presence of Na2”5S04, and the labeled PCS was extracted and chromatographed on a 1% agarose column. Figure la represents the chromatographic profile of PCS synthesized by normal embryonic sterna. Three distinct peaks are resolved. The first two, designated as peaks Ia and Ib, represent approximately 90% of the eluted radioactivity, and the last, designated as peak II, the remaining 10%. Subsequent studies on PCS synthesized by embryonic chick limb bud cultures have shown that an aggregate-subunit relationship exists between peaks Ia and Ib, as peak Ia can be dissociated reversibly by high ionic strength conditions (Goetinck et al., 1974; Palmoski

1. Elution profile of proteochondroitin sulfate chromatographed from sterna of normal (a) and nanomelic (b) 14-day embryos

after

on 1% agarose. labeling with

The material was Na,WO, for 4 hr.

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et al., in preparation). A similar relationship between peaks Ia and Ib was observed during this study. The 1% agarose profile of PCS synthesized by mutant sterna is shown in Fig. Ib. Nanomelic cartilage does synthesize a small, but significant amount of peak Ia, which represents 8-10% of the eluted material. This observation is different from a previous communication from this laboratory (Palmoski and Goetinck, 1972) in which it was reported that peak I could not be detected in the elution profile of the mutant. Peak II material in the mutant is equivalent to normal in elution volume, and it has been previously demonstrated that it is also quantitatively similar to normal (Palmoski and Goetinck, 1972). Collagen Synthesis in Normal tant Sternal Cartilage

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FIG. 2. Elution profile of collagen chromatographed on carboxymethylcellulose. The collagen was obtained from sterna of normal embryonic chicks after labeling with [3Hlglycine for 24 hr.

and Mu-

The hydroxyproline concentration in normal and nanomelic cartilage is equivalent (Mathews, 1967) indicating that collagen synthesis and accumulation is quantitatively normal. To identify the type of collagen synthesized, sterna from the normal and mutant embryos were pulsed with r3H]glycine. The labeled collagen was extracted, carrier lathyritic chick skin collagen was added, and the sample was partially purified for CMC chromatography. The CMC elution profile of the radioactive collagen extracted from normal sternal cartilage is shown in Fig. 2. Essentially all of the radioactive collagen coeluted with the al(I) subunit of the carrier. The absence of a radioactive a2 peak implies that only cartilage-type collagen is being synthesized. An identical profile is observed if [3Hlproline is used to label the newly synthesized collagen. Figure 3 shows the elution profile of the radioactive collagen synthesized by mutant sterna. The chromatogram is essentially identical to that of Fig. 2 and suggests that collagen synthesis in the mutant cartilage is qualitatively similar to normal.

FIG. 3. Elution profile of collagen chromatographed on carboxymethylcellulose. The collagen was obtained from sterna of nanomelic embryonic chicks after labeling with [3H]glycine for 24 hr.

As a further means of identifying the type of collagen synthesized, the percentage of lysine residues oxidized to hydroxylysine in the collagen of nanomelic cartilage was determined (Miller, 1972). A value of 54.5% was found for the hydroxylation of lysine residues in the normal sternum and 57.6% for the mutant (Table 1). Both values are very close to the 56.4% value reported (Trelstad et al., 1970) for purified cartilage collagen and add further support to the conclusions drawn from the CMC chromatographic profiles. It, therefore, appears that collagen synthesis is unaffected by the mutation involving PCS in nanomelic cartilage. Ultrastructural Nanomelic

Studies Cartilage

on Normal

and

Chondrocytes in the normal sternum are polygonal with their periphery scalloped owing to numerous cellular processes (Fig.

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4). The nuclei of these cells are large, occupying a major portion of the total cellular volume. They display prominent nucleoli and densely staining chromatin. Particularly distinctive in normal cartilage is the large amount of extracellular matrix that separates the cells. The matrix contains numerous electron-dense granules and a mesh of fine, intersecting fibrils. The granules, based upon their sensitivity to trypsin, hyaluronidase, and guanidineHCl extraction (Matukas et al., 1967; Anderson and Sajdera, 1970; Anderson et al., 19701, are considered to be representations of PCS in the cartilage matrix. The fibrils, although lacking the distinctive periodicity, are thought to be collagen, based upon their strong affinity for the binding of phosphotungstic acid (Anderson et al., 1970). The extracellular matrix of the embryonic sternum is typical of cartilage from many sources. No differences from normal are observed in the general morphology of the nanomelic chondrocytes (Fig. 5); however, there is a significant reduction in the amount of extracellular space. At scan magnification there is a 50-60% increase in the number of chondrocytes observed per unit area. This finding is in agreement with the observation that there is no difference between normal and mutant sterna in either cell number or DNA content, while the mutant sterna are approximately twothirds the size of normal. The most prominent features of the mutant matrix are the dense mesh of collagen fibrils (Fig. 5) and the severe reduction in the number of matrix granules. Although some granules are present, they are obscured at this magnification by the fibrils.

Normal

and Nanomelic

Cartilage

Both normal and nanomelic chondrocytes display all the structural features of cartilage cells actively synthesizing and exporting matrix constituents (Figs. 6 and 7). They have well-developed rough endoplasmic reticulum and large Golgi complexes. Numerous mitochondria are also observed. At high magnification, the architecture of the normal matrix is clearly resolved (Fig. 8). The matrix granules are variable in shape, appearing either rounded or spindle-shaped. The granules also have fine processes, which in some cases extend into very thin filaments. The granules are generally associated with collagen fibrils; however, this association is random with no pattern to the granules along the fibrils. The mutant extracellular matrix differs from the normal matrix in two important aspects (Fig. 9). The collagen fibers are the prominent feature of this matrix. They form a dense mesh, criss-crossing in every direction. The other important difference is that the mutant matrix is almost completely devoid of any electron dense matrix granules. While the mutant extracellular matrix differs from normal in the density of the collagen fibrils and the reduction in PCS granules, there are several important similarities with respect to the collagen. There is no difference in the diameter of fibrils as compared to normal, and the fibrils in the mutant fail to display periodicity. The collagen fibrils in both normal and mutant cartilage range from 150-200 A in diameter. This suggests that the decrease in extracellular PCS has had little effect on collagen fibril formation. DISCUSSION

TABLE LYSINE

Type

AND HYDROXYLYSINE IN COLLAGEN NORMAL AND NANOMELIC STERNA

of sternum

Normal Nanomelic

1 FROM

Lysine (dpm)

Hydroxylysine (dpm)

Lysine hydroxyl. ated (o/c)

320,091 116,505

384,027 158,164

54.5 57.6

An understanding of the molecular basis for the increase in PCS synthesis during chondrogenesis is required for the eventual understanding of the means by which this developmental event is regulated. The observation that the PCS synthesized by embryonic chick chondrocytes in culture is

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FIG. 4. Sternal cartilage of normal chick embryos matrix (EM) are the electron-dense, proteochondroitin of collagen fibrils ( x 9350).

chromatographically heterogeneous and that one chromatographic fraction is affected by inhibitors of chondrogenic expression suggested the possibility that the affected fraction might represent a cartilage specific species of PCS (Palmoski

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at 14 days of age. Most prominent sulfate granules. Also observable

in the extracellular is the fine network

and Goetinck, 1972; Levitt and Dorfman, 1973). However, analysis of embryonic chick limb-bud cells, cultured under conditions in which chondrogenic expression takes place, suggested that the cartilagespecific event might not be the appearance

PENNYPACKER

AND GOETINCK

of a new species of PCS but rather the preferential increase of a preexisting speties (Goetinck et al., 1974). Low levels of the cartilage-related fraction were found in these cultures prior to the onset of chondrogenesis. Furthermore, this fraction was also identified in intact limb buds before

FIG. 5. Sternal cartilage of nanomelic chick embryos mutant extracellular matrix (EM) is the dense network extracellular space ( x 9350).

Normal

and Nanomelic

Cartilage

41

any indication of cartilage expression is evident (stages 18-19). The presence of small quantities of the cartilage-related PCS fraction in precartilaginous tissues supports a model for chondrogenesis in which there is a preferential increase in a preexisting species of PCS. However, the

at 14 days of age. The most prominent feature of the of collagen fibrils. Note particularly the reduction in

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FIG. 6. Sternal cartilage of normal chick embryos at 14 days of age. Normal developed rough endoplasmic reticulum (R) and Golgi complexes (G). The portion of the intracellular volume (X 13,930).

possibility that the small amount synthesized prior to overt chondrogenesis is the product of a few precociously differentiated cells and not the entire population makes this conclusion less than absolute. In this study PCS synthesized by normal and nanomelic sternal cartilage was analyzed by molecular sieve chromatography, and it was observed that the mutant cartilage synthesized only small quantities of the fraction which represented 90% of the material extracted from normal cartilage. The amount synthesized was approximately equivalent to that synthesized by precartilaginous mesenchyme (Goetinck et al., 1974). These observations make it unlikely that the mutation affects the appearance of a new species of PCS. Rather, it is more reasonable to assume that at the time of onset of chondrogenesis, the mutant is unable to regulate the preferential increase of the syntheis of PCS.

chondrocytes nucleus (N)

have both welloccupies a major

Previously, it was reported that PCS I could not be detected in nanomelic cultures. In that study (Palmoski and Goetinck, 1972) only the material in the medium was examined because under these conditions of culture (Falcon plates No. 10071, 90-95% PCS was found in the medium. The cell associated fraction was not examined. In tissue-culture dishes (Falcon No. 3002) in which the majority of PCS remains associated with the cells, PCS I is also absent in the medium of nanomelic cultures but it is present in small amounts in association with the cells. /3-n-Xylosides stimulate the synthesis of chondroitin sulfate (CS) in cell cultures of precartilaginous mesenchyme and chondrocytes inhibited by BrdUrd (Schwartz et al., 1974). These results have been interpreted to mean that precartilaginous mesenchyme possesses the enzymatic machinery to synthesize CS prior to overt chon-

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drogenesis and that the inability to synthesize PCS is due to limiting amounts of protein backbone that acts as a receptor for the glycosyltransferase system (Levitt and Dorfman, 1974). The mutant synthesizes normal amounts of PCS II, the glycosaminoglycan of which is indistinguishable from normal PCS II (Palmoski et al., in preparation). This would suggest that the glycosyltransferase system is unaffected by the mutation. Furthermore, studies currently in progress indicate that /3-nxylosides stimulate CS synthesis in the mutant to levels similar to those found in stimulated normal cells (Stearns and Goetinck, unpublished observations). The data presently available thus suggest that the absence of increased levels of PCS I in nanomelic cartilage could result from a failure of PCS I protein backbone to become available in increased quantities at the time of chondrogenesis. Identification of the type of collagen syn-

FIG. 7. Sternal prominent rough

cartilage of nanomelic endoplasmic reticulum

Normal

and Nanomelic

Cartilage

43

thesized by the nanomelic cartilage was accomplished by CMC chromatography and determination of the percentage hydroxylation of lysine residues. Both procedures suggested that only cartilage-type collagen is being synthesized. It therefore appears that collagen synthesis is unaffected by the mutation involving PCS I regulations and implies that the mutation does not involve a regulatory gene, which might control overall expression of the cartilage phenotype. Previous investigations have considered the possibility that the synthesis of collagen and PCS might be coregulated (Bhatnagar and Prockop, 1966; Rokosova-Cmuchalova and Bentley, 1968). Impetus for these investigations came from the observation that the synthesis of these two macromolecules is simultaneously affected when chondrogenic expression is altered in vitro (Prockop et al., 1964). To determine whether these two major constitu-

chick embryos at 14 days of age. Mutant chondrocytes also display CR), Golgi complexes (G), and a large nucleus (N) (X 13,930).

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FIG. granules granules

DEVELOPMENTAL

BIOLOGY

VOLUME

8. Sternal cartilage of normal chick embryos at 14 days are rounded or spindle-shaped (arrow). The collagen fibrils are usually in association with the fibrils. (x 100,000).

ents of the matrix could be synthesized independently of one another, the synthesis of one was selectively inhibited. It was found that inhibition of PCS by 6-diazo-5 oxonorleucine, an analog of glutamine, does not affect the synthesis of collagen and, conversely, the synthesis of PCS was not affected by cr-a’-dipyridyl, a chelator of ferrous ions which inhibits collagen synthesis. On the basis of these results it was concluded that the regulation of these two macromolecules need not be inter-r-related (Bhatnagar and Prockop, 1966). It is important to point out, however, that the above investigation was carried out prior to the discovery of tissue-specific collagens and, therefore, the possibility of a transition from [(rl(II)l, to [al(I)l,~2 was not considered. More recent studies on the effect of BrdUrd and H-fraction could be interpreted to mean that collagen and PCS

50,

1976

of age. (F) lack

The proteochondroitin any detectable periodicity.

sulfate The

synthesis are coordinated since the synthetic pattern of both macromolecules is altered with the treatments (Schiltz et al., 1973). However, under the experimental conditions used, the chondrocytes are altered from their characteristic polygonal morphology to a fibroblastic form. In the cartilage of the nanomelic mutant, where cellular morphology is not affected, collagen synthesis was found to be both quantitatively and qualitatively normal. Since only PCS is affected in the mutant, this provides more conclusive evidence for the independence of collagen and PCS synthesis. The fine structure of the nanomelic chondrocytes is very similar, if not identical, to normal chondrocytes. The mutant cells have the same polygonal shape, large nuclei, and prominent rough endoplasmic reticulum and Golgi complexes. Signifi-

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cant differences were observed only in the extracellular matrix of the mutant cartilage. While the normal matrix is composed of numerous electron dense granules and a fine network of intersecting tibrils, the PCS granules are almost entirely absent in the mutant. Furthermore, the collagen fibrils are much more densely packed in the matrix and are, therefore, more prominent. Both observations are consistent with what is known from biochemical studies on the mutant. Because collagen concentration in the mutant is equivalent to normal, this suggests that the reduced extracellular space has resulted in a greater density of fibrils. However, the individual fibrils are similar in their ultrastructure and diameter to normal cartilage collagen fibrils. They lack the 640 A periodicity and range from 150-200 A in diameter. On this basis it is suggested that the reduction in PCS has not influenced

FIG. 9. Sternal the collagen fibrils

Normal

and Nanomelic

45

Cartilage

the assembly of the fibrils. Previous studies that suggested a relationship between PCS and collagen fibril formation were of two types. One approach was to correlate an observed fibril pattern with the presence of a particular type of proteoglycan in different tissues (Jackson and Bentley, 1968). The precipitation of collagen from salt solutions provided a second means of studying the effects of proteoglycans on fibril formation (Wood, 1960; Toole and Lowther, 1968; Mathews and Decker, 1968; Mathews, 1965). However, a fundamental assumption in both approaches was that the collagen molecule in various tissues is identical. As this is now known not to be the case, one must consider the possibility that differences in the fibril size and structure might reflect differences in the type of collagen involved. While the observations on the nanomelic mutant do not rule out the possibility that

cartilage of nanomelic chick embryos at 14 days of age. There (F) of the mutant, as compared to normal (X 100,000).

is no apparent

difference

in

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BIOLOGY

other factors might affect the formation of cartilage-type collagen fibrils, the fact that the reduction in PCS did not affect fibril formation suggests that the formation of small diameter fibrils might be intrinsic to [al(I collagen. Mutant mice homozygous for the autosoma1 recessive gene chondrodysplasia (cho) have large collagen fibrils, with 640 A periodicity, in the matrix of their cartilage. It was originally suggested (Seegmiller et al., 1971, 19721, based on histochemical techniques, that the defect in these mice involved PCS and that this change resulted in the formation of abnormal collagen fibrils. However, recent biochemical studies indicate that PCS in the cartilage of the cholcho mice is unaltered (Seegmiller, personal communication). Therefore, as an alternative explanation one has to consider the possibility that collagen metabolism in the cholcho cartilage is defective. If this is the case, as preliminary evidence suggests (Seegmiller, personal communication), it would further support the conclusion, drawn from the present study on nanomelic cartilage, that the structure and dimension of collagen fibers are at least partially dependent upon the type of collagen molecules involved. An important feature of the sternal cartilage of the nanomelic embryo is its similarity to normal cartilage. It has been observed previously that addition of exogenous PCS to cultured chondrocytes results in the stimulation of further PCS synthesis (Nevo and Dorfman, 1972) and that exogenous PCS can substitute effectively for notochord in promoting somite chondrogenesis (Kosher et al., 1973). On this basis, it has been suggested that the matrix proteoglycans may be involved in the induction or maintenance of chondrogenic expression. While this may be the case, the results of this study suggest that active synthesis of PCS is not required for overt chondrogenic expression. Despite the severe reduction in PCS levels, the fine structure of the mutant chondrocytes is

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indistinguishable from normal and the cells are capable of synthesizing cartilage related products. Therefore, it appears that while the altered matrix of the mutant cartilage affects the longitudinal growth of cartilaginous limb rudiments, it has had no other obvious effect on the cartilage phenotype. This work was supported by Grant HD 09174 from the NICHHD and by Training Grant 5-TOl-GM00317 to the first author from the NIGMS. This work was also supported by NC1 CA 14733. The authors are grateful to Dr. A. Wachtel for his assistance with the ultrastructural studies. This paper is Scientific Contribution No. 630 of the Storrs Agricultural Experiment Station, the University of Connecticut. REFERENCES ANDERSON, H. C., CHACKO, S., ABBOTT, J., and HOLTZER, H. (1970). The loss of phenotype traits by differentiated cells in uitro. Amer. J. Path. 60, 289-311. ANDERSON, H. C., and SAJDERA, S. (19701. Extraction as a technique for the electron microscopic study of protein-polysaccharides and collagen in cartilage matrix. Fed. Proc. 49, 554. BHATNAGAR, R. S., and PROCKOP, D. J. (1966). Dissociation of the synthesis of sulphated mucopolysaccharides and the synthesis of collagen in embryonic cartilage. Biochim. Biophys. Actu. 130, 383392. FRASER, R. A., and GOETINCK, P. F. (19711. Reduced synthesis of chondroitin sulfate by cartilage from the mutant, nanomelia. Biochem. Biophys. Res. Comm. 43, 494-503. GOETINCK, P. F., PENNYPACKER, J. P., and ROYAL, P. D. (1974). Proteochondroitin sulfate synthesis and chondrogenic expression. Exp. Cell Res. 87, 241-248. HASCALL, V., and HEINEG~RD, D. (1974a). Aggregation of cartilage proteoglycans. I. The role of hyaluronic acid. J. Biol. Chem. 249, 4232-4241. HASCALL, V., and HEINEG~RD, D. (1974bl. Aggregation of cartilage proteoglycan. II. Oligosaccharide competitors of the proteoglycanhyaluronic acid interaction. J. Biol. Chem. 249, 4242-4249. HEINEGARD, D., and HASCALL, V. (1974). Aggregation of cartilage proteoglycan. III. Characteristics of the proteins isolated from trypsin digests of aggregates. J. Biol. Chem. 249, 4250-4256. JACKSON, D. S., and BENTLEY, J. P. (19681. Collagenglycosaminoglycan interactions. In “Treatise on Collagen” (B. S. Gould, ed.), pp. 189-214. Academic Press, New York. KANG, A. H., NAGAI, Y., PIEZ, K. A., and GROSS, J.

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(1966). Studies on the structure of collagen utilizing a collagenolytic enzyme from tadpole. Biochemistry 5, 509-515. KOSHER, R. A., LASH, J. W., and MINOR, R. R. (19’73). Environmental enhancement of in vitro chondrogenesis. IV. Stimulation of somite chondrogenesis by exogenous chondromucoprotein. Develop. Biol. 35, 210-220. LANDAUER, W. (1965). Nanomelia, a lethal mutation of the fowl. J. Heredity 56, 131-138. LINSENMAYER, T. F., TOOLE, B. P., and TRELSTAD, R. L. (1973). Temporal and spatial transitions in collagen types during embryonic chick limb development. Develop. Biol. 35, 232-239. LEVITT, D., and DORFMAN, A. (1973). Control of chondrogenesis in limb-bud cultures by bromodeoxyuridine. Proc. Nat. Acad. Sci. USA 70, 22012205. LEVITT, D., and DORFMAN, A. (1974). Concepts and mechanisms of cartilage differentiation. In “Current Topics of Developmental Biology” (A. A. Moscona and A. Monroy, eds.), Vol. 8, pp. 103149. Academic Press, New York. MATHEWS, M. B. (1965). The interaction of collagen and acid mucopolysaccharides. Biochem. J. 96, 710-716. MATHEWS, M. B. (1967). Chondroitin sulfate and collagen in inherited skeletal defects of chickens. Nature (London) 213, 1255-1256. MATHEWS, M. B., and DECKER, L. (1967). The effect of acid mucopolysaccharides and acid mucopolysaccharide-proteins on flbril formation from collagen solutions. Biochem. J. 109, 517-526. MATUKAS, V. J., PANNER, B. J., and ORBISON, J. L. (1967). Studies on ultrastructural identification and distribution of protein-polysaccharide in cartilage matrix. J. Cell Biol. 32, 365-377. MILLER, R. L. (1972). Rapid assay for lysyl-protocollagen hydroxylast activity. Anal. Biochem. 45, 202-210. NEVO, Z., and DORFMAN, A. (1972). Stimulation of chondromucoprotein synthesis in chondrocytes by extracellular chondromucoprotein. Proc. Nat. Acad. Sci. USA 69, 2069-2072.

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