Sulfated mucopolysaccharide synthesis during the development of Rana pipiens

DEVELOPMENTAL

Sulfated

BIOLOGY

32,50-68

(1973)

Mucopolysaccharide

Synthesis

of Rana pipiens’? ROBERT

during

the Development

’

A. KOSHER~ AND L. SEARLS

ROBERT Department

of Biology,

Temple University, Accepted

November

Kiladelphia,

Pennsylvania

19122

3, 1972

Sulfated mucopolysaccharide (MPS) synthesis during the development of Rano pipiens was studied autoradiographically and biochemically following injection of embryos with 35S-sulfate. a5S-sulfate incorporation can be detected in unfertilized and fertilized eggs. The sulfate-incorporating material accumulates along the periphery of yolk platelets of eggs. During cleavage, the 35S-sulfate-incorporating material accumulates on cell surfaces as well as along the periphery of yolk platelets. Biochemical analysis utilizing the enzymes chondroitinase ABC and AC and nitrous acid degradation indicates that the MPS synthesized during cleavage is approximately 82% heparin and/or heparan sulfate and 18% chondroitin 4-sulfate. During gastrulation, a greatly enhanced incorporation of Y+sulfate is observed in the invaginating chordamesoderm and lateroventral mesoderm, and by the end of gastrulation enhanced incorporation can be detected in neural tissue. During this period, chondroitin B-sulfate synthesis is initiated. Incorporation of %-sulfate is observed in all tissues of the embryo from the beginning of neurulation through hatching. This ubiquitous incorporation is accompanied by an increase in the relative amount of chondroitin 6-sulfate synthesized. During the period following hatching, incorporation is suppressed in some tissues and enhanced in others so that by the late feeding tadpole stage a very high incorporation is observed only in cartilaginous tissue. These results indicate that sulfated MPS synthesis occurs in all stages of development of Rana pipiens, but that significant changes in the rate of synthesis occur in various cell types during gastrulation and after hatching. The ubiquity of sulfated MPS synthesis during the critical early stages of development and the changes in the pattern of synthesis in various cell types suggest that these molecules are involved in a number of embryonic processes.

Abbott and Holtzer, 1966; Nameroff and Holtzer, 1967; Cal-m and Lasher, 1967). However, it has been demonstrated that prechondrogenic cells synthesize chondroitin sulfate long before they form morphologically distinguishable matrix (although an enhancement of synthesis occurs concomitant with overt chondrogenesis) (France-Browder et al., 1963; Searls, 1965a,b; Medoff, 1967; Marzullo and Lash, 1967; Lash, 1968a-c; Ellison and Lash, 1971). Furthermore, a large number of embryonic cells that are not precursors of cartilage cells synthesize sulfated MPS including chondroitin sulfate during early stages of development (Marzullo and Lash, 1967; Lash, 1968a-c; Kvist and Finnegan. 1970a,b; Manasek, 1970). Incorporation of TS-sulfate suggesting sulfated MPS syn-

INTRODUCTION

Sulfated mucopolysaccharides (MPS) often have been considered to be specific products of connective tissue cells. In particular, the synthesis of the sulfated MPS chondroitin sulfate has been used as evidence of cartilage function in studies in ouo (e.g., Amprino, 1955; Searls, 1965a,b), in organ culture (e.g., Holtzer, 1961; Lash et al., 1960), and in cell culture (e.g., ’ This material was included in a dissertation submitted by RAK to the Temple University Graduate Board in partial fulfillment of the requirements for the degree of Doctor of Philosophy. 2Supported by National Aeronautics and Space Administration Grant NGT39-012-007 and Temple University Predoctoral Fellowship to RAK. 3 Present address: Department of Anatomy, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania 19104. Copyright All rights

0 1973 by Academic Press. Inc. of reproduction in any form reserved.

50

KOSHER AND SEARLS

Mucopolysaccharide

thesis has been observed in chick embryos as early as stage 3+ in the primitive streak and the floor of the primitive groove (Johnston and Comar, 1957), and chondroitin sulfate synthesis has been detected biochemically in chick embryos as early as stage 11 (France-Browder et al., 1963). Thus, sulfated MPS synthesis starts very early in embryonic development, and occurs in precursor cells to a large number of different cell types. The widespread synthesis of these molecules suggests that they may be functionally important during embryonic development. In the present study, sulfated MPS synthesis during the development of the frog Rana pipiens was investigated autoradiographically and biochemically following injection of embryos at all stages of development from the unfertilized egg to the feeding tadpole stage with 35S-sulfate. This study was undertaken to determine (1) how early in development sulfated MPS synthesis can be detected, (2) what changes in rates of sulfated MPS synthesis occur during embryonic development in various regions of the embryo, (3) whether these changes in the rates of synthesis are accompanied by alterations in the types or relative amounts of sulfated MPS synthesized, or can be correlated with significant developmental events, and (4) whether the pattern of sulfated MPS synthesis at various stages and in various cell types will give some insight into the functional significance of these molecules. We have found that sulfated MPS including chondroitin sulfate are synthesized at all stages of development of Rana pipiens embryos, but that significant changes in the rate of synthesis in various cell types occur during gastrulation and after hatching. MATERIALS

AND

METHODS

Mature male and female Rana pipiens collected in the spring and fall immediately before or after hibernation were purchased from J. M. Hazen and Co., Alburg, Vermont. Frogs were maintained at 4°C in

Synthesis

during

Development

51

spring water (Great Bear, Philadelphia, Pennsylvania) or dechlorinated tap water (DiBerardino, 1967) until used. Ovulation was induced in mature female frogs, and eggs were artificially inseminated according to methods outlined by DiBerardino (1967). Embryos were reared at 1%20°C in groups of 20-30 in finger bowls filled with spring water or dechlorinated tap water. The embryos were staged according to Shumway (1940, 1942). Embryos at all 25 Shumway stages of development (from the unfertilized egg through feeding tadpole stages) were injected with approximately 0.02 &i of 35S-sulfate (0.04 ~1 of HZ3%0,, 5 mCi/ml, carrier free; New England Nuclear Corp., Boston, Massachusetts) by the procedure of King (1967). The isotope was injected into the animal hemisphere of eggs and cleaving embryos, into the blastocoel or archenteron of blastulae and gastrulae, and into the gut of older embryos. Injection of the isotope had no observable effect on normal embryonic development. Control embryos injected at stage 2 (fertilized eggs) developed normally at least through stage 25 (feeding tadpoles). Autoradiography. Embryos injected with 35S-sulfate were allowed to develop for 2 hr at 18-20°C in finger bowls. The jelly of the embryos was then removed manually, and the embryos were fixed in Smith’s fluid for 2 hr. Embryos were dehydrated with a graded series of ethyl alcohols, cleared in amyl acetate, embedded in 56-57°C paraplast, and sectioned at 5 and occasionall> 10 pm. The paraplast was removed with xylene, and the sections were rehydrated to water with a graded series of ethyl alcohols. So that pigment granules would not subsequently be mistaken for silver grains. sections were bleached in a solution of potassium chlorate containing HCl according to the method of Brunet and Small (1959). The sections were stained by the Feulgen reaction following an 11-min hydrolysis in 1 N HCl. The slides were then dipped in Kodak NTB-3 nuclear track

52

DEVELOPMENTAL BIOLOGY

VOLUME 32, 1973

emulsion (diluted 1: 1 with water), ex- The samples were then dialyzed for 2 days posed in the dark at 4°C for 6 weeks in the against 4% sodium sulfate and for 2 days presence of Drierite, Developed in Kodak against distilled water. The dialyzates D-19, and fixed with Kodak Rapid Fixer were supplied with 1 mg each of carrier (Caro and van Tubergen, 1962). The auto- chondroitin 4- and 6-sulfate (Miles Laboraradiographs were dehydrated with a tories Inc., Kankakee, Illinois), made 1.0 graded series of ethyl alcohols, lightly M with respect to potassium acetate bufcounterstained with Fast Green, cleared fer, pH 4.5, and MPS were precipitated by in xylene, and mounted with histoclad. the addition of 3 volumes of absolute ethExtraction of 35S-sulfate-labeled MPS. anol. The precipitates that formed after Fertilized eggs (stage 2), late gastrulae 24 hr at 4°C were collected by centrifuga(stage 12), late neurulae (stage 16), and tion, washed once with 75% ethanol, twice feeding tadpoles (stage 25) were injected with absolute ethanol, dried in a desicwith approximately 0.04 ~1 HZ3”S0, cator with P205, and dissolved in distilled (5 mCi/ml, carrier free; New England Nu- water. Enzymatic analysis. The relative clear Corp., Boston, Massachusetts) as previously described. Approximately 50 amounts of labeled chondroitin 4-sulfate, embryos of each stage were injected. The chondroitin g-sulfate, and dermatan sulembryos injected at stages 2, 12, and 16 fate extracted from each stage were deterwere allowed to develop through stages 8, mined by the enzymatic method of Saito 14, and 17, respectively, at temperatures et al. (1968) using chondroitinase ABC and between 18” and 23°C that enabled them chondroitinase AC (Miles Laboratories to attain the appropriate stage in approxi- Inc., Kankakee, Illinois) (Yamagata et al., mately 24 hr. The embryos injected at 1968). Aliquots (20 ~1) of the MPS samples stage 25 were exposed to the isotope for 24 of each stage were incubated at 37°C for hr at room temperature. When embryos 30 min to 3 hr with either 0.1 unit chonhad attained the appropriate stage, their droitinase ABC or 0.3 unit chondroitinase jelly was removed, and they were usually AC in the presence of 10 ~1 of enriched frozen at - 20°C prior to analysis. Tris buffer, pH 8.0 (prepared as described The injected embryos and an equal num- by Saito et al., 1968). Control samples were ber of equivalent-aged unlabeled embryos, incubated with buffer alone. The reaction added to supply carrier MPS, were thor- mixtures were applied in a stream of hot oughly disrupted in 1.0-1.5 ml of distilled air to Whatman No. 1 filter paper (46 x 57 cm) along with 0.1 pmole each of two unwater with a Sonifier Cell Disruptor, Model ~1850 (Heat Systems-Ultrasonics, saturated disaccharide standards, ADi-4S Inc., Plainview, New York). The sonicates and ADi-6S (Miles Laboratories Inc., Kanwere made 0.1 M with respect to sodium kakee, Illinois). 4 Descending chromatogacetate, pH 5.0, 0.005 M with respect to raphy was carried out at room temperature L-cysteine-HCl (General Biochemicals, overnight in l-butanol-acetic acid-l N amChagrin Falls, Ohio), 0.005 M with respect monia (2 : 3 : 1, v/v). After chromatography to ethylenediaminetetraacetic acid, and ’ The ADi-4S is 2-acetamido-2-deoxy-3-0-(&n supplied with 2.9 mg/ml papain (twice gluco-4-enepyranosyluronic acid)-4-O-sulfo-ngalaccrystallized; Worthington, Freehold, New tose, and is the product of chondroitinase ABC and Jersey). Digestion was carried out for 24 hr AC digestion of chondroitin 4-sulfate and of chonat 63°C. The papain digests were then droitinase ABC digestion of dermatan sulfate. The is 2-acetamido-2-deoxy-3-0-(P-n-gluco-4made 0.5 M with respect to NaOH. After ADi-6S enepyranosyluronic acid)-6-O-sulfo-n-galactose, and exposure to NaOH for 4 hr at 4°C the is the product of chondroitinase ABC and AC digesNaOH was neutralized with acetic acid. tion of chondroitin 6-sulfate.

KOSHER AND SEARLS

Mucopolysaccharide

the disaccharides could be visualized with ultraviolet light. The distribution of radioactivity along the chromatographs was determined by cutting strips of the chromatographs into O.&inch sections, and placing each section in a scintillation vial containing 6 ml of scintillation fluid (Saito et al., 1968). Radioactivity was determined with a Packard Tri-Carb liquid scintillation spectrometer. The amount of radioactivity associated with the 6-sulfated disaccharide (ADi-6s) was used as a measure of labeled chondroitin g-sulfate. The difference in radioactivity in the 4-sulfated disaccharide region (ADi-4s) after treatment with each enzyme was used to determine the relative amount of labeled chondroitin 4-sulfate and dermatan sulfate, since chondroitinase ABC will degrade chondroitin 4-sulfate and dermatan sulfate, while chondroitinase AC will not degrade dermatan sulfate into disaccharide residues. Nitrous acid degradation. Aliquots of the MPS samples of each stage were subjected to nitrous acid degradation, since HNOz will specifically degrade the N-sulfated MPS, heparin and heparan sulfate (Lagunoff and Warren, 1962; Cifonelli, 1968a,b; Lindahl, 1970; Kraemer, 1971a,b). Nitrous acid degradation was carried out as described by Dische and Borenfreund (1950). To 0.25 ml of the MPS samples, 0.25 ml of 5% sodium nitrite and 0.25 ml of 33% acetic acid were added. The reaction mixtures were shaken, stoppered, and allowed to remain at room temperature for 90 min. Undegraded MPS was then precipitated by the addition of 5 mg of cetylpyridinium chloride (Scott, 1960; Bates and Levene, 1971; Toole and Gross, 1971). The precipitates were collected by centrifugation and washed with distilled water. Aliquots of the supernatants (HNO ,-sensitive material) were dried in scintillation vials and supplied with scintillation fluid, and radioactivity was determined. To dissociate the water-insoluble cetylpyridinium-MPS complexes, the pre-

Synthesis

during Development

53

cipitates (HNO ,-resistant material) were agitated in saturated ethanolic KCNS (Scott, 1960). The precipitates were washed with 95% ethanol, dried in a desiccator with P,O,, dissolved in distilled water, and radioactivity determined as previously described. In this manner, the amount of HNO,-sensitive material (material soluble in CPC following HNO, treatment, i.e., heparin and/or heparan sulfate) was compared with the amount of HNO *-resistant material (material precipitable with CPC following HNO, treatment). In addition, the HNO,-resistant material was analyzed with chondroitinase ABC as previously described. The presence of keratan sulfate would have been indicated by the presence of material resistant to both chondroitinase ABC and HNOZ. RESULTS

Autoradiographic

Analysis

Embryos were exposed to 35S-sulfate for 2 hr at 18-20°C prior to fixation and subsequent autoradiography. Between 5 and 20 embryos of each stage were subjected to autoradiographic analysis. Incorporation was judged subjectively for the most part. Grain counts were made only when incorporation was not clearly in excess of background. No silver grains in excess of background were observed in autoradiographs of embryos of various stages that had not been injected with 35S-sulfate or that had been fixed immediately after having been injected with the isotope. In addition, in a series of preliminary experiments we found that the number of silver grains were dependent upon the amount of YS-sulfate injected, the length of exposure, and the thickness of the section. The pattern of ?S-sulfate incorporation observed in autoradiographs of unfertilized eggs activated by injection of the isotope and of fertilized eggs was identical. Silver grains were distributed through-

54

DEVELOPMENTALBIOLOGY

FIGS. l-4

VOLUME 32, 1973

KOSHER

AND SEARLS

Mucopolysaccharide

out the eggs, but significantly more radioactive sulfate was incorporated in the vegetal hemisphere than in the animal hemisphere (Fig. la). Virtually all silver grains were found along the periphery of yolk platelets, and often long strings of silver grains were seen, particularly in the vegetal hemisphere, which almost completely surrounded yolk platelets (Figs. lb,c). During cleavage (stages 3 through 7), silver grains were observed not only intracellularly, but also along the outer surfaces of cells (Fig. 2). The intracellular pattern of YS-sulfate incorporation observed in cleaving embryos was very similar to that observed in eggs. All blastomeres incorporated 3”S-sulfate; silver grains were distributed throughout the blastomeres, but were more concentrated in the larger macromeres in the vegetal regions of the embryos than in the smaller macromeres in the animal regions (Fig. 2) ; and, virtually all intracellular silver grains were found along the periphery of yolk platelets. Silver grains were observed along the surfaces of cells in both the animal hemisphere and the vegetal hemisphere. At the blastula stage (stages 8 and 9), 35S-sulfate incorporation was observed throughout the cells of the vegetal hemisphere below the blastocoel (Fig. 3), and a somewhat lower but nevertheless significant incorporation was

Synthesis

during Development

55

observed in the equatorial marginal zones. In these regions, strings of silver grains were seen which appeared to be along the periphery of yolk platelets. In contrast, there was little incorporation in the cells comprising the roof and sides of the blastocoel (Fig. 3). The small amount of 35Ssulfate incorporated by the blastocoel roof cells could be detected only by counting silver grains. Therefore, it could not be determined whether the silver grains were localized at the surfaces of cells or intracellularly. During the early stages of gastrulation (stage 10 and early stage ll), the dorsal lip of the blastopore appeared as a slight indentation or notch slightly below the equator of the embryo. During these stages, the pattern of incorporation was virtually identical with the pattern observed at the blastula stage in that only the cells of the yolky presumptive endoderm below the blastocoel showed extensive incorporation of 35S-sulfate, and the incorporation was almost exclusively peripheral to yolk platelets. During the period of gastrulation when the chordamesoderm had invaginated significantly but not completely and a slitlike archenteron and large yolk plug were visible (late stage 11 to early stage 12), substantial incorporation of 3”S-sulfate was observed in the cells of the invaginated chordameso-

FIG. 1. (a) Section through an egg, which was exposed to YS-sulfate for 2 hr, photographed in dark field. The area shown is about at the equator between the animal (A) and vegetal (V) hemispheres. Note the higher concentration of silver grains in the vegetal hemisphere. x 250. (b) An area in the vegetal hemisphere of the section shown in (a). Note the strings of silver grains along the periphery of yolk platelets. x 250. (c) An enlargement of the yolk platelets indicated by arrows in (b) demonstrating the accumulation of silver grains along the periphery of yolk platelets. x 700. FIG. 2. Section through several cells of a cleaving embryo. Intracellular incorporation is higher in the macromeres in the vegetal hemisphere than in the micromeres in the animal hemisphere, and intracellular silver grains are along the periphery of yolk platelets. Note the accumulation of silver grains along cell surfaces. Exposure to 35S-sulfate was for 2 hr. (a) Dark field. (b) Bright light. x 120. FIG. 3. Section through a blastula. There is little incorporation of 9-sulfate in the cells of the roof of the blastocoel, and the high incorporation in the cells below the blastocoel is almost exclusively peripheral to yolk platelets. Exposure to 36S-sulfate was for 2 hr. (a) Dark field. (b) Bright light. x 120. FIG. 4. Section through a gastrula (early stage 12) demonstrating incorporation of 3”S-sulfate in the invaginated chordamesoderm. Note that there is little incorporation in the presumptive chordamesoderm that has not yet invaginated. The silver grains in the presumptive endoderm are along the periphery of yolk platelets. (a) Dark field. x 200. (b) Bright light. x 220.

56

DEVELOPMENTAL BIOLOGY

derm (Fig. 4). Little incorporation was observed in the presumptive chordamesoderm that had not yet invaginated or in the presumptive neural ectoderm. At the ventral lip of the blastopore, incorporation of 35S-sulfate was observed in the invaginated lateroventral mesoderm. At late stage 12, when invagination of the chordamesoderm was essentially complete and the chordamesoderm had completed contact with the presumptive neural tissue, incorporation was observed throughout the neural tissue as well as throughout the underlying invaginated chordamesoderm (Fig. 5). Little incorporation was observed throughout the neural tissue until gastrulation was virtually complete. Throughout gastrulation, as at all earlier stages, the yolky endoderm incorporated 35S-sulfate, and the silver grains in the endoderm appeared to be almost exclusively along the periphery of yolk platelets (Figs. 4 and 5). At stage 13, when the neural ectoderm had thickened to form the neural plate, the pattern of 35S-sulfate incorporation was identical to the pattern observed at late stage 12. By stages 14 and 15 lateral neural folds were present, and by stage 16 the lateral neural folds had met to form the neural tube. Throughout neurulation (Figs. 6 and 7), a uniform high incorporation was observed throughout the neural tube, the neural crests, notochord, somitic mesoderm, intermediate mesoderm, lateral plate mesoderm, and epidermis. The yolky endoderm and cells surrounding the gut incorporated 35S-sulfate, and the silver grains appeared to be associated with the yolk platelets of the endoderm.

VOLUME 32, 1973

The pattern of incorporation observed throughout neurulation persisted with only slight modification during stages 17 through 19 (the tail bud stage through hatching). During stages 17 through 19 (Figs. 8 and 9), a uniform incorporation of 35S-sulfate was observed in the neural tube and neural crests, although incorporation was somewhat lower than at previous stages. A uniform incorporation was also observed throughout the somites of tail bud embryos. By stage 19, the somites consisted of a loose inner layer of sclerotoma1 cells that clustered around the neural tube and notochord, a large myotome, and an outer layer of dermatomal cells. Incorporation was observed in the sclerotome and dermatome, while lower but nevertheless significant incorporation was observed in the myotome (Fig. 9). Throughout stages 17-19, the highest concentration of label was over the notochord, particularly the notochordal sheath, and over the subchorda. Incorporation was also observed in the cell-free perinotochordal space (Fig. 8). Label continued to be observed over the lateral plate mesoderm, including at stage 19 the somatic and splanchnic layers, the intermediate mesoderm, and epidermis. By stage 19, the myocardium and endocardium of the heart were distinguishable, and silver grains were distributed throughout the myocardium (Fig. 10). Throughout stages 17 and 18 the yolky endoderm incorporated 35S-sulfate, but by stage 19 incorporation in this tissue was much depressed. After hatching, during stages 20 through 22 (swimming tadpole stages), changes in

FIG. 5. Section through a late gastrula (late stage 12). Incorporation can now be detected in the presumptive neural tissue as well as in the invaginated chordamesoderm. Note also continuing incorporation in the yolky endoderm and lateroventral mesoderm. (a) Dark field. (b) Bright light. x 120. FIG. 6. Section through an early neurula. Incorporation can be observed throughout the lateral neural folds, axial mesoderm, notochord, epidermis, and endoderm. (a) Dark field. (b) Bright light. x 200. Fro 7. Section through a late neurula. The lateral neural folds have met fo form the neural tube. Incorporation can be seen throughout the neural tube, somitic mesoderm, intermediate mesoderm, notochord, and epidermis. (a) Dark field. (b) Bright light. x 220. FIG. 8. Section through a tail bud embryo (stage 17). Incorporation can be observed throughout all tissues. (a) Dark field. (b) Bright light. x 220.

KOSHER AND SEARLS

Mucopolysaccharide

FIGS. 5-8

Synthesis

during Development

57

58

DEVELOPMENTAL BIOLOGY

FIGS. 9-12

VOLUME 32, 1973

KOSHER

AND SEARLS

Mucopolysaccharide

the pattern of sulfate incorporation were evident, particularly in the axial regions of the embryo (Figs. 11 and 12). Throughout stages 20 to 22, incorporation in the neural tube was negligible in contrast to the high incorporation observed in the neural tube from the end of gastrulation through stage 19. Although incorporation continued in the sclerotome and dermatome during stages 20 to 22, there was negligible incorporation in the myotome. Silver grains were still observed over the notochord although incorporation in that region was lower than at previous stages. Similarly, although there was some incorporation by the epidermis during stages 20-22, it was much depressed from the incorporation observed during previous stages. Throughout stages 20-22, a moderate incorporation of 35S-sulfate was observed in the myocardium, pronephric tubules, liver, and lateral plate mesoderm. During stages 23 through 25 (feeding

Synthesis

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59

tadpole stages), cartilage was observed in the embryos for the first time, and in these embryos the highest incorporation of 35Ssulfate was in cartilaginous tissue. An extremely high concentration of silver grains parawas observed over sclerotomic chordal, trabecular, and visceral cartilage (Fig. 13). Throughout stages 23-25, moderate incorporation of 35S-sulfate was observed in the notochord, sclerotome, and myocardium, but incorporation in all other tissues, including the neural tube, myotome, and epidermis, was either very low or negligible. Enzymatic

Analysis

The autoradiographic analysis had indicated that significant changes occur in the pattern of 35S-sulfate incorporation during gastrulation and after hatching. The sulfated MPS that had been synthesized before and after each of these changes was extracted, and analyzed for

FIG. 13. Section through the head region of a stage 25 embryo. Note the extremely of label over cartilaginous tissue. (a) Dark field. (b) Bright light. x 120.

high concentration

FIG. 9. Section through a stage 19 embryo. There is high incorporation of 35S-sulfate by the notochord and sclerotome, and moderat e incurporation by the myotome. Incorporation by the neural tube is somewhat lower than at the previous stages. (a) Dark field. x 200. (b) Bright light. x 250. FIG. 10. Section through the heart of a stage 19 embryo demonstrating incorporation by the myocardium. (a) Dark field. (b) Bright light. x 200. FIG. 11. Section through the pronephric region of a stage 20 embryo. There is negligible incorporation by the neural tube and myotome, although there is significant incorporation by the notochord and pronephric tubules. (a) Dark field. (b) Bright light. x 200. FIG. 12. Section through the pronephric region of a stage 22 embryo. Negligihle incorporation by the neural tube and myotome, and moderate incorporation by the notochord and pronephric tubules. (a) Dark field. (b) Bright light. x 200.

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DEVELOPMENTAL BIOLOGY

ABC, while 17% was degraded by AC. This result suggested that little, if any, dermatan sulfate had been synthesized, and that the radioactivity in the ADi-4S region must represent degraded chondroitin 4-sulfate. By comparing the radioactivity in the ADi-4S region with that remaining at the origin (chondroitinase-resistant MPS), it could be concluded that chondroitin 4-sulfate represents approximately 18% of the sulfated MPS synthesized between fertilization and the blastula stage, while approximately 82% is chondroitinase-resistant material (Table 1). To determine the nature of the sulfated MPS synthesized during gastrulation, particularly during the period when the autoradiographic pattern of ?3-sulfate incorporation was changing, embryos were injected with ?3-sulfate at the beginning of stage 12 and allowed to complete gastrulation prior to extraction. After chondroitinase digestion, radioactivity could be detected in the ADi-6S region as well as in the ADi-4S region and at the origin. Therefore, during gastrulation chondroitin 6-sulfate synthesis is initiated. No significant difference in radioactivity could be detected in the ADi-4S region after treatment with each enzyme indicating the absence of dermatan sulfate synthesis (Table 1). Comparison of the radioactivity

the presence of chondroitin 4-sulfate, chondroitin 6-sulfate, and dermatan sulfate by the enzymatic method of Saito et al. (1968) using chondroitinase ABC and AC. A chondroitinase analysis of MPS extracted from embryos exposed to 35S-su1fate from fertilization to the blastula stage is given in Fig. 14. When a MPS aliquot that had not been treated with enzyme was chromatographed, all radioactivity remained at the origin. After treatment with chondroitinase ABC, a radioactive area could be detected corresponding to the 4-sulfated disaccharide standard (ADi-4s) as well as at the origin. A virtually identical chromatograph was obtained after treatment with chondroitinase AC. Since no radioactivity could be detected in the 6-sulfated disaccharide region ( ADi-6S), this result indicated the absence of chondroitin 6-sulfate synthesis. To determine whether the radioactivity in the ADi-4S region resulted from degraded chondroitin 4-sulfate or dermatan sulfate, the amount of radioactivity in this region was compared after treatment with each enzyme (Table 1). Essentially the same amount of radioactivity was present in the ADi-4S region after treatment with either chondroitinase ABC or AC: approximately 19% of the MPS was degraded by 9

CONTROL

VOLUME 32, 1973

9-

8

S-

7

7-

N 6 b x5 g4

5-

CHASE

ABC

64-

3

3-

2

12I I' -

/ 0

'@

ADi-6S

ADi-4s

FIG. 14. Paper chromatograph of untreated and chondroitinase ABC (CHase ABC)-treated MPS extracted from embryos exposed to W-sulfate from fertilization through blastula stage. 0 = origin; dotted line = solvent front. The ADi-4S and ADi-6S digestion products and the method of determining the distribution of radioactivity along the chromatographs are described in Materials and Methods.

KOSHER AND SEARLS

Mucopolysaccharide

Synthesis

61

during Development

TABLE 1 RELATIVE AMOUNT OF RADIOACTIVITY IN VARIOUS PRODUCTS AVER CHONDROITINASE(CHASE) AND NITROUS ACID TREATMENT OF MPS EXTRACTED FROM EMBRYOS EXPOSED TO 35S-S~~~~~~ AT VARIOUS STAGES OF DEVELOPMENT Approximate Sample

Products

12-14

16-17

25

Resistant” ADi-4S ADi-6S Resistant ADi-4S ADi-6S Soluble in CPCb Insoluble in CPC Resistant Sensitive

81 19 0 83 17 0 85 15 3 97

82 8 10 83 7 10 81 19 4 96

68 8 24 68 12 20 63 37 5 95

62 21 17 65 19 16 61 39 4 96

Treatment

MPS aliquot

CHase ABC

MPS aliquot

CHase AC

MPS aliquot

HNO,

HNO1-resistant’

CHase ABC

n Material remaining at the origin after chondroitinase digestion b 90-100% of untreated MPS could be precipitated by CPC. ’ Material insoluble in CPC after HNO, degradation.

321 241 S-

%

Stages 2-9

CONTROL

Br

and paper chromatography.

CHASE

ABC

I71 8

7y. 6 X5E 0432I-

L(

in ADI-6S

&Di-4S

FIG. 15. Paper chromatograph of untreated and chondroitinase ABC-treated MPS extracted from embryos exposed to W-sulfate from the late neurula stage (late stage 16) through the tail bud stage (stage 17). 0 = origin; dotted line = solvent front. Details in Materials and Methods.

at the origin and in the disaccharide regions indicated that during gastrulation chondroitin 6-sulfate represented approximately 10% of the sulfated MPS synthesized, while approximately 8% was chondroitin 4-sulfate and 82% was chondroitinase-resistant (Table 1). A chondroitinase analysis of MPS extracted from embryos exposed to 35Ssulfate from the late neurula stage (stage 16) through the tail bud stage (stage 17) is given in Fig. 15. Radioactive areas could be detected at the origin and in the 4- and

g-sulfated disaccharide regions (Fig. 15), and there was no significant difference in radioactivity in the ADi-4S region after treatment with each enzyme (Table 1). Comparison of radioactivity in the three regions indicated an increase in the relative amount of chondroitin 6-sulfate synthesized. The 35S-sulfate-labeled MPS of embryos exposed to YS-sulfate from late stage 16 through stage 17 consisted of 68% chondroitinase-resistant material, 10% chondroitin 4-sulfate, and 22% chondroitin 6-sulfate (Table 1).

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DEVELOPMENTALBIOLOGY

Chondroitinase analysis of MPS extracted from late tadpoles (stage 25) indicated further quantitative changes in the relative amounts of the various sulfated MPS synthesized. Radioactivity could again be detected at the origin and in the 4- and 6-sulfated disaccharide regions after treatment with each enzyme. Comparison of radioactivity in the three regions indicated that chondroitinase-resistant material represented 62% of the label, while 20% was chondroitin 4-sulfate and 18% was chondroitin 6-sulfate (Table 1). Nitrous Acid Degradation Enzymatic analysis had indicated the presence at all stages of a chondroitinaseresistant sulfated MPS. In order to characterize this material, which could have been heparin, heparan sulfate, or keratan sulfate, the labeled MPS samples were subjected to nitrous acid degradation. HNOz will specifically degrade the N-sulfated MPS, heparin and heparan sulfate (Lagunoff and Warren, 1962; Cifonelli, 1968a,b; Lindahl, 1970; Kraemer, 1971a,b). Nitrous acid degradation of MPS extracted from embryos exposed to Y3-sulfate from fertilization to the late blastula stage indicated that approximately 85% of the labeled MPS was HNO ,-sensitive (soluble in CPC following HNO, treatment, i.e., heparin and/or heparan sulfate), while approximately 15% was HNO ,-resistant (precipitable with CPC following HNO, treatment) (Table 1). There was, therefore, a close correspondence between the amount of chondroitinase-resistant MPS found after enzymatic analysis and the amount of HNO,-sensitive MPS (Table 1). When the HNO,-resistant material was subjected to digestion with chondroitinase ABC, significant radioactivity could be detected only in the 4sulfated disaccharide region (Table 1). Very little, if any, radioactivity remained at the origin, indicating that virtually all the chondroitinaseresistant material could be degraded by nitrous acid.

VOLUME 32. 1973

Similarly, nitrous acid degradation of MPS made during gastrulation, between late stage 16 and stage 17, and at stage 25 indicated that in each case the amount of HNOz-sensitive MPS corresponded closely to the amount of chondroitinaseresistant MPS, and virtually all the HNO 2resistant material could be degraded by chondroitinase ABC (Table 1). In general, the results of nitrous acid degradation indicated that at all stages the chondroitinase-resistant material detected after enzymatic analysis could be degraded by HNO,, and, therefore, was heparin and/or heparan sulfate. A significant amount of material resistant to both chondroitinase and HNO, could not be detected at any stage indicating the absence of keratan sulfate synthesis. A summary of the relative amounts of the various sulfated MPS synthesized during the four periods of development examined is given in Table 2. DISCUSSION

35S-Sulfate Incorporation as an Assay for Sulfated MPS Synthesis The incorporation of 35S-sulfate into material detectable autoradiographically has been used in this investigation as an assay for the synthesis of sulfated MPS. Although little is known about the metabolism of inorganic sulfate in amphibians, it has been established that in other vertebrates 35S-sulfate that is detectable in autoradiographs is almost exclusively in soulfated MPS (for reviews see Bostrijm and Aqvist, 1952; Amprino, 1955; DziewiatkowTABLE 2 RELATIVE PROPORTIONSOF THE VARIOUS SULFATED MPS SYNTHEHZED DURING FOUR PERIODS OF DEVELOPMENT Approximate Stages

Heparan sulfate

Chondroitin 4-sulfate

2-9 12-14 16-17 25

82 82 68 62

18 8 :z 20

% Chondroitin &sulfate 0 10 22 18

KOSHER

AND SEARLS

Mucopolysaccharide

ski, 1958). Inorganic sulfate is not utilized for the synthesis of methionine or cystine in vertebrates (Tarver and Schmidt, 1939, 1942; Bostrijm and Aqvist, 1952; Machlin et al., 1955; Lowe and Roberts, 1955; Dziewiatkowski, 1958; Huovinen and Gustafsson, 1967), so 35Sderived from sulfate is not incorporated into protein. Sulfated lipids have been demonstrated only in myelin (Rouser and Yamamoto, 1969). In chick embryos, sulfate may be utilized for the synthesis of taurine (Machlin et al., 1955) and for certain low molecular weight aryl and alkyl sulfates, such as methyl sulfate, 1,2-propanediol sulfate, and isopropyl sulfate (Adams, 1963; Yagi, 1966). However, all these compounds are water soluble and would not remain in sections following the extensive washing that is done prior to autoradiography. Although the possibility of 35S-sulfate incorporation into sulfated glycoproteins (Slomiany and Meyer, 1972) cannot conclusively be eliminated, all of the SsS-sulfate-labeled, protease-resistant, nondialyzable material, we have been able to detect biochemically in the present study has been characterized as sulfated MPS. It has been established that sulfation occurs concomitant with chain elongation during the synthesis of the polysaccharide chains of chondroitin sulfate (Telser et al., 1966; Meezan and Davidson, 1967; DeLuca and Silbert, 1968; Derge and Davidson, 1972). Therefore, 35S-sulfate incorporation can serve as an assay for the synthesis of chondroitin sulfate. However, detailed information on the biosynthesis of sulfated MPS other than chondroitin sulfate is unavailable. The possibility that sulfation can occur long after chain elongation of other sulfated MPS (e.g., heparan sulfate) cannot be eliminated (Lindahl et al., 1972). Association Platelets opment

of Sulfated MPS with Yolk during Early Stages of Devel-

The intracellular 35S-sulfate-incorporating material that was observed autoradio-

Synthesis

during Development

63

graphically in unfertilized and fertilized eggs, cleaving embryos, and blastulae was associated predominantly with yolk platelets. During these stages, silver grains were always more concentrated in regions of the embryo where yolk platelets were larger and more concentrated, and virtually all intracellular silver grains were seen along the periphery of yolk platelets. Amphibian yolk platelets including those of Rana pipiens consist of an inner crystalline core and a fibrillar and granular outer superficial layer (Karasaki, 1963a). Ohno et al. (1964) have observed that the periphery of yolk platelets stains metachromatically with toluidine blue, and suggested that the superficial layer of yolk platelets contains acid polysaccharide. Tandler and La Torre (1967) have isolated an acidic polysaccharide from the superficial layers of Bufo arenarum yolk platelets. Thus, the intracellular sulfate incorporation observed in the autoradiographs probably represents synthesis of sulfated MPS which is a component of the superficial layer of yolk platelets. There is a correlation between the presence or absence of 35S-sulfate-incorporating material along the periphery of yolk platelets and the presence or absence of superficial layers of yolk platelets in various regions of the embryo as determined by Karasaki (1963b). Little incorporation of 3”S-sulfate was observed in the cells of the roof of the blastocoel of late blastulae, and Karasaki (1963b) has observed a disappearance of the superficial layer from many of the yolk platelets in these cells. Similarly, sulfate-incorporating material continues to be present peripheral to yolk platelets in the yolky endoderm throughout gastrulation and through the tail bud stage. The superficial layers of yolk platelets in the yolky endoderm are still present as late as the tail bud stage (Karasaki, 1963b). Biochemical analysis of MPS extracted from embryos exposed to 35S-sulfate from fertilization to the blastula stage indicates that chondroitin 4-sulfate represents 18%

64

DEVELOPMENTAL BIOLOGY

of the sulfated MPS synthesized, while 82% is heparin and/or heparan sulfate. It cannot be determined conclusively whether both of these sulfated MPS are present in superficial layers of yolk platelets, since during cleavage there is accumulation of sulfated MPS along cell surfaces as well. However, since the predominant MPS synthesized is heparin/heparan sulfate and since most of the sulfated MPS is associated with yolk platelets, it is likely that the heparin/heparan sulfate is predominantly, if not entirely, in superficial layers of yolk platelets. This suggestion is consistent with the observation of Ohno et al. (1964) that the metachromasy observed along the periphery of yolk platelets after toluidine blue staining is not affected by treatment with testicular hyaluronidase, an enzyme that will degrade chondroitin 4-sulfate, but not heparin or heparan sulfate. The suggestion that 35S-sulfate is incorporated into sulfated MPS present in the superficial layer of yolk platelets indicates that a component of the superficial layer of yolk platelets may be continuously synthesized throughout cleavage and synthesized in the yolky endoderm as late as the tail bud stage. Therefore, the superficial layer of yolk platelets does not appear to be simply a residue of the surface coat of the oolemma that is incorporated into the oocyte during micropinocytosis of the yolk protein precursor molecule, SLPP (Wallace and Jared, 1969), but rather appears to be metabolically active, being continuously synthesized and presumably continuously destroyed. The presence of a sulfated MPS in the superficial layer of yolk platelets is of interest in view of the recent suggestion that yolk platelets may sequester calcium and sodium ions (Barth and Barth, 1972). Negatively charged molecules such as sulfated MPS may provide sites for the binding and sequestering of these ions.

VOLUME 32, 1973

Changes in Sulfated ing Development

MPS Synthesis

dur-

During the early stages of gastrulation, there is little incorporation of 35S-sulfate in the cells of the roof of the blastocoel (including the presumptive chordamesoderm and presumptive neural ectoderm). The autoradiographs indicate that during stage 12, the chordamesoderm begins incorporating substantial 35S-sulfate as it invaginates. Similarly, incorporation is observed in the lateroventral mesoderm as it invaginates. When gastrulation is essentially complete and the chordamesoderm has completed contact with the presumptive neural tissue, incorporation is seen in the neural tissue. The sulfated MPS synthesized by the invaginating chordamesoderm and lateroventral mesoderm during gastrulation probably accumulates along cell surfaces. Tarin (1971) has observed that as the chordamesoderm and lateroventral mesoderm invaginate during gastrulation in Xenopus laevis, toluidine blue-staining metachromatic material accumulates in the intercellular spaces between the mesodermal cells. In addition, the sulfated MPS synthesized by the invaginating chordamesoderm, lateroventral mesoderm, and neural tissue cannot be associated with the superficial layer of yolk platelets, since Karasaki (1963b) has observed that the superficial layers have completely disappeared from the yolk platelets of these tissues by stage 12. The synthesis of chondroitin 6-sulfate is initiated during the period of development when the invaginating chordamesoderm, lateroventral mesoderm, and neural tissue are observed autoradiographically to begin incorporating 35S-sulfate. Since the pattern of 3”S-sulfate incorporation in the yolky endoderm during gastrulation is identical to that observed during previous stages in that all silver grains are peripheral to yolk platelets, we suggest that the sulfated MPS made in the yolky endoderm

KOSHER AND SEARLS

Mucopolysaccharide

are the same MPS made at previous stages (heparin/heparan sulfate and chondroitin 4-sulfate). Therefore, the chondroitin 6sulfate synthesized during gastrulation is probably synthesized by the chordamesoderm, lateroventral mesoderm, and/or neural tissue. From neurulation through stage 19 (hatching), incorporation of 35S-sulfate is observed in virtually every tissue of the embryo including the neural tube, neural crests, somites (including at stage 19 the sclerotome, myotome, and dermatome), notochord, subchorda, lateral plate mesoderm, pronephros, endoderm, epidermis, and myocardium. This ubiquitous incorporation of “S-sulfate is accompanied by an increase in the relative amount of chondroitin 6-sulfate which is synthesized. Therefore, several nonchondrogenic cell types, as well as prechondrogenic cells, synthesize sulfated MPS including chondroitin sulfate. Similar results indicating that sulfated MPS production is not limited to prospective chondrogenic or connective tissue cells have been obtained in the chick embryo (France-Browder et al., 1963; Marzullo and Lash, 1967; Lash, 1968a-c; Kvist and Finnegan, 1970a,b; Manasek, 1970). During stages 20-22, incorporation of 35S-sulfate remains high in some tissues (e.g., notochord, sclerotome), while in other tissues it is either depressed (e.g., epidermis, endoderm) or completely suppressed (e.g., neural tube, myotome). By stage 25, a very high incorporation is observed only in cartilaginous tissue, although some incorporation is observed in other tissues. Therefore, during development there is apparently a selective restriction in sulfated MPS synthesis to certain cell types. The gradual limitation of a previously ubiquitous pathway to certain cell types during embryogenesis has also been observed in the chick embryo (Fabro and Rinaldi, 1965; Searls, 1965a; Lash, 1968a--c; Manasek, 1970).

Synthesis

65

during Development

During stage 25, when cartilage is present in the embryos, the amount of chondroitin 4-sulfate synthesized relative to chondroitin 6-sulfate increases (20% chondroitin 4-sulfate and 18% chondroitin 6-sulfate at stage 25 compared to 10% chondroitin 4-sulfate and 22% chondroitin 6-sulfate between stage 16 and stage 17). This may be a reflection of the fact that there is a progressive increase in the proportion of chondroitin 4-sulfate in cartilage with age (Mathews and Hinds, 1963; Robinson and Dorfman, 1969). Keratan sulfate synthesis has not been detected even at stage 25. However, keratan sulfate is not present in young embryonic cartilage (Kaplan and Meyer, 1959; Shulman and Meyer, 1968). Function of Sulfated velopment

MF’S

during

De-

The present study indicates that during early stages of development a large number of different cell types synthesize sulfated MPS including chondroitin sulfate. It has been suggested that if nonchondrogenie cell types synthesize chondroitin sulfate during early stages of development, this would indicate that these embryonic cells are passing through a chondrogenic phase before their own differentiative biosynthetic pattern is stabilized (Holtzer and Matheson, 1970). However, we feel it is more reasonable to assume that the ubiquitous synthesis of sulfated MPS during the critical early stages of development indicates that these molecules perform some significant function(s). We suggest that the sulfated MPS that accumulates along the surfaces of cells during cleavage may participate in the cleavage process by mediating the formation of cell-to-cell junctions. MPS have been implicated in cellular adhesions (Overton, 1969; Khan and Overton, 1969, 1970; Pessac and Defendi, 1972), and the formation of cleavage furrows in amphibians requires, in addition to constriction and the local growth of new cell surfaces, the formation of cell-to-cell

66

DEVELOPMENTAL BIOLOC Y

junctions (Selman and Perry, 1970; Bluemink, 1971a,b). Similarly, the sulfated MPS synthesized during gastrulation by the invaginating chordamesoderm and lateroventral mesoderm could possibly be involved in the changes in cell surface properties and cell contact behavior that occur during gastrulation in Rana pipiens and that appear to be necessary for gastrulation to take place (Johnson, 1970). In addition to the proposed role of extracellular MPS in cellular adhesions and morphogenetic movements, these molecules could be involved in other embryonic processes. Since the chordamesoderm synthesizes sulfated MPS as it invaginates and synthesis of sulfated MPS by neural tissue begins upon completion of contact between the chordamesoderm and neural tissue, one must consider the possibility that sulfated MPS is involved in the process of primary induction. In addition, numerous investigators have demonstrated the importance of extracellular materials in the cellular interactions that are necessary for normal morphogenesis and differentiation (for review see Grobstein, 1967), and MPS have been implicated in these interactions (Kallman and Grobstein, 1966; Slavkin et al., 1969; Bernfield and Wessells, 1970; Bernfield and Banerjee, 1972; Bernfield et al., 1972). The authors are pleased to thank Dr. Marie DiBerardino for her technical advice and for supplying us with frogs during critica. periods of these experiments. The excellent technical assistance of Miss Joyce Williams is gratefully acknowledged. REFERENCES ABBOTT, J., and HOLTZER, H. (1966). The loss of phenotypic traits by differentiated cells. III. The reversible behavior of chondrocytes in primary cultures. J. Cell Biol. 28, 473-487. ADAMS, J. B. (1963). Sulfate metabolism in avian and mammalian cartilage extracts. Arch. Biochem. Biophys. 101, 478-488. AMPRINO, R. (1955). Autoradiographic research on the Sgs-sulphate metabolism in cartilage and bone differentiation and growth. Acta Anat. 24, 121163. BARTH, L. G., and BARTH, L. J. (1972). %odium and

VOLUME 32, 1973

‘%alcium uptake during embryonic induction in Ram pipiem. Deuelop. Biol. 28, 18-34. BATES, C. J., and LEVENE, C. I. (1971). The synthesis of sulphated glycosaminoglycans by the mouse fibroblastic line, 3T6. Biochim. Biophys. Acta 237, 214-226. BERNFIELD, M. R., and BANERJEE, S. D. (1972). Acid mucopolysaccharide (glycosaminoglycan) at the epithelial-mesenchymal interface of mouse embryo salivary glands. J. Cell Biol. 52, 664-673. BERNFIELD, M. R., and WESSELLS, N. K. (1970). Intraand extracellular control of epithelial morphogenesis. In “Changing Synthesis in Development” (M. N. Runner, ed.), pp. 195-249. Academic Press, New York. BERNFIELD, M. R., BANERJEE, S. D., and COHN, R. H. (1972). Dependence of salivary epithelial morphology and branching morphogenesis upon acid mucopolysaccharide-protein (proteoglycan) at the epithelial surface. J. Cell Biol. 52, 674-689. BLUEMINK, J. G. (1971a). Cytokinesis and cytochalasin-induced furrow regression in the first-cleavage zygote of Xenopus laevis. Z. Zellforsch. Microsk. Anut. 121, 102-126. BLUEMINK, J. G. (1971b). Effects of cytochalasin B on surface contractility and cell junction formation during egg cleavage in Xenopus laevis. Cytobiology 3, 176187. BOSTR~M, H., and AQVIST, S. (1952). Utilization of Sas-labeled sodium sulphate in the synthesis of chondroitin sulphuric acid, taurine, methionine, and cystine. Acta Chem. Stand. 6, 1557-1559. BRUNET, P. C. J., and SMALL, P. L. (1959). An improved radioautographic method for demonstrating tyrosine uptake and tyrosinase activity in melanocytes. Quart. J. Micros. Sci. 100, 591-598. CAHN, R. D., and LASHEP., R. (1967). Simultaneous synthesis of DNA and specialized cellular products by differentiating cartilage cells in vitro. Z’roc. Nut. Acad. Sci. U.S. 58, 1131-1138. CARO, L. G., and VAN TUBERGEN, R. P. (1962). High resolution autoradiography. I. Methods. J. Cell Biol. 15, 173-188. CIFONELLI, J. A. (1968a). Reaction of heparitin sulfate with nitrous acid. Carbohydr. Res. 8, 233-242. CIFONELLI, J. A. (1968b). Structural features of acid mucopolysaccharides. In “The Chemical Physiology of Mucopolysaccharides” (G. Quintarelli, ed.), pp. 91-105. Little, Brown, Boston, Massachusetts. DELUCA, S., and SILBERT, J. E. (1968). Biosynthesis of chondroitin sulfate. II. Incorporation of sulfate-s6S into microsomal chondroitin sulfate. J. Biol. Chem. 243, 2725-2729. DERGE, J. G., and DAVIDSON, E. A. (1972). Proteinpolysaccharidebiosynthesis. Membrane-bound saccharides. Biochem. J. 126, 217-223. DIBERARDINO, M. A. (1967). Frogs. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 53-74. Crowell, New York.

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DISCHE, Z., and BORENFREUND, E. (1950). A spectrophotometric method for the microdetermination of hexosamines. J. Biol. Chem. 184, 517-522. DZIEWIATKOWSKI, D. D. (1958). Autoradiographic studies with Ssh-sulfate. Znt. Rev. Cytol. 7, 159193. ELLISON, M. L., and LASH, J. W. (1971). Environmental enhancement of in oitro chondrogenesis. Deuelop. Biol. 26, 486-496. FABRO, S. P., and RINALDI, L. M. (1965). Loss of ascorbic acid synthesis in embryonic development. Deuelop. Biol. 11, 468-488. FRANCO-BROWDER,S., DERYDT, J., and DORFMAN, A. (1963). The identification of a sulfated mucopolysaccharide in chick embryos, stages 11-23. Proc. Nat. Acad. Sci. U.S. 49, 643-647. GROBSTEIN, C. (1967). Mechanism of organogenetic tissue interaction. Nat. Cancer Inst. Mongr. 26, 279-299. HOLTZER, H. (1961). Aspects of chondrogenesis and myogenesis. In “Molecular and Cellular Synthesis” (D. Rudnick, ed.). pp. 35-86. Ronald, New York. HOLTZER, H., and MATHESON, D. W. (1970). Induction of chondrogenesis in the embryo. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), Vol. 3, pp. 1753-1770. Academic Press, New York. HUOVINEN, J. A., and GUSTAFSSON,B. E. (1967). Inorganic sulphate, sulphite, and sulphide as sulphur donors in the biosynthesis of sulphur amino acids in germ-free and conventional rats. Biochim. Biophys. Acta 136, 441-447. JOHNSON, K. E. (1970). The role of changes in cell contact behavior in amphibian gastrulation. J. Exp. 2001. 175, 391-428. JOHNSTON, P. M., and COMAR, C. L. (1957). Autoradiographic studies of the utilization of Ss5-sulfateby the chick embryo. J. Biophys. Biochem. Cytol. 3,231-245. KALLMAN, F., and GROBSTEIN, C. (1966). Localization of glucosamine-incorporating materials at epithelial surfaces during salivary epithelio-mesenchyma1 interaction. Deuelop. B~ol. 14, 52-67. KAPLAN, D. and MEYER., K. (1959). Ageing of human cartilage. Nature (London) 183, 1267-1268. KARASAKI, S. (1963a). Studies on amphibian yolk. I. The ultrastructure of the yolk platelet. J. Cell Biol. 18, 135-151. KARASAKI, S. (196313). Studies on amphibian yolk. II. Electron microscopic observations on the utilization of yolk platelets during embryogenesis. J. Ukrastruct. Res. 9, 225-247. KHAN, T., and OVERTON, J. (1969). Staining of intercellular material in reaggregating chick liver and cartilage cells. J. Exp. Zool. 171, 161-174. KHAN, T. and OVERTON, J. (1970). Lanthanum staining of developing chick cartilage and reaggregating cartilage cells. J. Cell Biol. 44, 433-438. KING, T. J. (1967). Amphibian nuclear transplanta-

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tion. In “Methods in Developmental Biology” (F. H. Wilt and N. K. Wessells, eds.), pp. 737-751. Crowell, New York. KRAEMER, P. M. (1971a). Heparan sulfates of cultured cells. I. Membrane-associated and cell-sap species in Chinese hamster cells. Biochemistry 10, 1437-1445. KRAEMER, P. M. (1971b). Heparan sulfates of cultured cells. II. Acid-soluble and -precipitable species of different cell lines. Biochemistry 10, 14451451. KVIST, T. N., and FINNEGAN, C. V. (1970a). The distribution of glycosaminoglycans in the axial region of the developing chick embryo. I. Histochemical analysis. J. Exp. Zool. 175, 221-240. KVIST, T. N., and FINNEGAN, C. V. (1970b). The distribution of glycosaminoglycans in the axial region of the developing chick embryo. II. Biochemical analysis. J. Exp. Zool. 175, 241-258. LAGUNOFF, D., and WARREN, G. (1962). Determination of 2-deoxy-2sulfoaminohexose content of mucopolysaccharides. Arch. Biochem. Biophys. 99, 396-400. LASH, J. W. (1968a). Chondrogenesis: genotypic and phenotypic expression. J. Cell Physiol. 72, Suppl. 1, 35-46. LASH, J. W. (1968b). Phenotypic expression and differentiation: in vitro chondrogenesis. In “The Stability of the Differentiated State” (H. Ursprung, ed.), pp. 17-24. Springer-Verlag, New York. LASH, J. W. (1968c). Semitic mesenchyme and its response to cartilage induction. In “Epithelial-Messenchymal Interactions” (R. F. Fleischmajer and R. E. Billingham, eds.), pp. 165-172. Williams & Wilkins, Baltimore, Maryland. LASH, J. W., HOLTZER, H., and WHITEHOUSE, M. W. (1960). In vitro studies on chondrogenesis: the uptake of radioactive sulfate during cartilage induction. DeueEop. Biol. 2, 76-89. LINDAHL, U. (1970). Structure of heparin, heparan sulfate and their proteoglycans. In “Chemistry and Molecular Biology of the Intercellular Matrix” (E. A. Balazs, ed.), Vol. 2, pp. 943-960. Academic Press, New York. LINDAHL, U., BXCKSTR~M, G., MALMSTR~M, A., and FRANSSON,L.-A. (1972). Biosynthesis of L-iduronic acid in heparin: epimerization of n-glucuronic acid on the polymer level. Biochem. Biophys. Res. Commun. 46, 985-991. LOWE, I. P., and ROBERTS, E. (1955). Incorporation of radioactive sulfate sulfur into taurine and other substances in the chick embryo. J. Biol. Chem. 212, 477-483. MACHLIN, L. J., PEARSON, P. B., and DENTON, C. A. (1955). The utilization of sulfate sulfur for the synthesis of taurine in the developing chick embryo. J. Biol. Chem. 212, 469-475. MANASEK, F. J. (1970). Sulfated extracellular matrix production in the embryonic heart and adjacent

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NAMEROFF,M., and HOLTZER,H. (1967). The loss of phenotypic traits by differentiated cells. IV. Changes in polysaccharides produced by dividing chondrocytes. Develop. Biol. 16, 250-281. OHNO, S., KARASAKI, S., and TAKATA, K. (1964). Histo- and cytochemical studies on the superficial layer of yolk platelets in the Triturus embryo. Exp. Cell Res. 33, 310-318. OVERTON,J. (1969). A fibrillar intercellular material between reaggregating embryonic chick cells. J. Cell Biol. 40, 136-143. PESSAC,B., and DEFENDI,V. (1972). Cell aggregation: role of acid mucopolysaccharides. Science 175, 898-900.

ROBINSON,H. C., and DORFMAN,A. (1969). The sulfation of chondroitin sulfate in embryonic chick cartilage epiphyses. J. Biol. Chem. 244, 348-352. ROUSE& G., and YAMAMOTO, A. (1969). Lipids. In “Handbook of Neurochemistry” (A. Lajtha, ed.), Vol. 1, pp. 121-169. Plenum, New York. SAITO, H., YAMAGATA, T., and SUZUKI, S. (1968). Enzymatic methods for the determination of small quantities of isomeric chondroitin sulfates. J. Biol. Chem. 243, 1536-1542. SCO?T,J. E. (1960). Aliphatic ammonium salts in the assay of acidic polysaccharides from tissues. In “Methods of Biochemical Analysis” (D. Glick, ed.), Vol. 8, pp. 145-195. Wiley (Interscience), New York. SEARLS,R. L. (1965a). An autoradiographic study of the uptake of SaJ-sulfate during the differentiation of limb bud cartilage. Develop. Biol. 11, 155168. SEARLS, R. L. (1965b). Isolation of mucopolysaccharide from the precartilaginous embryonic chick limb bud. Proc. Sot. Exp. Biol. Med. 118, 11721176.

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SELMAN,G. G., and PERRY,M. M. (1970). Ultrastructural changes in the surface layers of the newt’s eggs in relation to the mechanism of cleavage. J. Cell Sci. 6, 207-227.

SHLJLMAN, H. J., and MEYER, K. (1968). Cellular differentiation and the ageing process in cartilaginous tissues. J. Exp. Med. 128, 1353-1362. SHUMWAY,W. (1940). Stages in the normal development of Rana pipiens. I. External form. An&. Rec. 78, 139-146. SHUMWAY,W. (1942). Stages in the normal development of Rand pipiens. II. Identification of the stages from sectioned material. Anat. Rec. 83, 309-315. SLAVKIN,H. C., BRINGAS,P., CAMERON, J., LEBARON, R., and BAVETTA,L. A. (1969). Epithelial and mesenchymal cell interactions with extracellular matrix material in vitro. J. Embryol. Exp. Morphol. 22, 395-405.

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amphibian yolk. VIII. The estrogen-induced hepatic synthesis of a serum lipophosphoprotein and its selective uptake by the ovary and transformation into yolk platelet proteins in Xenopus laevis. Develop.

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S. (1968). Purification and properties of bacterial chondrotinases and chondrosulfatases. J. Biol. Chem. 243, 1523-1535.