Acid mucopolysaccharide metabolism, the cell surface, and primary mesenchyme cell activity in the sea urchin embryo

DEVELOPMENTAL

BIOLOGY

41, 110-123 (1374)

Acid Mucopolysaccharide Primary

Mesenchyme

Metabolism, Cell Activity

in the Sea Urchin

GERALD C. KARP AND MICHAEL Department

of Zoology,

the Cell Surface,

Embryo

SOLURSH

University of Florida, Gainesville, Florida 32601 and Department University of Iowa, Iowa City, Iowa 52242 Accepted

and

of Zoology,

July 8, 1974

The relationship between YSO, incorporation into acid mucopolysaccharides and the appearance and activity of the primary mesenchyme cells has been studied in the sea urchin, Lytechinus pictus. The ratio of the uptake of 36SO, to its incorporation into cetylpyridinium chloride precipitable material varies over a wide range during early development, with the smallest ratio, therefore the greatest sulfation activity, being found at the early mesenchyme blastula stage. The types of mucopolysaccharides produced have not been identified, but are heterogeneous. At the mesenchyme blastula stage nearly 90% of the polysaccharides produced become sulfated. When embryos develop in sulfate-free sea water to the mesenchyme blastula stage there is a 70% decrease in the incorporation of W-acetate into polysaccharides and a 13-fold decrease in the ratio of sulfated to nonsulfated polysaccharides produced. Embryos raised in sulfate-free sea water develop normally to the mesenchyme blastula stage at which time there is an accumulation in the blastocoel of primary mesenchyme cells that do not migrate. The surface of the primary mesenchyme cells of sulfate-deficient embryos has a smooth appearance in the scanning electron microscope, while the surface of these cells in control embryos is rough, possibly reflecting the presence of an extracellular coat. It is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology and cell movement. INTRODUCTION

During the development of the sea urchin, migratory cells are released from the vegetal wall of the blastula into the blastocoel. These primary mesenchyme cells collect for a period on the inner surface of the vegetal cells and then migrate to form a characteristic pattern along the inner surfaces of the cells of the blastula wall. These migrations have been studied by time-lapse cinematography (Gustafson, 1963; Gustafson and Wolpert, 1961) and it is believed that the direction these movements take and the ultimate locations to which these cells migrate is determined by the selective interactions of the filopodial processes of the migratory cells and the substrate cell surfaces to which contacts are made. Earlier studies suggest that sulfate metabolism may be of particular importance in the activities of the primary mesenthyme cells. This is based on two earlier observations. The first was the demonstra110 Copyright All rigbta

0 1974 by Academic Press, Inc. of reproduction in any form reserved.

tion that the primary mesenchyme cells contain an accumulation of sulfated polysaccharides as judged by histochemical procedures (Immers, 1961; Sugiyama, 1972) and the second, that these cells selectively incorporatesSO, before their release from the blastula wall (Sugiyama, 1972). We now report on various quantitative aspects of sulfated polysaccharide metabolism during sea urchin development, the effects of withholding sulfate on the migratory properties of the primary mesenchyme cells, and the morphology of these cells in the light and electron microscopes. On the basis of these results, it is suggested that there is a correlation between sulfated polysaccharide synthesis, cell surface morphology, and cell movement. MATERIALS

AND METHODS

Handling of gametes and embryos. Throughout this study sea water was made following the formula of Hinegardner

KARP AND SOLURSH

AMPS and Cell Migration

:1967). Sulfate-free sea water was made ‘ram the same formula substituting MgCl, for MgSO,. Embryos to be raised in sulfate-free sea water had developed from eggs that had been spawned into sulfate-free sea water and fertilized by sperm that had been diluted in sulfate-free sea water. Labeling of embryos with 3”S0,. Embryos of Lytechinus pi&us (Pacific BioMarine) were cultured to the desired stage in complete sea water plus streptomycin G sulfate (100 Fg/ml). Embryos were washed three times by light centrifugation in sulfate-free sea water containing streptomycin G sulfate and suspended in 1.0 ml (final volume) of this solution. Ten ~1 of carrier-free H,WO, (5 mCi/ml, New England Nuclear) was added and incubation was carried out at 18°C for 30 min on a rotary shaker. At the end of the incubation, the embryos were washed rapidly with seven changes of ice cold complete sea water. Total washing time was approximately 20 min. At the end of the last wash, 1 ml of 1% NaCl was added to the pelleted embryos and 3 ml of 95% ethanol, and the embryos stored at 4°C. If embryo counts were performed, five equal aliquots of this suspension were counted under a dissecting microscope. The ethanol suspensions were sonicated for 30 set at minimal power with a Biosonik sonicator. The sonicates were centrifuged at 12,000g and the supernatant removed. To 0.5 ml of this supernatant was added 10 mls of Cocktail D (5 g of 2,5diphenyloxazol, 100 g of naphthalene, and 1,4 dioxane to 1 liter), and the radioactivity determined. These counts provide the ethanol soluble figure of sulfate uptake. To determine the ethanol insoluble radioactivity, the precipitates from the above centrifugation were washed three times with cold 70% ethanol and suspended in 1.0 ml of 0.2 M Tris, pH 8.0. One-tenth milliliter of this suspension was removed for protein determination by the method of Lowry et al. (1951). To the remainder of the Tris suspension was added 100 ~1 of pronase (10 mg/ml) and incubation was

111

carried out at 55°C for 48 hr. An equal volume of ice cold 20% trichloracetic acid (TCA) was added to the digested embryos and the suspension allowed to precipitate. The TCA supernatant containing the polysaccharides was dialyzed at 4°C against several changes of distilled water. To a 0.1 ml aliquot of the dialysate was added 0.9 mls of 0.1 M NaCl-0.1 M NaAcetate, pH 5.3, 0.1 ml of a solution of hyaluronic acid and chondroitin sulfate as carriers to a final concentration of 250 pg/ml and 200 pg/ml respectively, and 0.1 ml of 1% cetylpyridinium chloride (CPC). After 5 min at room temperature the precipitate was collected by centrifugation at 10,OOOgfor 15 min. The precipitate was washed with cold water, dissolved in methanol, and counted in cocktail D. At each step of the procedure volumes were determined and radioactivity monitored. Labeling embryos with SH-acetate. Embryos were cultured in either sulfate-free sea water or complete sea water to the desired stage, packed by hand centrifugation, and suspended in 1.0 ml of sulfatefree sea water or complete sea water respectively. To each tube was added 20 ~1 of SH-acetate (1290 Ci/mM, New England Nuclear, sodium salt from which the ethanol was evaporated and the salt dissolved in sulfate-free sea water at a final concentration of 1 mCi/ml), and the embryo suspension was incubated at 18°C for 30 min on a rotary shaking platform. Embryos were washed with the appropriate sea water (sulfate-free sea water or complete sea water) and frozen until polysaccharides could be purifed as described above. Barium sulfate determinations. To 0.5 ml of the ethanol soluble sonicates described above was added 0.5 ml of 0.025 M Na,SO, and then 0.5 ml of 0.05 M BaCl,. Solutions were mixed, centrifuged at 10,OOOgfor 5 min, and the supernatant removed and counted in cocktail D. Controls were run by barium precipitation of comparable ethanolic solutions to which known amounts of HZ3YS0, were added.

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DEVELOPMENTALBIOLOGY

Polysaccharide electrophoresis. Aliquots of dialyzed 3H-labeled polysaccharide solutions in water were dried, dissolved in 20 ~1 of 0.1 N HCl, containing 2 fig each of authentic samples of hyaluronic acid (Sigma), chondroitin-4-sulfate (Mann) and heparin (Nutritional Biologicals), applied to cellulose acetate membranes (Gelman), and electrophoresed using 0.1 N HCl and a constant current of 5 mA/membrane for 50 min, as described by Hsu et al. (1972). Dried membranes were stained for 15 min in 0.1% Alcian blue in aqueous solution containing 5% acetic acid and 10% ethanol and destained in 5% acetic acid. After drying, the strips were cut into 0.5 cm pieces, corresponding to the known samples, and counted in the scintillation counter. Approximately 50-75% of CPC precipitable counts were recovered on the membranes after the entire procedure.

VOLUME 41, 1974

were fixed in 2% glutaraldehyde in sulfatefree sea water or complete sea water washed with complete sea water, postfixed in 1% osmium tetroxide in complete sea water, washed in complete sea water, dehydrated, embedded in epon-araldite, sectioned at 0.5 micron, and stained with Richardson’s stain (Richardson et al.: 1960). Scanning electron microscopy. Embryos were raised to the desired stage in either sulfate-free sea water or complete sea water, fixed in Karnovsky’s fixative (Karnovsky, 1965). Embryos were then washed three times in Millonig’s phosphate buffer, postfixed for 2 hr in 1% osmium tetroxide and dehydrated to 70% ethanol. The embryos were cracked open by freezing in liquid nitrogen as described by Lim (1971), dehydrated in 100% ethanol, and dried by the critical point drying method (AnderFractionation of sulfated polysacchason, 1951) using liquid CO, as a transirides. Aqueous solutions of 35S-labeled tional fluid. Some embryos were broken polysaccharides were dried and dissolved open manually with a mounted eye lash in 50 ~1 of water containing 2 rg of va- after critical point drying and mounting. rious authentic standards including The embryos were coated with palladium: chondroitin-4-sulfate (Mann), dermatan gold (40:60) approximately 100 A thick, sulfate and keratan sulfate-l (both the and observed with a Cambridge S4 scan-‘ generous gifts of Dr. J. A. Cifonelli), and ning electron microscope at 20,000 V. heparitin sulfate (a gift from the Upjohn RESULTS Co.). The sample was spotted on silicaSulfate Uptake and Sulfated Polysacchachromatographic impregnated paper ride Synthesis during Development (ITLC-SA, Gelman Instrument Co.) and The uptake of exogenous 3”SO, and its chromatography was done in system II of into polysaccharides has Lippiello and Mankin (1971). Chromato- incorporation grams to be stained were immersed in 0.1% been studied during the early development Alcian blue, destained, dried, cut in 1 cm of Lytechinus pictus. The uptake of 3”S0, in the presence of varying concentrations of pieces, and counted in the scintillation counter. Over 75% of the initial counts were unlabeled sulfate is shown in Fig. 1. In the recovered from the unstained chromato- following study of uptake and incorporation of 35S0, (Fig. 2), embryos were raised graphs. Prior to drying and chromatography, some samples were treated with tes- in complete artificial sea water to the ticular hyaluronidase (100 pg/ml, Worth- desired stage, washed in artificial sea water ington Corp.) for 3 hr at 37°C in 0.1 M containing MgCl, as a substitute for MgSO, (sulfate-free sea water), and laNaCl, 0.1 M Na-Acetate, pH 5.3. Light microscopy. Embryos raised to the beled in sulfate-free sea water containing early mesenchyme blastula stage in either streptomycin G sulfate (100 pg/ml) and sulfate-free sea water or complete sea water carrier-free 35S0, (50 pCi/ml). Sulfate-free

KARP AND SOLURSH

113

AMPS and Cell Migration

. .Ol

.02 moler,

I

.03 so,

FIG. 1. Uptake of YSO, in the presence of increasing concentrations of unlabeled sulfate. Embryos were grown in complete sea water to the late gastrula stage, washed three times with sulfate-free sea water and incubated with 36SO, (50 &i/ml) in varying sulfate concentrations. Counts per minute represents the isotope retained by the living embryos after a rapid and extensive wash regime in complete sea water.

sea water containing streptomycin sulfate was chosen as the incubation medium during the short pulse to insure maximal uptake of isotope (Fig. l), yet provide a minimal level of sulfate to avoid artifacts resulting from complete sulfate deprivation. The sulfate content of sulfate-free sea water is estimated to be approximately 0.000006 M as a result of the sulfate contamination in the other chemicals and the addition of the antibiotic raises it to

0.0002 M. In this study sulfate uptake is defined as the radioactivity retained by the embryos after incubation in sulfate-free sea water plus streptomycin sulfate and extensive washing in complete sea water. Incorporation represents radioactivity incorporated into polysaccharides that are at first ethanol precipitable, soluble in cold 10% TCA after pronase digestion but nondialyzable, and precipitable by 1% cetylpyridinium chloride (CPC). Unfertilized eggs do not take YSO, up from the sea water in detectable amounts (Fig. 2). The uptake values are no higher than unfertilized eggs that had been killed by heat or freezing prior to incubation. By the two to four cell stage, low levels of YSO, appear to be taken up, although

c,

b

10 E

d

20

e

f

30 g tfO”rS

h4’

,50

60

70 J

FIG. 2. Uptake and incorporation into polysaccharides of 3”SO, at different developmental stages. Embryos were cultured in complete sea water to the desired stage, washed three times with sulfate-free sea water, and incubated for 30 min in sulfate-free sea water containing 100 fig/ml streptomycin sulfate and Y!40, (50 pCi/ml). The results are typical of four experiments. Open circles shows the radioactivity retained by living embryos after extensive washing in cold complete sea water. Closed circles shows the radioactivity incorporated into sulfated polysaccharides. The scale for uptake is 20 times that of incorporation. Stages are represented by the following letters: a, unfertilized egg; b, four cell stage; c, late cleavage; d, hatching blastula; e, late blastula; f, early mesenchyme hlastula; g, late mesenchyme blastula; h, gastrula (one-third complete); i, postgastrulated embryo; j, prism.

there is no detectable incorporation of this sulfate into polysaccharides. At this stage the washed ethanol precipitates retain radioactivity prior to pronase digestion indicating sulfate incorporation into nonpolysaccharide materials. By the end of cleavage, 3sS0, uptake has increased and its incorporation into polysaccharide is just detectable (32 cpm above background per pg protein). By the hatched blastula stage, incorporation has increased to approximately 800 cpm per Mg protein. Embryos just prior to and just after hatching were compared and found to have similar levels of uptake and incorporation. From the late blastula to the early mesenchyme blastula there is approximately a twofold rise in uptake and a 13-fold rise in incorporation into CPC pre-

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DEWLOPMENTAL

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cipitates. It is during this period of development when the most dramatic change in sulfation activity is seen. From the mesenthyme blastula through the end of gastrulation, uptake values rise very steeply while incorporation fails to undergo a corresponding increase. By the prism stage uptake is approximately nine times that of the mesenchyme blastula while incorporation has less than doubled. In some experiments (as is shown in Fig. 2) there was a drop in incorporation after the mesenthyme blastula stage. If the assumption is made that there are no changes in the size of the sulfate pools available for polysaccharide sulfation, then the ratio of incorporation to uptake should provide a measure of the sulfation of polysaccharides. In Fig. 3 these ratios are plotted and it is evident that the early

FIG. 3. Ratios of incorporation the values given in Fig. 2.

to uptake based on

TABLE ELE~TROPHORESIS

Stage

CPM co-electrophoresing luronic acid With chondroitin sulfate Sulfated/nonsulfated rides

polysaccha-

1 POLYSACCHARIDES

blastula

(30 hr.)

Gastrula

(38 hr.)

SFSW

csw

SFSW

49

221

363

321

332

108

1291

90

csw with hya-

mesenchyme blastula has the greatest sulfation activity during early development. Barium sulfate precipitation indicates that at least 99.5% of the YSO, taken up during the 30 min incubation remains as free sulfate at all stages and therefore is presumably free to be utilized for polysaccharide sulfation. The use of an alternate measure of sulfation of polysaccharides supports the contention that the mesenchyme blastula stage is followed by a drop in the sulfation of polysaccharides. In this second method embryos were labeled with 3H-acetate (30 &i/ml) for 30 min and the polysacchairdes isolated in the same way as above. There is a dramatic increase in the incorporation of 3H-acetate into CPC precipitates in the early mesenchyme blastula (118 cpm/pg protein) relative to earlier stages (late blastula: 23 cpm/pg protein). Electrophoresis of polysaccharides was done in 0.1 N HCl, which separates polysaccharides primarily on the basis of the degree of their sulfation (Hsu et al., 1972). Under these conditions 3H-acetate-labeled material coelectrophoreses with carrier hyaluronic acid (a nonsulfated polysaccharide) or chondroitin sulfate (a sulfated polysaccharide), as shown in Table 1. At this point in the discussion only the values for control embryos raised in CSW are considered. First it is evident from the ratios of radioactivity in the sulfated and the nonsulfated polysaccharides that a large majority of the polysaccharides synthesized at these stages

OF SH-A~~~~~-~~~~~~

Mid-mesencbyme

Treatment

VOLUME41, 1974

6.8

0.49

3.7

0.28

KARP AND SOLUFLSH

AMPS

are sulfated. Second, a comparison of the mid-mesenchyme blastula to the gastrula shows that this ratio drops from 6.8 to 3.7 indicating a drop in the percentage of polysaccharides that are sulfated. To assess the possible role of sulfated mucopolysaccharides in sea urchin development, it is necessary to have some idea of their heterogeneity. As an initial approach, isolated sulfate-labeled polysaccharides were separated by thin layer chromatography by the method Lippiello and Mankin (1971), which separates acidic polysaccharides on the basis of the solubilities of their calcium salts in ethanolic solutions. By this method embryonic sea urchin sulfated mucopolysaccharides are separated into three fractions, as shown in Fig. 4. Fractions 1, 2, and 3 cochromatograph with dermatan sulfate, chondroitin 4-sulfate (or heparatin sulfate), and keratan sulfate, respectively. However, the polysaccharides isolated from the embryos do not fit other criteria established for these particular sulfated polysaccharides. When the embryonic polysaccharides are treated with chondroitinase AC or chondroitinase ABC (Saito, et al., 1968) there are no hydrolysis products identifiable by paper chromatography, suggesting the absence of chondroitin sulfates A and C, as well as dermatan sulfate. Nevertheless, about 45% of the counts in peak 2 in the late mesenchyme blastula stage are removed by treatment with testicular hyaluronidase prior to chromatography, suggesting the presence of endo-N-acetylhexosamine bonds resemTABLE

115

and Cell Migration

bling those of chondroitin sulfate. Peak 3 is soluble in 5% acetic acid and is thus removed by the procedure used to stain the chromatograph, unlike authentic keratan sulfate. The material of peak 3 is excluded by Sephadex G-15 and is nondialyzable. It probably represents an acid soluble glycoprotein fraction. In Table 2 a comparison is presented between the percentage of counts in the three fractions and various stages of development. It can be seen that in the blastula stage, the rapidly migrating, acid soluble fraction predominates. From the mesenthyme blastula stage on, the synthesis of the other two fractions becomes more prevalent.

FIG. 4. Migration pattern of SsSO,-labeled POlYsaccharides after thin layer chromatography with ethanolic solutions of calcium acetate as solvent. Three distinct peaks which cochromatograph with authentic standards are seen at all stages of development. Peak 1 cochromatographs with dermatan sulfate, peak 2 with chondroitin+sulfate (or heparatin sulfate), and peak 3 with keratan sulfate. 2

RELATTVE AMOUNTS OF SULFATED-P• LYSACCHARIDEFRACTIONS WITH DEVELOPMENT Peak stage

Hatched blastula Early mesenchyme blastula Late mesenchyme blastula Gastrula (l/3) Postgastrula

1

2

3

CPM

90

CPM

7%

CPM

%

91 102 423 560 1096

4 15 19 20 24

312 239 820 969 2271

14 23 37 34 49

1851 695 950 1292 1196

80 67 43 46 26

116 Effects of Sulfate Development

DEVELOPMENTALBIOLOGY VOLIJME41, 1974

Deprivation

on

Another approach to the study of sulfate metabolism during sea urchin development is the analysis of development in the absence of this -anion in the culture medium. Figure 5 illustrates the dramatic effect of the lack of sulfate on the development of Lytechinus pictus. Embryos shown in Fig. 5a-5care control embryos grown in complete sea water, while those of Fig. 5d-5f are embryos from the same female raised in sulfate-free sea water from a time prior to fertilization. No effect on development in sulfate-free sea water is observed up to the stage when mesenchyme cells collect along the vegetal plate within the blastocoel. During development in complete sea water these primary mesenchyme cells emerge from the wall of the embryo and are soon found to be migrating along the wall of the blastula (Fig. 5a). In contrast, within the embryos developing in the sulfate-free sea water there is a normal emergence of the primary mesenchyme cells into the blastocoel but a complete lack of migration. As a result these cells collect in a mass toward the vegetal pole within the blastocoel (Fig. 5d). As the controls gastrulate (Fig. 5b) with continued primary mesenchyme cell migration, the embryos in sulfate-free sea water remain blocked at the mesenchyme blastula stage (Fig. 5e). Eventually the morphological development of the sulfate-free sea water embryos progresses. Figure 5c shows an embryo of the prism stage raised in complete sea water and Fig. 5f, an embryo of a corresponding hour raised in sulfatefree sea water. By this late hour an abortive gastrulation has occurred, producing an archenteron that is approximately onethird the length of controls and is collapsed along the vegetal wall of the blastocoel. A pair of triradiate spicules is eventually formed. Since the most conspicuous effect of sulfate-free sea water is on the activity of

the primary mesenchyme cells, it is of interest to determine if any morphological alteration of these cells occurs in embryos raised in a sulfate deficient environment. Figure 6 shows the appearance of thick epon sections of primary mesenchyme cells of embryos raised in complete sea water (Fig. 6a) and sulfate-free sea water (Fig. 6b). In both embryos the primary mesenthyme cells have a characteristic appearance with a uniformly densely staining protoplasm and a large number of vacuoles. No difference is seen in cellular structure at the light microscopic level with Richardson’s stain between embryos raised in complete sea water or sulfate-free sea water. In order to examine the surfaces of the primary mesenchyme cells under these different, growth conditions, a technique was developed whereby frozen, fixed embryos were broken into fragments to provide access to the interior of the embryo with the scanning electron microscope. If one of these embryos is cut approximately perpendicularly to the animal-vegetal axis and the vegetal fragment is examined, the primary mesenchyme cells can be seen emerging from the wall of the embryo and migrating along the inner surface of the wall (Fig. 7). The pseudopodia of these cells are evident and both the migrating primary mesenchyme cells and the columnar cells of the wall have numerous zygotic blebs. If the surface of the primary mesenthyme cells of a control embryo is examined at higher magnification (Fig. 8), it is evident that the surface itself is very rough, possibly a result of a covering layer of extracellular material. This material is seen on both the blebs and the surface of the cell itself. Figures 9 and 10 show the surfaces of primary mesenchyme cells of an embryo raised in sulfate-free sea water. Development in a sulfate deficient medium results in the surface of the primary mesenthyme cells and its blebs having a very smooth appearance with no evidence of the rough extracellular coat. The ultrastruc-

FIG. 5. Photomicrographs of living embryos of Lytechinus &us. (a-c) are control embryos raised in complete sea water at 29,38, and 68 hr after fertilization. (d-f) are of embryos raised from prior to fertilization in sulfate free sea water at 29, 38, and 68 hr after fertilization respectively. x 400. 117

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DEVELOPMENTALBIOLOGY

VOLUME 41, 1974

6b FIG. 6. Light micrographs of 0.5 pm epon sections of mesenchyme blastulae after fixation in glutaraldehyde and post-fixation in osmium tetroxide, both buffered in sea water. (a) is a control embryo raised in complete sea water and (b) an embryo raised for the same time in sulfate-free sea water. x 788.

ture of the cells following ruthenium red staining will be the subject of a subsequent report. If the appearance and properties of the cells of the sea urchin embryo are significantly altered by the sulfate content of their environment, it is important to know to what extent sulfate metabolism is restricted in the absence of added exogenous sulfate. For embryos raised in sulfate-free sea water two sources of sulfate are still available. One is from sulfate that was present as a contaminant in the chemicals used in making sulfate-free sea water, provided these embryos possessa transport system capable of concentrating sulfate against a highly adverse concentration gradient. The second source of sulfate for these embryos is an endogenous store derived from that originally present in the unfertilized egg. The actual effect on sulfate metabolism of withholding sulfate from the sea water has been examined in the analyses of sulfated and nonsulfated SH-acetatelabeled polysaccharides by electrophoresis. In Table 1 is presented a comparison of the ratio of these two groups of polysaccharides between embryos grown in complete sea water and sulfate-free sea water in both the mid-mesenchyme blastula and the gastrula. At both stages there is a 13- to

14-fold reduction in the ratio of sulfated to nonsulfated polysaccharides in embryos raised in sulfate-free sea water as compared to controls. In addition, development in the absence of sulfate results in a marked decrease in the incorporation of aH-acetate into the total polysaccharide fraction. At the mesenchyme blastula stage total polysaccharide synthesis per microgram protein was reduced at least 70% (complete sea water: 118 cpm/pg protein; sulfate-free sea water: 34 cpm/pg protein). The effect of sulfate deprivation on the ratio of sulfated to nonsulfated polysaccharides is partially reversible. When embryos developing in sulfate-free sea water to the mid-mesenchyme blastula are put in complete sea water during the incubation with sH-acetate, the ratio increases from 0.49 to 1.2. DISCUSSION

Recent studies on amphibian embryos indicate that a change in the ionic charge at the surface of cells of the dorsal lip and invaginating chordamesoderm occurs during gastrulation (Schaeffer et al., 1973) and this agrees with an activation of sulfation in these cells at this time (Kosher and Searls, 1973). The stage of development when these cells begin to incorporate YSO, is marked by changes in intercellular adhe-

KARPANDSOLURSH

AMPS

and Cell Migration

119

FIG. 7. Scanning electron micrograph of the vegetal fragment of a control embryo with the primary mesenchyme cells in the process of emergence from the vegetal wall and migration in an animal direction. Large zygotic blehs are seen on the columnar cells of the wall and smaller blebs on the mesenchyme cells. x 850. FIG. 8. Scanning electron micrograph of a primary mesenchyme cell of a control embryo. The surface of this cell, including that of the blebs, is covered with a coat of extracellular material that gives the surface a very rough appearance. This is particularly evident on the surface of the adjacent cell. x 7820. FIG. 9. Scanning electron micrograph of a cluster of primary mesenchyme cells of an embryo raised in sulfate-free sea water. The surfaces of these cells, and their blebs, are smooth, indicating the absence of material that was present in the controls. x 3825. FIG. 10. Scanning electron micrograph of one primary mesenchyme cell of an embryo raised in sulfate-free sea water. x 7820.

sions and the movements of these cells to a position within the embryo. In the sea urchin, as in the frog, it appears that sulfated polysaccharide metabolism is closely linked with the activity of a migratory class of cells. In the early blastula stage, Alcian blue selectively stains the cytoplasm of the cells of micromere origin (Sugiyama, 1972). As

these cells approach the stage at which they are liberated into the blastocoel, there is material at the circumference of these cells that is stained by Hale’s reaction and later this material is seen to cover these cells (Sugiyama, 1972). Correlated with the histochemistry is the finding that the presumptive primary mesenchyme cells are the only cells of the late blastula that

120

DEVELOPMENTAL

BIOLOGY

incorporate YSO, (Sugiyama, 1972). This label is concentrated in the intercellular space and moves with the mesenchyme cells as they migrate. In the present study the determination of YSO, uptake and incorporation into polysaccharides indicates that the time of appearance and migration of the primary mesenchyme cells represents the stage of greatest synthesis of sulfated acid mucopolysaccharides during early sea urchin development. The ability of the sea urchin embryo to take up 35S0, varies approximately 40 fold from the time of the hatched blastula to the prism. If data on the incorporation of exogenous sulfate is to provide an estimate of the acid mucopolysaccharide synthetic activity of the embryo, then these differences in uptake must be taken into account. The ratio of uptake to incorporation is shown in Fig. 3 and indicates that the mesenchyme blastula stage has the greatest level of polysaccharide sulfation. This assumes that differences in sulfate pools do not exist, though no direct evidence on the size of sulfate pools has been obtained for sea urchin embryos. In an attempt to confirm the findings with 35S0,, an alternative method was utilized to measure the level of sulfation of polysaccharides. The incorporation of 3H-acetate into sulfated and nonsulfated polysaccharide fractions after electrophoresis in 0.1 N HCl similarly suggests a drop occurs in the sulfation of polysaccharides after the mesenchyme blastula stage. Since this conclusion is based only on the differences in the ratio of nonsulfated to sulfated polysaccharides at different stages, it assumes that there are no significant changes in overall polysaccharide synthesis at these times, which could be determined only after the measurement of the uptake and incorporation of 3H-acetate. The finding that at the mesenchyme blastula stage that nearly 90% of the polysaccharides synthesized become sulfated emphasizes the importance of sulfation at

VOLUME

41, 1974

this stage. Since these electrophoretic ratios measure 8H-acetate incorporation using a separation based on sulfation, which occurs after the SH-acetate has been incorporated, these ratios represent a minimal value of sulfated to nonsulfated polysaccharides. Attempts to identify the nature of the sulfated polysaccharides have served to eliminate from consideration those molecules familiar from work on vertebrate systems. Thin layer chromatography separates the purified polysaccharides into three fractions (Fig. 4), which cochromatograph with authentic standards from vertebrate tissues. However, enzymatic analysis indicates that the embryonic sea urchin molecules are not chondroitin sulfate (A or C), dermatan sulfate, or keratan sulfate. The nature of the component sugars and the types of linkages have not been determined. Heparan sulfates are known to be membrane-associated in Chinese hamster cells (Kraemer, 1971). In the present study, in spite of technical difficulties, there was no consistent nitrous acid degradation of sea urchin material, suggesting the absence of heparan sulfate, as well. Kinoshita (1971) has claimed that heparan is made by sea urchin gastrulae based on its solubility in 1.2 M NaCl and the pressence of hexosamine. No enzymatic, electrophoretic, chromatographic or other analytic procedure was used to verify this claim, and this point remains in question. When the percentages of incorporated YSO, in each of the three chromatographic peaks of Fig. 4 is determined for the various stages examined, there appears to be a relative decline during development in the rapidly migrating, acid soluble material of peak 3. No clear correlation can be made between any of the peaks and a given developmental event, and it is likely that some of the three classes are themselves heterogeneous. This is clearly the case for peak 2 which is reduced approximately in half by testicular hyaluronidase.

KARP AND SOLURSH

AMPS and Cell Migration

For several reasons the study of the sea urchin embryo provides a unique opportunity for the study of the relationship of acid mucopolysaccharides to the activities of a migratory cell. These embryos are transparent and the migratory cells are easily followed. At the stage when significant levels of sulfation are beginning it appears that the cells engaged in this activity are the primary mesenchyme cells (Sugiyama, 1972); therefore, quantitative studies of sulfate incorporation by the whole embryo should reflect largely the activity of these cells. In contrast, in the frog embryo the incorporation of sulfate into cells of the invaginating archenteron represents only a fraction of the total sulfate incorporated, the bulk being found associated with the yolk platelets and having no morphogenetic role (Kosher and Searls, 1973). If it is true that the primary mesenchyme cells are the main source of sulfated polysaccharides in the late blastula, then the results of the present study indicate that these cells synthesize a variety of acid mucopolysaccharides, some of which are destined for the cell surface. An additional advantage of the sea urchin embryo is its dependence upon an exogenous sulfate source, which can be easily withheld. The effect on various aspects of development of sea urchins raised in sulfate free sea water has been studied in several laboratories (Herbst, 1904; Lindahl, 1942; Immers, 1956; Immers and RunnstrGm, 1965; Runnstrijm et al., 1964; Sugiyama, 1972). In the studies of Immers and Runnstriim on the species Paracentrotus liuidus, the effect of sulfate deprivation is interpreted as causing an alteration in the normal animal-vegetal gradient. This alteration produces an animalization of the embryo, with one of the effects being a disruption of the normal activities of the primary mesenchyme cells and the inhibition of archenteron formation. The effect of the lack of sulfate on sulfated mucopolysaccharides was suggested as one of the

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major developmental effects. In Purucentrotus the first sign of abnormality is an over-development of the ciliated apical tuft in the animal region of the blastula, reflecting the apparent animalization. We have found a similar effect of sulfate-free sea water on Arbacia punctulata, but the development of Lytechinus pictus in sulfatefree sea water is not characterized by any evidence of animalization through the formation of the mesenchyme blastula. At this stage there appears a dramatic arrest of development in all embryos, as mesenthyme cells pile up within the vegetal region of the blastocoel. For some reason this effect of sulfate deprivation has not been clearly described in any of the above studies. Whether these differences are the result of species specific effects is not known, although it is not unlikely that other species may retain a greater sulfate content in the absence of the exogenous ion. Since the ratios of sulfated to nonsulfated polysaccharide synthesis are found to be reduced to approximately 5% of their normal value (Table 1) and it is the primary mesenchyme cells that appear to be the major detectable cell incorporating sulfate (Sugiyama, 1972), it is not surprising that the activity of these cells is disturbed. Examination of the primary mesenchyme cells in sulfate-free embryos in the light microscope failed to reveal any obvious structural abnormalities. A study of the primary mesenchyme cells in the electron microscope (Gibbins et al., 1969) has already indicated the role of microtubules in the maintenance of the elongated processes of these cells. Primary mesenchyme cells of sulfate deficient embryos remain rounded and an effect on microtubule orientation would be expected, unless these abnormal cells simply have a reduced affinity for the substratum. To date, ultrastructural studies have revealed no intracellular abnormalities. Embryos deprived of exogenous sulfate maintain normal swimming activity for several days,

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suggesting the sulfate deficiency has little effect on the energy metabolism or ciliary function of these embryos. When the surfaces of the primary mesenthyme cells are examined with the scanning electron microscope, a distinct difference is found between embryos raised in the presence and absence of sulfate. The mesenchyme cells of control embryos possess a surface with a rough appearance in the scanning electron microscope due to what appears to be a covering of extracellular material. In contrast, the mesenchyme cells of sulfate-deficient embryos have a very smooth surface. The basis for this difference in appearance in the scanning electron microscope may reflect the absence of the cell coat on the mesenchyme cells of the sulfate-deficient embryos. Further studies are underway to determine whether or not the migratory activity of these cells is related to the presence or absence of a normal acid mucopolysaccharide-containing cell coat. This work was supported by grants GB 28228 from NSF and HD-05505 from NIH. We thank Rebecca S. Reiter for her skilled technical assistance in various aspects of this work and Gene C. Shih for his help with the scanning electron microscope. REFERENCES T. (1951). Technique for the preservation of 3-dimensional structure in preparing specimens for the E.M. Trans. N.Y. Acad. Sci. 13, 130-134. GIBBINS, J. R., TILNEY, L. G., and PORTER, K. R. (1969). Microtubules in the formation and development of the primary mesenchyme in Arbacia punctulata. I. The distribution of microtubules. J. Cell

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