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Polysaccharides sulfated at the time of gastrulation in embryos of the sea urchin Clypeaster japonicus
Experimental Cell Research 159 (1985) 353-365
Polysaccharides Sulfated at the Time of Gastrulation in Embryos of the Sea Urchin Clypeasterjaponicus MASAAKI YAMAGUCHI* and SEIICHIRO KINOSHITA Misaki Marine Biological Station, University of Tokyo, Kanagawa-ken 238-02, Japan
Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeasterjaponicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extraceUular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation. © 1985AcademicPress, lnc.
Increasing attention has been paid to the biological significance of sulfated PSs and proteoglycans, particularly in morphogenesis during embryonic development, such as cell migration and gastrulation. In sea urchins, the arrest of development at the blastula stage under sulfate-deficient conditions was shown first in 1897 by Herbst [1], and subsequently many investigations have been devoted to elucidation of the physiological roles of sulfate in development. Immers drew attention to the sulfation of PSs by using histochemical and autoradiographic procedures [2, 3]. Sulfated PSs were observed to be distributed in the hyaline layer, basement membrane and blastocoelic cavity [2-5], suggesting a possible participation of these structures in morphogenesis. Sulfated PSs in the hyaline layer of early embryos were secreted by the cortical granules of eggs during fertilization [24]. Recently the presence of an acellular fibrous structure in the blastocoelic cavity was shown by electron microscopy. This structure is considered to provide mesenchyme ceils with orientation and guidance during gastrulation [6-9], though whether this structure is composed of proteoglycans remains to be elucidated. The plausible participation in the gastrulation process of proteoglycans containing sulfated PSs was suggested by the facts that inhibitors of proteo* To whom offprint requestsshould be sent. Address: Misaki Marine Biological Station, University of Tokyo, Misaki, Kanagawa-ken 238-02, Japan.
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glycan synthesis prevent the gastrulation of sea urchin embryos [10] and that th~ activity of PS-sulfating systems increase at the time of gastrulation [11-13]. On the other hand, the presence of heparan sulfate [14], dermatan sulfate [14-17] and the complex of sulfated fucan and unidentified glycosaminoglycan [18] in sea urchin embryos has already been reported. The present study was designed to compare acid PSs prepared from embryos at the blastula and gastrula stages with those reared under sulfate-free conditions, with the aim of distinguishing those PSs that are specifically related to gastrulation. Another objective was to analyse the specific PSs which are coincidentally sulfated at the time of gastrulation, by means of [35S]sulfate labeling and subsequent autoradiography. MATERIAL AND METHODS
Material Eggs of the sea urchin, Clypeasterjaponicus, obtained by introducing 0.5 M KCI into the body cavity, were washed twice with sea water, After insemination, embryos were allowed to develop at 20°C with gentle stirring. In the present study, artificial normal sea water (NSW) and sulfate-free sea water (SFSW), purchased from Jamarine Laboratory (Osaka, Japan) were used throughout the experiment. The ionic composition (g/I) of NSW is: C1, 17.099; Na, 9.409; Mg, 1.137; S, 0.774; Ca, 0.395; K, 0.344 pH 8.2. SFSW is produced by exchanging MgSO4 (the only S source) for MgClz.
Dissociation of Embryonic Cells Embryos were collected by centrifugation and washed twice with 3 vol of 1 M glycine containing 2 mM EDTA (pH 8.2) [19]. Embryonic cells were dissociated completely by shearing in 10 vol of the glycine-EDTA solution using a loose-fitting hand-operated Dounce homogenizer. The dissociated cells were sedimented by centrifugation at 6400 g for 5 min. The sedimented material was mainly composed of naked embryonic ceils and is referred to as the 'dissociated cell' fraction. The supernatant and previous washings were pooled. This was composed mainly of extracellular materials and is referred to as the 'glycine extract' fraction. All procedures were performed at 0°C.
Preparation of Acid PSs Acid PSs were prepared from both the 'glycine extract' and 'dissociated cell' fractions, and also from whole embryos in some experiments. The 'glycine extract' was dialysed against water at 4°C. Three volumes of ethanol in the presence of 1% sodium acetate were added and kept at -20°C overnight in order to precipitate PSs. The precipitate thus obtained was defatted by sequential extraction with ethanol, ethanol--ether (3 : 1), and ether. The dried precipitate obtained from 107 embryos was suspended in 8 ml of 0.5 N NaOH and incubated for 24 h at 4°C for the purpose offl-elimination. After incubation, the pH of the solution was re-adjusted to 7.6 with HCI in the presence of 50 mM Tris. Twenty mg of Pronase E (106 tyrosine units/g; Kaken Kagaku, Tokyo) dissolved in 0.2 ml of water was added and the mixture was incubated further for 3 days at 37°C in the presence of 0.02 % sodium azide and a few grains of thymol. Each day the pH of the solution was re-adjusted and 20 mg of fresh Pronase E was added. The complete digestion with Pronase, one-fifth volume of 60 % trichloroacetic acid (TCA) was added, the mixture was left to stand for 30 min at 0°C and then centrifuged. The supernatant was neutralized with NaOH and PSs were precipitated by adding 3 vol of ethanol as described above. The precipitate was dissolved in 2.5 ml of 10 mM Tris-HCl (pH 7.4), 4 mM MgC12 containing 2 mg each of DNase I and RNase A (Sigma, St. Louis, Mo.). After incubation for 12 h at 37°C, the mixture was diluted up to 9 ml with 0.02 M sodium sulfate, and then l ml of 10 % cetylpyridinium chloride (CPC) was added. After incubation at 37°C overnight, the complex formed was spun down, dissolved in 2,5 ml of 4 M MgCI2 and precipitated again with ethanol in order to remove CPC. The precipitate thus obtained was washed with ethanol, dried and used as acid PSs in the present study. Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation
355
1. Recoveries of [35S]sulfate incorporated into embryos and authentic chondroitin sulfate C at several steps of the preparation of acid PSs
Table
Embryos [35S]sulfate-labelled for 8 h during gastrula
Chondroitin sulfate C
Step of the preparation of acid PSs
Radioactivity ~
Recovery
Recoveryb
Defatted powder Ethanol precipitate CPC precipitate Ethanol precipitate
4.67x 105 cpm 3.21 x 105 1.97× 105 1.56x 105
100 % 70 43 35
100 Ixg (%) 75 75 62
a Calculated from the radioactivity of an aliquot. b Estimated according to the method of Dische [20].
The 'dissociated cell' preparations and packed whole embryos were homogenized in 3 vol of water at 0°C using a motor-driven Teflon-glass homogenizer. To the homogenate, 3 vol of ethanol was added in the presence of 1% sodium acetate. The succeeding procedures were the same as described above. The recovery ratd of PSs by the procedure employed in the present study was determined using authentic chondroitin sulfate C and PSs obtained from [35S]sulfate-labelled Clypeasterembryos (table 1).
Electrophoresis PSs were subjected to electrophoresis on cellulose acetate sheets (10× 10 cm, Juko Sangyo, Tokyo) in 0.47 M formic acid -0.1 M pyridine (pH 3.0) for the first dimension under a constant current of 1 mA/cm for 40 min, and in 0.1 M barium acetate for the second dimension under the same current for 3 h according to the method of Hata & Nagai [25]. The spots were contrasted by staining with 0.1% Alcian blue, pH 3.0 in 1% acetic acid. The amount of acid PSs applied on the sheet for each electrophoresis was equivalent to that prepared from 3 × 103 embryos.
Enzymatic Digestion of Acid PSs Digestion of acid PSs with chondroitinase AC and ABC was carried out according to the method of Yamagata et al. [21], and digestion with Streptomyces hyaluronidase was done by the method of Ohya & Kaneko [22]. All enzymes were products of Seikagaku Kogyo (Tokyo). After digestion, the reaction was stopped by heating the mixture at 100°C for 2 min and centrifuged at 14000 g for 20 min. The supernatant was used for electrophoresis.
Incorporation of [35S]sulfate Incorporation into whole embryos. Embryos
at the desired developmental stage (3.5x 104 embryos each stage) were washed twice with SFSW and then transferred to 2 ml of SFSW, in which 1 ixCi of cartier-free [35S]sulfate (Japan Atomic Institute, Tokyo) and 200 units of penicillin were contained. The labelling was continued for 1 h at 20°C. Sampling was made at intervals of 2 h. At the end of the labelling period, 2 ml of 20 % TCA was added to the embryo suspension and the mixture left to stand for 30 min at 0°C. The precipitate was washed twice with ice-cold 10% TCA and twice defatted by successive extraction with ethanol, ethanol-ether (3 : 1), and ether. The radioactivity in the dried precipitate was estimated using a liquid scintillation counter after dissolving it into Soluene 350 (Packard, Downers Grove, I11.). For histological observation, labelling of embryos was done at a higher concentration of [35S]sulfate (50 ixCi/ml SFSW for 1 h). Embryos were fixed in Carnoy's solution and sectioned as usual in paraffin at 6--8 laan. Sections were dipped in Sakura NR-M2 emulsion (Konishiroku Photo Inc., Japan) for autoradiographic observation.
Exp CellRes 159(1985)
356 Yamaguchi and Kinoshita
Fig. 1. Development of the sea urchin, Clypeasterjaponicus embryos reared in NSW and SFSW at 20°C. Embryo reared in (a) NSW for 21 h (mesenchyme blastula stage); (b) SFSW for 21 h (note that no retardation in development is observed); (c) NSW for 38 h (gastrula stage); (d) SFSW for 38 h (note that the assemblage of primary mesenchyme cells and the second step of gastrulation are prevented). × 113.
Incorporation into PSs. Embryos were washed twice with SFSW and then allowed to develop for 8 h at 20°C in 50 ml NSW-SFSW (1 : 9), in which 100 ~Ci of [35S]sulfate and 5 000 units of penicillin were contained. At the end of the labelling, one-fifth volume of 60% TCA was added to the embryo suspension and the mixture was left to stand for 30 min at 0°C. PSs were prepared as described above, making this precipitate the starting material. Labelled PSs were electrophoresed on cellulose acetate sheets and exposed to a Kodak X-omat AR film for autoradiographic analysis.
RESULTS
Development of Sea Urchin Embryos Fig. 1 shows the development of sea urchin embryos under the conditions employed in the present study. Embryos, both in NSW and SFSW, develop normally up to the stage at which primary mesenchyme cells are shed into the blastocoel (fig. 1 a, b). Differences were observed from this stage onward. In normal embryos, primary mesenchyme cells initially migrated on the inner surface of the ectodermal wall to form 'ring-' and 'cable-shaped' structures which constituted partial matrices for skeletal rudiments later, and this was followed by initial invagination and stretching of the primitive gut (fig. 1 c). However, in the embryos reared in SFSW, primary mesenchyme cells appeared to be prevented from forming the above-mentioned structures and the second step of the gastrulation process was inhibited, although the initial step of invagination did occur (fig.
ld). Electrophoretic Analysis of Acid PSs Acid PSs were prepared from the 'glycine extract' and 'dissociated cell' fractions and were compared by means of two-dimensional electrophoresis on Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 357 cellulose acetate sheets. Comparison was made between hatching blastula (15 h after fertilization), gastrula (30 h) and embryos reared in SFSW for 30 h. Acid PSs stained with Alcian blue were classified according to their mobilities in the first and second dimensions of the electrophoresis. Each PS was noted in a coordinate manner by the combination of numerals (1--6; first dimension as abscissa) and letters (A-F; second dimension as ordinate). Acid PSs in the ' glycine extract' fraction. Acid PSs prepared from the 'glycine extract' fraction, composed mainly of extracellular materials, were compared electrophoretically. At blastula stage (fig. 2 a), two major spots (2A, 2B) and one minor spot (4F) were detected. At gastrula stage (fig. 2 b), two additional spots (3B, 3C) were observed. As is seen in fig. 2 g, 3B and 3C became major spots in late gastrula stage (38 h) and another new spot (3A) appeared by this stage. On the other hand, these new spots were never observed in the embryos reared in SFSW, which were prevented from gastrulation, while other labelled spots (5E, 6D) appeared instead (fig. 2c). The mobilities of 5E and 6D in the first dimension of electrophoresis (formic acid-pyridine) were as low as that of authentic hyaluronic acid, suggesting that both PSs may be less or non-sulfated PSs, as appeared in extracellular materials under the sulfate-deficient condition. Acid PSs in the "dissociated cell' fraction. Acid PSs prepared from the"dissociated cell' fraction, composed mainly of intracellular substances, were next compared. Six spots IF, 2A, 2B, 2F, 4D, 4F) were detected at blastula stage (fig. 2d). Of these spots, 2A, 2B and 4F were common between the 'glycine extract' and the 'dissociated cell' fractions. Therefore, IF, 2F and 4D were to be regarded as PSs characteristic of the 'dissociated cell' fraction. The electrophoretic pattern of acid PSs obtained from gastrula (fig. 2 e) was essentially the same as that from blastula, provided the common spots between the two fractions (2A, 2B, 3B, 3C, 4F) were excluded. The same can be said for the acid PSs of the embryos reared in SFSW with the only exception of 6F (fig. 2J), which showed very low mobility in the first dimension of electrophoresis, suggesting again that this may be less or nonsulfated PS formed within cells under sulfate-deficient conditions.
Enzymatic Digestion o f Acid PSs Embryos were reared in NSW and SFSW for 30 h. Acid PSs were prepared from whole embryos and subjected to digestion by Streptomyces hyaluronidase and chondroitinases AC and ABC. Electrophoretic patterns of acid PSs were compared before and after digestion (fig. 3). It was found that hyaluronidase (normal substrate, hyaluronic acid) digested none of the PSs in the embryos reared either in NSW or SFSW. Chondroitinase AC (normal substrate, hyaluronic acid, chondroitin, chondroitin sulfate) digested 6D, and chondroitinase ABC (normal substrate, those of chondroitinase AC and Exp Cell Res 159 (1985)
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Polysaccharides sulfated during gastrulation
359
Fig. 3. Enzymatic susceptibility of acid PSs as analysed by means of electrophoresis. (a, b) Acid PSs obtained from normal embryos (30 h, gastrula stage) were subjected to the action of chondroitinase ABC (a) before; (b) after digestion (note that 3C is susceptible to chondroitinase ABC.); (c, d) Acid PSs obtained from embryos reared in SFSW for 30 h were subjected to the action of chondroitinase AC: (c) before; (d) after digestion. (Note that 6D is susceptible to chondroitinase AC.)
dermatan sulfate) digested 3C and 6D. The other spots were resistant to chondroitinase digestion. In other words, 3C (a PS that appeared extracellularly in normal gastrula) was resistant to hyaluronidase and chondroitinase AC but susceptible to chondroitinase ABC, suggesting that 3C may be dermatan sulfate isomer; and 6D (a PS which appeared extracellularly only in permanent blastula reared in SFSW, and thus corresponded electrophoretically to authentic hyaluronic acid) was resistant hyaluronidase but susceptible to both chondroitinase AC and ABC, suggesting that 6D may be related to non-sulfated chondroitin.
Incorporation of ffSS]suifate into Sea Urchin Embryos Embryos were labelled with [35S]sulfate separately for 1 h at 2-h intervals from fertilization to late gastrula stage (38 h) and the radioactivity incorporated into the TCA-insoluble, defatted fraction was measured on each sampling. As is seen in fig. 4, the rate of incorporation increased gradually until early gastrula stage (28 h) with no conspicuous peaks. Thereafter it increased sharply, nearly quadrupling in value by the end of gastrulation. It is interesting to note that this steeply ascending phase in the incorporation rate (30-38 h) corresponded to the second step of gastrulation.
Fig. 2. Two-dimensional electrophoretograms of acid PSs prepared from (a, b, c, g) 'glycine extract', (d, e, J)'dissociated cell' fractions which were obtained from (a, d) blastula; (b, e, g) gastrula; (c,J), embryos reared in SFSW. The electrophoresis in the first dimension was carded out in 0.47 M formic acid-0.1 M pyridine (pH 3.0), and the second in 0.1 M barium acetate. Authentic spots: Ha, Hyaluronic acid; D, dermatan sulfate; C, chondroitin sulfate C; Hp, heparin. Exp Cell Res 159 (1985)
360 Yamaguchi and Kinoshita 20-
:~ ~15.
~to. .9o
]
mBl
[.
Gs
>
u
% o
1'0 2'0 3'0 time after fertilization (hr)
3'8
Fig. 4. Incorporation of [35S]sulfate into sea urchin embryos at different stages of development. 3.5×104 embryos were reared in NSW and washed twice with SFSW before labelling. They were incubated in 2 ml of SFSW containing 1 ~tCi of [35S]sulfate for I h. Radioactivity of TCAinsoluble, defatted fraction of the labelled embryos was measured using a liquid scintillation counter, mBI, Mesenchyme blastula; Gs, gastrula.
Autoradiographic Analysis of Acid PSs Labelled with [35S]sulfate Acid PSs were prepared from sea urchin embryos labelled with [35S]sulfate at various stages of development and analysed electrophoretically. Embryos were labelled for 8 h in NSW-SFSW (1 : 9) at early blastula (10-18 h), mesenchyme blastula (18-26 h) and gastrula (26-34 h) stages. As seen in fig. 5, radioactivity was detected to be very weak in spot 2AB (2A and 2B were combined here) at early blastula stage, while for other spots no radioactivity was observed; at mesenchyme blastula stage, radioactive spots were found to increase in number and five spots were detected (2B, 2F, 3A, 3B, 3C); at gastrula stage, radioactive spots did not increase in number, but 3B and 3C became predominant. The incorporation of [3SS]sulfate into each spot on the electrophoretogram was measured quantitatively by measuring radioactivity directly by scintillation counter after cutting out the corresponding spots on the cellulose acetate sheet and dissolving them in dimethyl sulfoxide (DMSO). As seen in fig. 6, the amount of radioactivity incorporated into total PSs increased as development proceeded and the greater part of the incorporated radioactivity was recovered in 3B and 3C, which accounted for 22 and 65 % of the total incorporation at gastrula stage, respectively. It should be noted that the recovery rate of polysaccharides is limited at about 60 % at the maximum due to the inefficiency of recovery at the step of ethanol precipitation (table 1). The rate would be much lower when the amount of polysaccharides is smaller in the solution. Therefore, it is plausible that the total amount of laSS]sulfate actually incorporated is greater than that estimated, and that this tendency is more pronounced in the earlier stages of development.
Distribution of [~SS]sulfate in Histological Sections of Embryos Distribution of incorporated [35S]sulfate was examined by comparing the autoradiogram and the stainability with Alcian blue on the same sections of sea urchin embryo at the time of gastrulation (fig. 7). Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation
361
Fig. 5. Incorporation of [35S]sulfate into PSs as analysed by means of electrophoresis. PSs were
prepared from embryos at (a, b), early blastula; (c, d) mesenchyme blastula; (e,J) gastrula stages. Just prior to the preparation of PSs, embryos were labelled with [35S]sulfate for 8 h. (a, c, e) Electrophoretograms stained with Alcian blue, pH 3.0; (b, d, J) autoradiograms.
Exp Cell Res 159 (1985)
362 Yamaguchi and Kinoshita 2AB 2F 3A
o >, 15
x
~
5
3C Fig. 6. Incorporation of [35S]sulfate into PS spots of two-
dimensional electrophoretograms shown in fig. 5. Each spot was cut out separately, dissolved in dimethylsulfoxide (DMSO), and the radioactivity was measured.
10-18 eBI
18-26 mBI
26-34(hr) Gs
Fig. 7 a-d shows the histological sections and the corresponding autoradiograms of the embryos at early gastrula stage (30 h) fixed just after labelling for 1 h. Grains due to radioactivity were observed to be distributed on the ectodermal wall and mesenchyme cells in the blastocoel. In the ectodermal walls, especially in the animal pole region which is thickened, grains were localized mainly on the inner and outer surfaces (fig. 7 a, b). It will be noted that the same pattern of positive Alcian blue staining was recognizable. Fig. 7 e,fshows the distribution pattern of [35S]sulfate in an embryo which was labelled for I h at the 30-h stage and subsequently subjected to chase for 6 h under aSS-free conditions until the 37 h stage (note that gastrulation has proceeded). It is obvious that the localization of grains was comparatively more dispersed in the blastocoelic cavity and that Alcian blue-positive structures spread throughout the blastocoelic cavity. The grains in the blastocoelic cavity seemed to coincide with the fibrous structures. The correspondance between the grains and the histological structures was also observed in fig. 7 a - d rather clearly as the structures existed partially in the blastocoelic cavity.
DISCUSSION
Changes o f Acid PSs Observed at the Time o f Gastrulation In this study, PSs were prepared separately from extracellular materials which constituted the 'glycine extract' and intracellular substances referred to as the 'dissociated cell' fraction. Comparing progressive changes in these two groups of PSs during development, it was observed that the PSs of extracellular origin changed remarkably at the time of gastrulation. Three kinds of PS, designated 3A, 3B and 3C in the present study, were not detected in embryos at hatching blastula stage, but they did appear at mesenchyme blastula to early gastrula stage Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 363
Fig. 7. Distribution of incorporated [3SS]sulfate in sea urchin embryos. Embryos were labelled with
[sSS]sulfate for 1 h at early gastrula stage (30 h), and fixed (a-d) at the end of the labelling period or (e,j) after a chase of 6 h. (a, c, e) Sections stained with Alcian blue; (b, d, j0 autoradiograms observed in dark field (b, d, fcorrespond to a, c, e, respectively). × 170.
and then increased conspicuously at late gastrula stage. Such changes were never observed in embryos prevented from gastrulating by rearing in SFSW. In the PS electrophoretic patterns, several spots were observed in common between the 'glycine extract' and the 'dissociated cell' fractions. Since PSs that appear de novo can be considered to have been originally synthesized intraceUulady, probably in the form of complexes of PSs and proteins such as proteoglycans, these common PSs could naturally occur within cells before extracellular secretion. Aside from these common PSs, it is remarkable that the remaining Exp Cell Res 159 (1985)
364
Yamaguchi and Kinoshita
kinds of PSs of specific intracellular origin exhibited practically no changes during development.
Partial Identification of 3C and 6D and Their Possible Relationship in Biosynthesis Among the specific acid PSs which were synthesized de novo at the time of gastrulation, 3C was identified as dermatan sulfate on the basis of its mobility in electrophoresis and its sensitivity to chondroitinase ABC. In the permanent blastula reared in SFSW, none of these PSs was detected, while, instead, different PSs, 5E and 6D, were observed. Of these two PSs, 6D was considered very likely to be non-sulfated chondroitin, on the basis of its electrophoretic mobility and its susceptibility to both of the chondroitinases AC and ABC. Apart from still other unidentified PSs, an interesting hypothesis can be formulated, that two PSs, dermatan sulfate (3C) and chondroitin (6D), may be mutually related with respect to their synthetic pathways. As was demonstrated by Lindahl's group in a cell-free human fibroblast system [23], chondroitin is a precursor of dermatan sulfate at the time of its synthesis. According to them, sulfation of N-acetylgalactosamine adjacent glucuronic acid (GIcUA) in chondroitin molecules is necessary for the inversion of GlcUA to iduronic acid which is a constituent sugar of dermatan sulfate. If this step in the reaction (called C-5 epimerization) occurs in sea urchin embryos, the sugar chains which will become dermatan sulfate in NSW remain in the chondroitin state under sulfate-free conditions, for lack of C-5 epimerization. There are several reports on the presence of dermatan sulfate in sea urchin embryos [14-17].
[SSS]Sulfate Incorporation into PSs and Histological Distribution of Radioactivity Labelling experiments using [SSS]sulfate demonstrated after electrophoretic analysis that most of the radioactivity is recoverable in two PSs, 3B and 3C, of the "glycine extract" fraction at the time of gastrulation. In autoradiograms of corresponding histologically sectioned embryos, grains were distributed mainly in extracellular matrices, including the hyaline layer, basement membrane and fibrous structures in the blastocoelic cavity. It may be concluded that the bulk of [35S]sulfate incorporated during gastrulation is to be found in extracellular structures probably composed of sulfated PS-containing substances such as proteoglycans.
The nature of the inner surface of the ectodermal wall may be important for determining the distribution pattern of primary mesenchyme cells according to the animal-vegetal gradient. On the other hand, the fibrous structures in the blastocoel have been considered recently to provide a matrix for the guidance of the invaginating primitive gut [6--9]. It was revealed by electron microscopy that these structures are composed of fibrous and granular components, and the latter Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 365 resemble morphologically sulfated proteoglycans identified in some other systems [7, 14] and they are susceptible to sulfate deprivation [7]. In conclusion, it was demonstrated in this study that the synthesis of new kinds of PSs and their sulfation may be indispensable for the achievement of gastrulation and that these sulfated PSs may take part in morphogenetic movements within extraceUular matrices. We are indebted to Professor A. Gorbman of Washington University for kindly reading the manuscript, and also to Dr T. Yamagata for valuable suggestions during this investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Herbst, C, Wilhelm Roux's arch 5 (1897) 649. Immers, J, Exp cell res 10 (1956) 546. - - Ibid 24 (1961) 356. Motomura, I, Bull mar biol Asamushi 10 (1960) 165. Sugiyama, K, Dev growth differ 14 (1972) 63. Endo, Y & Noda, Y D, Zool mag 86 (1977) 309. Katow, H & Solursh, M, Exp cell res 136 (1981) 233. Akasaka, K, Amemiya, S & Terayama, H, Exp cell res 129 (1980) 1. Kawabe, T T, Armstrong, P B & Pollock, E G, Dev bioi 85 (1981) 509. Kinoshita, S & Saiga, H, Exp cell res 123 (1979) 229. Kinoshita, S, Exp cell res 87 (1974) 382. Nozawa, A & Kinoshita, S, J fac sci univ Tokyo see IV 14 (1977) 11. Saotome, K & Yanagisawa, T, Dev growth differ 21 (1979) 401. Solursh, M & Katow, H, Dev biol 94 (1982) 326. Yamagata, T & Okazaki, K, Biochim biophys acta 372 (1974) 469. Oguri, K & Yamagata, T, Biochim biophys acta 541 (1978) 385. Saotome, K & Yanagisawa, T, Dev growth differ 21 (1979) 413. Akasaka, K & Terayama, H, Exp cell res 146 (1983) 177. Kane, R E, Exp cell res 81 (1973) 301. Dische, Z, J biol chem 167 (1947) 189. Yamagata, T, Saito, H, Habuchi, O & Suzuki, S, J biol chem 243 (1968) 1523. Ohya, T & Kaneko, Y, Biochim biophys acta 198 (1970) 607. Malmstrfm, A, Fransson, L-/~, H66k, M & Lindahl, U, J biol chem 250 (1975) 3419. Schuel, H, Kelly, J W, Berger, E R & Wilson, W L, Exp cell res 88 (1974) 24. Hata, R & Nagai, Y, Anal biochem 45 (1972) 462.
Received November 20, 1984 Revised version received March 5, 1985
Exp Cell Res 159 (1985)
Polysaccharides Sulfated at the Time of Gastrulation in Embryos of the Sea Urchin Clypeasterjaponicus MASAAKI YAMAGUCHI* and SEIICHIRO KINOSHITA Misaki Marine Biological Station, University of Tokyo, Kanagawa-ken 238-02, Japan
Based on the fact that the development of sea urchin embryos is arrested at the blastula stage in sulfate-free sea water (SFSW), we attempted in the present study to elucidate the nature of sulfated polysaccharides (PSs) which appear at the time of gastrulation in embryos of the sea urchin Clypeasterjaponicus. Electrophoretic analysis of PSs prepared from embryos at different developmental stages revealed that three kinds of PSs (3A, 3B, 3C) appear de novo at the gastrula stage, and that these PSs are not found in embryos at the hatching blastula stage, nor are they found in permanent blastula reared in SFSW. These, three PSs were mostly of extraceUular matrix origin. Among them, 3C was identified as dermatan sulfate on the basis of its electrophoretic mobility and sensitivity to enzymatic digestion. 3A and 3B remained to be identified. Further, a plausible precursor of 3C, which was sulfated under normal conditions, was detected as 6D in the embryos reared in SFSW. Autoradiographic analysis using [35S]sulfate revealed that these three PSs, accounted for more than 90% of [35S]sulfate incorporated into the acid PS fraction during gastrulation. © 1985AcademicPress, lnc.
Increasing attention has been paid to the biological significance of sulfated PSs and proteoglycans, particularly in morphogenesis during embryonic development, such as cell migration and gastrulation. In sea urchins, the arrest of development at the blastula stage under sulfate-deficient conditions was shown first in 1897 by Herbst [1], and subsequently many investigations have been devoted to elucidation of the physiological roles of sulfate in development. Immers drew attention to the sulfation of PSs by using histochemical and autoradiographic procedures [2, 3]. Sulfated PSs were observed to be distributed in the hyaline layer, basement membrane and blastocoelic cavity [2-5], suggesting a possible participation of these structures in morphogenesis. Sulfated PSs in the hyaline layer of early embryos were secreted by the cortical granules of eggs during fertilization [24]. Recently the presence of an acellular fibrous structure in the blastocoelic cavity was shown by electron microscopy. This structure is considered to provide mesenchyme ceils with orientation and guidance during gastrulation [6-9], though whether this structure is composed of proteoglycans remains to be elucidated. The plausible participation in the gastrulation process of proteoglycans containing sulfated PSs was suggested by the facts that inhibitors of proteo* To whom offprint requestsshould be sent. Address: Misaki Marine Biological Station, University of Tokyo, Misaki, Kanagawa-ken 238-02, Japan.
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glycan synthesis prevent the gastrulation of sea urchin embryos [10] and that th~ activity of PS-sulfating systems increase at the time of gastrulation [11-13]. On the other hand, the presence of heparan sulfate [14], dermatan sulfate [14-17] and the complex of sulfated fucan and unidentified glycosaminoglycan [18] in sea urchin embryos has already been reported. The present study was designed to compare acid PSs prepared from embryos at the blastula and gastrula stages with those reared under sulfate-free conditions, with the aim of distinguishing those PSs that are specifically related to gastrulation. Another objective was to analyse the specific PSs which are coincidentally sulfated at the time of gastrulation, by means of [35S]sulfate labeling and subsequent autoradiography. MATERIAL AND METHODS
Material Eggs of the sea urchin, Clypeasterjaponicus, obtained by introducing 0.5 M KCI into the body cavity, were washed twice with sea water, After insemination, embryos were allowed to develop at 20°C with gentle stirring. In the present study, artificial normal sea water (NSW) and sulfate-free sea water (SFSW), purchased from Jamarine Laboratory (Osaka, Japan) were used throughout the experiment. The ionic composition (g/I) of NSW is: C1, 17.099; Na, 9.409; Mg, 1.137; S, 0.774; Ca, 0.395; K, 0.344 pH 8.2. SFSW is produced by exchanging MgSO4 (the only S source) for MgClz.
Dissociation of Embryonic Cells Embryos were collected by centrifugation and washed twice with 3 vol of 1 M glycine containing 2 mM EDTA (pH 8.2) [19]. Embryonic cells were dissociated completely by shearing in 10 vol of the glycine-EDTA solution using a loose-fitting hand-operated Dounce homogenizer. The dissociated cells were sedimented by centrifugation at 6400 g for 5 min. The sedimented material was mainly composed of naked embryonic ceils and is referred to as the 'dissociated cell' fraction. The supernatant and previous washings were pooled. This was composed mainly of extracellular materials and is referred to as the 'glycine extract' fraction. All procedures were performed at 0°C.
Preparation of Acid PSs Acid PSs were prepared from both the 'glycine extract' and 'dissociated cell' fractions, and also from whole embryos in some experiments. The 'glycine extract' was dialysed against water at 4°C. Three volumes of ethanol in the presence of 1% sodium acetate were added and kept at -20°C overnight in order to precipitate PSs. The precipitate thus obtained was defatted by sequential extraction with ethanol, ethanol--ether (3 : 1), and ether. The dried precipitate obtained from 107 embryos was suspended in 8 ml of 0.5 N NaOH and incubated for 24 h at 4°C for the purpose offl-elimination. After incubation, the pH of the solution was re-adjusted to 7.6 with HCI in the presence of 50 mM Tris. Twenty mg of Pronase E (106 tyrosine units/g; Kaken Kagaku, Tokyo) dissolved in 0.2 ml of water was added and the mixture was incubated further for 3 days at 37°C in the presence of 0.02 % sodium azide and a few grains of thymol. Each day the pH of the solution was re-adjusted and 20 mg of fresh Pronase E was added. The complete digestion with Pronase, one-fifth volume of 60 % trichloroacetic acid (TCA) was added, the mixture was left to stand for 30 min at 0°C and then centrifuged. The supernatant was neutralized with NaOH and PSs were precipitated by adding 3 vol of ethanol as described above. The precipitate was dissolved in 2.5 ml of 10 mM Tris-HCl (pH 7.4), 4 mM MgC12 containing 2 mg each of DNase I and RNase A (Sigma, St. Louis, Mo.). After incubation for 12 h at 37°C, the mixture was diluted up to 9 ml with 0.02 M sodium sulfate, and then l ml of 10 % cetylpyridinium chloride (CPC) was added. After incubation at 37°C overnight, the complex formed was spun down, dissolved in 2,5 ml of 4 M MgCI2 and precipitated again with ethanol in order to remove CPC. The precipitate thus obtained was washed with ethanol, dried and used as acid PSs in the present study. Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation
355
1. Recoveries of [35S]sulfate incorporated into embryos and authentic chondroitin sulfate C at several steps of the preparation of acid PSs
Table
Embryos [35S]sulfate-labelled for 8 h during gastrula
Chondroitin sulfate C
Step of the preparation of acid PSs
Radioactivity ~
Recovery
Recoveryb
Defatted powder Ethanol precipitate CPC precipitate Ethanol precipitate
4.67x 105 cpm 3.21 x 105 1.97× 105 1.56x 105
100 % 70 43 35
100 Ixg (%) 75 75 62
a Calculated from the radioactivity of an aliquot. b Estimated according to the method of Dische [20].
The 'dissociated cell' preparations and packed whole embryos were homogenized in 3 vol of water at 0°C using a motor-driven Teflon-glass homogenizer. To the homogenate, 3 vol of ethanol was added in the presence of 1% sodium acetate. The succeeding procedures were the same as described above. The recovery ratd of PSs by the procedure employed in the present study was determined using authentic chondroitin sulfate C and PSs obtained from [35S]sulfate-labelled Clypeasterembryos (table 1).
Electrophoresis PSs were subjected to electrophoresis on cellulose acetate sheets (10× 10 cm, Juko Sangyo, Tokyo) in 0.47 M formic acid -0.1 M pyridine (pH 3.0) for the first dimension under a constant current of 1 mA/cm for 40 min, and in 0.1 M barium acetate for the second dimension under the same current for 3 h according to the method of Hata & Nagai [25]. The spots were contrasted by staining with 0.1% Alcian blue, pH 3.0 in 1% acetic acid. The amount of acid PSs applied on the sheet for each electrophoresis was equivalent to that prepared from 3 × 103 embryos.
Enzymatic Digestion of Acid PSs Digestion of acid PSs with chondroitinase AC and ABC was carried out according to the method of Yamagata et al. [21], and digestion with Streptomyces hyaluronidase was done by the method of Ohya & Kaneko [22]. All enzymes were products of Seikagaku Kogyo (Tokyo). After digestion, the reaction was stopped by heating the mixture at 100°C for 2 min and centrifuged at 14000 g for 20 min. The supernatant was used for electrophoresis.
Incorporation of [35S]sulfate Incorporation into whole embryos. Embryos
at the desired developmental stage (3.5x 104 embryos each stage) were washed twice with SFSW and then transferred to 2 ml of SFSW, in which 1 ixCi of cartier-free [35S]sulfate (Japan Atomic Institute, Tokyo) and 200 units of penicillin were contained. The labelling was continued for 1 h at 20°C. Sampling was made at intervals of 2 h. At the end of the labelling period, 2 ml of 20 % TCA was added to the embryo suspension and the mixture left to stand for 30 min at 0°C. The precipitate was washed twice with ice-cold 10% TCA and twice defatted by successive extraction with ethanol, ethanol-ether (3 : 1), and ether. The radioactivity in the dried precipitate was estimated using a liquid scintillation counter after dissolving it into Soluene 350 (Packard, Downers Grove, I11.). For histological observation, labelling of embryos was done at a higher concentration of [35S]sulfate (50 ixCi/ml SFSW for 1 h). Embryos were fixed in Carnoy's solution and sectioned as usual in paraffin at 6--8 laan. Sections were dipped in Sakura NR-M2 emulsion (Konishiroku Photo Inc., Japan) for autoradiographic observation.
Exp CellRes 159(1985)
356 Yamaguchi and Kinoshita
Fig. 1. Development of the sea urchin, Clypeasterjaponicus embryos reared in NSW and SFSW at 20°C. Embryo reared in (a) NSW for 21 h (mesenchyme blastula stage); (b) SFSW for 21 h (note that no retardation in development is observed); (c) NSW for 38 h (gastrula stage); (d) SFSW for 38 h (note that the assemblage of primary mesenchyme cells and the second step of gastrulation are prevented). × 113.
Incorporation into PSs. Embryos were washed twice with SFSW and then allowed to develop for 8 h at 20°C in 50 ml NSW-SFSW (1 : 9), in which 100 ~Ci of [35S]sulfate and 5 000 units of penicillin were contained. At the end of the labelling, one-fifth volume of 60% TCA was added to the embryo suspension and the mixture was left to stand for 30 min at 0°C. PSs were prepared as described above, making this precipitate the starting material. Labelled PSs were electrophoresed on cellulose acetate sheets and exposed to a Kodak X-omat AR film for autoradiographic analysis.
RESULTS
Development of Sea Urchin Embryos Fig. 1 shows the development of sea urchin embryos under the conditions employed in the present study. Embryos, both in NSW and SFSW, develop normally up to the stage at which primary mesenchyme cells are shed into the blastocoel (fig. 1 a, b). Differences were observed from this stage onward. In normal embryos, primary mesenchyme cells initially migrated on the inner surface of the ectodermal wall to form 'ring-' and 'cable-shaped' structures which constituted partial matrices for skeletal rudiments later, and this was followed by initial invagination and stretching of the primitive gut (fig. 1 c). However, in the embryos reared in SFSW, primary mesenchyme cells appeared to be prevented from forming the above-mentioned structures and the second step of the gastrulation process was inhibited, although the initial step of invagination did occur (fig.
ld). Electrophoretic Analysis of Acid PSs Acid PSs were prepared from the 'glycine extract' and 'dissociated cell' fractions and were compared by means of two-dimensional electrophoresis on Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 357 cellulose acetate sheets. Comparison was made between hatching blastula (15 h after fertilization), gastrula (30 h) and embryos reared in SFSW for 30 h. Acid PSs stained with Alcian blue were classified according to their mobilities in the first and second dimensions of the electrophoresis. Each PS was noted in a coordinate manner by the combination of numerals (1--6; first dimension as abscissa) and letters (A-F; second dimension as ordinate). Acid PSs in the ' glycine extract' fraction. Acid PSs prepared from the 'glycine extract' fraction, composed mainly of extracellular materials, were compared electrophoretically. At blastula stage (fig. 2 a), two major spots (2A, 2B) and one minor spot (4F) were detected. At gastrula stage (fig. 2 b), two additional spots (3B, 3C) were observed. As is seen in fig. 2 g, 3B and 3C became major spots in late gastrula stage (38 h) and another new spot (3A) appeared by this stage. On the other hand, these new spots were never observed in the embryos reared in SFSW, which were prevented from gastrulation, while other labelled spots (5E, 6D) appeared instead (fig. 2c). The mobilities of 5E and 6D in the first dimension of electrophoresis (formic acid-pyridine) were as low as that of authentic hyaluronic acid, suggesting that both PSs may be less or non-sulfated PSs, as appeared in extracellular materials under the sulfate-deficient condition. Acid PSs in the "dissociated cell' fraction. Acid PSs prepared from the"dissociated cell' fraction, composed mainly of intracellular substances, were next compared. Six spots IF, 2A, 2B, 2F, 4D, 4F) were detected at blastula stage (fig. 2d). Of these spots, 2A, 2B and 4F were common between the 'glycine extract' and the 'dissociated cell' fractions. Therefore, IF, 2F and 4D were to be regarded as PSs characteristic of the 'dissociated cell' fraction. The electrophoretic pattern of acid PSs obtained from gastrula (fig. 2 e) was essentially the same as that from blastula, provided the common spots between the two fractions (2A, 2B, 3B, 3C, 4F) were excluded. The same can be said for the acid PSs of the embryos reared in SFSW with the only exception of 6F (fig. 2J), which showed very low mobility in the first dimension of electrophoresis, suggesting again that this may be less or nonsulfated PS formed within cells under sulfate-deficient conditions.
Enzymatic Digestion o f Acid PSs Embryos were reared in NSW and SFSW for 30 h. Acid PSs were prepared from whole embryos and subjected to digestion by Streptomyces hyaluronidase and chondroitinases AC and ABC. Electrophoretic patterns of acid PSs were compared before and after digestion (fig. 3). It was found that hyaluronidase (normal substrate, hyaluronic acid) digested none of the PSs in the embryos reared either in NSW or SFSW. Chondroitinase AC (normal substrate, hyaluronic acid, chondroitin, chondroitin sulfate) digested 6D, and chondroitinase ABC (normal substrate, those of chondroitinase AC and Exp Cell Res 159 (1985)
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Polysaccharides sulfated during gastrulation
359
Fig. 3. Enzymatic susceptibility of acid PSs as analysed by means of electrophoresis. (a, b) Acid PSs obtained from normal embryos (30 h, gastrula stage) were subjected to the action of chondroitinase ABC (a) before; (b) after digestion (note that 3C is susceptible to chondroitinase ABC.); (c, d) Acid PSs obtained from embryos reared in SFSW for 30 h were subjected to the action of chondroitinase AC: (c) before; (d) after digestion. (Note that 6D is susceptible to chondroitinase AC.)
dermatan sulfate) digested 3C and 6D. The other spots were resistant to chondroitinase digestion. In other words, 3C (a PS that appeared extracellularly in normal gastrula) was resistant to hyaluronidase and chondroitinase AC but susceptible to chondroitinase ABC, suggesting that 3C may be dermatan sulfate isomer; and 6D (a PS which appeared extracellularly only in permanent blastula reared in SFSW, and thus corresponded electrophoretically to authentic hyaluronic acid) was resistant hyaluronidase but susceptible to both chondroitinase AC and ABC, suggesting that 6D may be related to non-sulfated chondroitin.
Incorporation of ffSS]suifate into Sea Urchin Embryos Embryos were labelled with [35S]sulfate separately for 1 h at 2-h intervals from fertilization to late gastrula stage (38 h) and the radioactivity incorporated into the TCA-insoluble, defatted fraction was measured on each sampling. As is seen in fig. 4, the rate of incorporation increased gradually until early gastrula stage (28 h) with no conspicuous peaks. Thereafter it increased sharply, nearly quadrupling in value by the end of gastrulation. It is interesting to note that this steeply ascending phase in the incorporation rate (30-38 h) corresponded to the second step of gastrulation.
Fig. 2. Two-dimensional electrophoretograms of acid PSs prepared from (a, b, c, g) 'glycine extract', (d, e, J)'dissociated cell' fractions which were obtained from (a, d) blastula; (b, e, g) gastrula; (c,J), embryos reared in SFSW. The electrophoresis in the first dimension was carded out in 0.47 M formic acid-0.1 M pyridine (pH 3.0), and the second in 0.1 M barium acetate. Authentic spots: Ha, Hyaluronic acid; D, dermatan sulfate; C, chondroitin sulfate C; Hp, heparin. Exp Cell Res 159 (1985)
360 Yamaguchi and Kinoshita 20-
:~ ~15.
~to. .9o
]
mBl
[.
Gs
>
u
% o
1'0 2'0 3'0 time after fertilization (hr)
3'8
Fig. 4. Incorporation of [35S]sulfate into sea urchin embryos at different stages of development. 3.5×104 embryos were reared in NSW and washed twice with SFSW before labelling. They were incubated in 2 ml of SFSW containing 1 ~tCi of [35S]sulfate for I h. Radioactivity of TCAinsoluble, defatted fraction of the labelled embryos was measured using a liquid scintillation counter, mBI, Mesenchyme blastula; Gs, gastrula.
Autoradiographic Analysis of Acid PSs Labelled with [35S]sulfate Acid PSs were prepared from sea urchin embryos labelled with [35S]sulfate at various stages of development and analysed electrophoretically. Embryos were labelled for 8 h in NSW-SFSW (1 : 9) at early blastula (10-18 h), mesenchyme blastula (18-26 h) and gastrula (26-34 h) stages. As seen in fig. 5, radioactivity was detected to be very weak in spot 2AB (2A and 2B were combined here) at early blastula stage, while for other spots no radioactivity was observed; at mesenchyme blastula stage, radioactive spots were found to increase in number and five spots were detected (2B, 2F, 3A, 3B, 3C); at gastrula stage, radioactive spots did not increase in number, but 3B and 3C became predominant. The incorporation of [3SS]sulfate into each spot on the electrophoretogram was measured quantitatively by measuring radioactivity directly by scintillation counter after cutting out the corresponding spots on the cellulose acetate sheet and dissolving them in dimethyl sulfoxide (DMSO). As seen in fig. 6, the amount of radioactivity incorporated into total PSs increased as development proceeded and the greater part of the incorporated radioactivity was recovered in 3B and 3C, which accounted for 22 and 65 % of the total incorporation at gastrula stage, respectively. It should be noted that the recovery rate of polysaccharides is limited at about 60 % at the maximum due to the inefficiency of recovery at the step of ethanol precipitation (table 1). The rate would be much lower when the amount of polysaccharides is smaller in the solution. Therefore, it is plausible that the total amount of laSS]sulfate actually incorporated is greater than that estimated, and that this tendency is more pronounced in the earlier stages of development.
Distribution of [~SS]sulfate in Histological Sections of Embryos Distribution of incorporated [35S]sulfate was examined by comparing the autoradiogram and the stainability with Alcian blue on the same sections of sea urchin embryo at the time of gastrulation (fig. 7). Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation
361
Fig. 5. Incorporation of [35S]sulfate into PSs as analysed by means of electrophoresis. PSs were
prepared from embryos at (a, b), early blastula; (c, d) mesenchyme blastula; (e,J) gastrula stages. Just prior to the preparation of PSs, embryos were labelled with [35S]sulfate for 8 h. (a, c, e) Electrophoretograms stained with Alcian blue, pH 3.0; (b, d, J) autoradiograms.
Exp Cell Res 159 (1985)
362 Yamaguchi and Kinoshita 2AB 2F 3A
o >, 15
x
~
5
3C Fig. 6. Incorporation of [35S]sulfate into PS spots of two-
dimensional electrophoretograms shown in fig. 5. Each spot was cut out separately, dissolved in dimethylsulfoxide (DMSO), and the radioactivity was measured.
10-18 eBI
18-26 mBI
26-34(hr) Gs
Fig. 7 a-d shows the histological sections and the corresponding autoradiograms of the embryos at early gastrula stage (30 h) fixed just after labelling for 1 h. Grains due to radioactivity were observed to be distributed on the ectodermal wall and mesenchyme cells in the blastocoel. In the ectodermal walls, especially in the animal pole region which is thickened, grains were localized mainly on the inner and outer surfaces (fig. 7 a, b). It will be noted that the same pattern of positive Alcian blue staining was recognizable. Fig. 7 e,fshows the distribution pattern of [35S]sulfate in an embryo which was labelled for I h at the 30-h stage and subsequently subjected to chase for 6 h under aSS-free conditions until the 37 h stage (note that gastrulation has proceeded). It is obvious that the localization of grains was comparatively more dispersed in the blastocoelic cavity and that Alcian blue-positive structures spread throughout the blastocoelic cavity. The grains in the blastocoelic cavity seemed to coincide with the fibrous structures. The correspondance between the grains and the histological structures was also observed in fig. 7 a - d rather clearly as the structures existed partially in the blastocoelic cavity.
DISCUSSION
Changes o f Acid PSs Observed at the Time o f Gastrulation In this study, PSs were prepared separately from extracellular materials which constituted the 'glycine extract' and intracellular substances referred to as the 'dissociated cell' fraction. Comparing progressive changes in these two groups of PSs during development, it was observed that the PSs of extracellular origin changed remarkably at the time of gastrulation. Three kinds of PS, designated 3A, 3B and 3C in the present study, were not detected in embryos at hatching blastula stage, but they did appear at mesenchyme blastula to early gastrula stage Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 363
Fig. 7. Distribution of incorporated [3SS]sulfate in sea urchin embryos. Embryos were labelled with
[sSS]sulfate for 1 h at early gastrula stage (30 h), and fixed (a-d) at the end of the labelling period or (e,j) after a chase of 6 h. (a, c, e) Sections stained with Alcian blue; (b, d, j0 autoradiograms observed in dark field (b, d, fcorrespond to a, c, e, respectively). × 170.
and then increased conspicuously at late gastrula stage. Such changes were never observed in embryos prevented from gastrulating by rearing in SFSW. In the PS electrophoretic patterns, several spots were observed in common between the 'glycine extract' and the 'dissociated cell' fractions. Since PSs that appear de novo can be considered to have been originally synthesized intraceUulady, probably in the form of complexes of PSs and proteins such as proteoglycans, these common PSs could naturally occur within cells before extracellular secretion. Aside from these common PSs, it is remarkable that the remaining Exp Cell Res 159 (1985)
364
Yamaguchi and Kinoshita
kinds of PSs of specific intracellular origin exhibited practically no changes during development.
Partial Identification of 3C and 6D and Their Possible Relationship in Biosynthesis Among the specific acid PSs which were synthesized de novo at the time of gastrulation, 3C was identified as dermatan sulfate on the basis of its mobility in electrophoresis and its sensitivity to chondroitinase ABC. In the permanent blastula reared in SFSW, none of these PSs was detected, while, instead, different PSs, 5E and 6D, were observed. Of these two PSs, 6D was considered very likely to be non-sulfated chondroitin, on the basis of its electrophoretic mobility and its susceptibility to both of the chondroitinases AC and ABC. Apart from still other unidentified PSs, an interesting hypothesis can be formulated, that two PSs, dermatan sulfate (3C) and chondroitin (6D), may be mutually related with respect to their synthetic pathways. As was demonstrated by Lindahl's group in a cell-free human fibroblast system [23], chondroitin is a precursor of dermatan sulfate at the time of its synthesis. According to them, sulfation of N-acetylgalactosamine adjacent glucuronic acid (GIcUA) in chondroitin molecules is necessary for the inversion of GlcUA to iduronic acid which is a constituent sugar of dermatan sulfate. If this step in the reaction (called C-5 epimerization) occurs in sea urchin embryos, the sugar chains which will become dermatan sulfate in NSW remain in the chondroitin state under sulfate-free conditions, for lack of C-5 epimerization. There are several reports on the presence of dermatan sulfate in sea urchin embryos [14-17].
[SSS]Sulfate Incorporation into PSs and Histological Distribution of Radioactivity Labelling experiments using [SSS]sulfate demonstrated after electrophoretic analysis that most of the radioactivity is recoverable in two PSs, 3B and 3C, of the "glycine extract" fraction at the time of gastrulation. In autoradiograms of corresponding histologically sectioned embryos, grains were distributed mainly in extracellular matrices, including the hyaline layer, basement membrane and fibrous structures in the blastocoelic cavity. It may be concluded that the bulk of [35S]sulfate incorporated during gastrulation is to be found in extracellular structures probably composed of sulfated PS-containing substances such as proteoglycans.
The nature of the inner surface of the ectodermal wall may be important for determining the distribution pattern of primary mesenchyme cells according to the animal-vegetal gradient. On the other hand, the fibrous structures in the blastocoel have been considered recently to provide a matrix for the guidance of the invaginating primitive gut [6--9]. It was revealed by electron microscopy that these structures are composed of fibrous and granular components, and the latter Exp Cell Res 159 (1985)
Polysaccharides sulfated during gastrulation 365 resemble morphologically sulfated proteoglycans identified in some other systems [7, 14] and they are susceptible to sulfate deprivation [7]. In conclusion, it was demonstrated in this study that the synthesis of new kinds of PSs and their sulfation may be indispensable for the achievement of gastrulation and that these sulfated PSs may take part in morphogenetic movements within extraceUular matrices. We are indebted to Professor A. Gorbman of Washington University for kindly reading the manuscript, and also to Dr T. Yamagata for valuable suggestions during this investigation.
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25.
Herbst, C, Wilhelm Roux's arch 5 (1897) 649. Immers, J, Exp cell res 10 (1956) 546. - - Ibid 24 (1961) 356. Motomura, I, Bull mar biol Asamushi 10 (1960) 165. Sugiyama, K, Dev growth differ 14 (1972) 63. Endo, Y & Noda, Y D, Zool mag 86 (1977) 309. Katow, H & Solursh, M, Exp cell res 136 (1981) 233. Akasaka, K, Amemiya, S & Terayama, H, Exp cell res 129 (1980) 1. Kawabe, T T, Armstrong, P B & Pollock, E G, Dev bioi 85 (1981) 509. Kinoshita, S & Saiga, H, Exp cell res 123 (1979) 229. Kinoshita, S, Exp cell res 87 (1974) 382. Nozawa, A & Kinoshita, S, J fac sci univ Tokyo see IV 14 (1977) 11. Saotome, K & Yanagisawa, T, Dev growth differ 21 (1979) 401. Solursh, M & Katow, H, Dev biol 94 (1982) 326. Yamagata, T & Okazaki, K, Biochim biophys acta 372 (1974) 469. Oguri, K & Yamagata, T, Biochim biophys acta 541 (1978) 385. Saotome, K & Yanagisawa, T, Dev growth differ 21 (1979) 413. Akasaka, K & Terayama, H, Exp cell res 146 (1983) 177. Kane, R E, Exp cell res 81 (1973) 301. Dische, Z, J biol chem 167 (1947) 189. Yamagata, T, Saito, H, Habuchi, O & Suzuki, S, J biol chem 243 (1968) 1523. Ohya, T & Kaneko, Y, Biochim biophys acta 198 (1970) 607. Malmstrfm, A, Fransson, L-/~, H66k, M & Lindahl, U, J biol chem 250 (1975) 3419. Schuel, H, Kelly, J W, Berger, E R & Wilson, W L, Exp cell res 88 (1974) 24. Hata, R & Nagai, Y, Anal biochem 45 (1972) 462.
Received November 20, 1984 Revised version received March 5, 1985
Exp Cell Res 159 (1985)