Further studies of the effects of deprivation of sulfate ... · Paracentrotus lividus by J.IMMERS...

/ . Embryol. exp. Morph., Vol. 14, Part 3, pp. 289-305, December 1965Printed in Great Britain

Further studies of the effects of deprivation of

sulfate on the early development of the sea urchin

Paracentrotus lividus

by J.IMMERS and j . RUNNSTROM1

From the Wenner-Gren Institute, University of Stockholm

WITH TWO PLATES

THE morphological effects of sulfate-free medium on sea urchin embryos weredescribed in detail by Herbst (1904). Further studies were carried out by Lindahl(1936,1942). He was the first to consider metabolic aspects of the role of sulfatein the development of the sea urchin. Immers (1956, 1959, 1961a and b, 1962)studied the distribution and function of acid mucopolysaccharides in earlydevelopmental stages of sea urchins, mainly Paracentrotus lividus. A dominantgroup of these acid polysaccharides are sulfated. Their location in the blastocoel,in the hyaline layer and in the lumen of the intestine could be demonstrated bystaining of sectioned specimens with the ferri-acetic reagent of Hale (1946). Inblastulae or gastrulae raised in sulfate-free sea water these regions are negativewith respect to Hale staining (Immers, 19616). On the other hand, the ectodermalnuclei of the animal region of the embryos are stained with the Hale reagentalthough the nuclei of the vegetal region remained unstained (I.e.).

Runnstrom et al. (1964) extended these studies and included observations on theeffect of sulfate-free medium on animal and vegetal halves. A survey and dis-cussion of previous work were made by Runnstrom et al (1964).

The morphological effect of the lack of sulfate involves an animalization of theembryo. The degree of animalization may vary with the batch of eggs used; itis further dependent on the degree of removal of the sulfate from the medium inwhich the embryos are raised and the stage and duration of treatment. At thelowest degrees of animalization, the archenteron attains its normal size; neverthe-less, an extension of the acron with its stereocilia takes place; the normal bilateralsymmetry of the embryo is replaced by a radial symmetry expressed both inectoderm and entoderm. The primary mesenchyme cells show no bilateralarrangement. As a consequence smaller supernumerary pieces of skeleton appearwhich to varying degrees manifest the radial symmetry of the embryos subjectedto sulfate-free medium. Increasing animalization leads to a stronger development

1 Authors' address: The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan16, Stockholm Va, Sweden.

290 J. IMMERS and J. RUNNSTROM

of the ectoderm with extension of the acron so that two-thirds of the embryo maybe covered by stereocilia; the entoderm becomes reduced or suppressed and themesenchyme loses to an increasing extent its capacity for migration and normaldistribution.

Runnstrom et ah (1964) demonstrated that lack of sulfate in the medium causesreduced incorporation of amino acids into whole embryos. Lack of sulfate alsoreduced the incorporation of amino acids into isolated animal halves (corre-sponding to the mesomere material). In vegetal halves (macromere + micromerematerial) on the other hand, lack of sulfate did not decrease the amino acid in-corporation, which even in the presence of sulfate is rather low (Markman, 1961a).Previous studies indicated strongly that lack of sulfate may interfere with proteinsynthesis within the embryos. The sulfated polysaccharides seem to be ofparticular importance for the maintenance of the integrity of the vegetal regionof the sea urchin embryo. The present paper is a continuation of those of Immersand of Runnstrom et ah referred to above. It includes a study of the incorporationof labelled nucleic acid precursors such as thymidine and uridine. Furthermore,the labelling was localized within the embryo by means of autoradiography.The previous experiments on amino acid incorporation have also been extended.

MATERIAL AND METHODS

Embryos of the sea urchin Paracentrotus lividus from the Gulf of Naples wereused in these experiments. The handling of gametes was the customary one (seeImmers, 1956). Fertilized eggs were raised under slow rocking in glass trayscovered with glass plates. The media were normal sea water (+ SO^) or sulfate-free sea water ( - SO^). The temperature was kept at 19° to 20° C.

At different times, 8-10 ml. suspensions were transferred from the trays tosmaller elongated containers to which labelled precursors were added. Thesuspensions were subject to rocking during the time of exposure which lasted fortritiated thymidine and tritiated uridine 5 hrs. and for 14C-labelled amino acids,30 min. at different final concentrations of the labelled precursors. Furtherdetails will be given below.

Tritiated thymidine (Thymidine-6-T; TRA. 61; 3 -9 C./mM.) tritiated uridine(Uridine-TG; TRA. 27; 1 -1 C./mM.) and 14C-labelled amino acids of algal proteinhydrolysate (14 different amino acids 100 /xC./mg.) were used. All isotopes weresupplied by the Radiochemical Centre, Amersham. The different labelled com-pounds were incorporated into embryos taken from one and the same eggsuspension, all embryos thus being in the same stage of development.

After incubation the embryos were fixed, washed and the radioactivity meas-ured in an automatic 'Nucleo' counter or in a 'Tracer lab.' windowless gas flowcounter. The details of procedures were the same as those used by Markman(1961&). The fixation in Carnoy's fluid and the subsequent washing in96 per cent and 70 per cent alcohol proved sufficient to eliminate interference

Lack ofsulfate and the development o/Paracentrotus 291

by lipids in the experiments with amino acid incorporation. Moreover, a treat-ment of the samples with 5 per cent trichloracetic acid (TCA) for 45 min. at80° C. was carried out in ten of the experiments in order to remove nucleic acids.In incorporation tests with labelled amino acids the TCA treatment influencedthe number of counts by only about 5 per cent, which is less than the error givenby the counters. Thus the method used must give a fairly accurate measure ofincorporation of the labelled amino acids into proteins. This was confirmed alsoin other ways (Markman, to be published).

Moreover, lipids do not seem to interfere with the incorporation of precursorsof nucleic acids.

Autoradiographs were made with Kodak autoradiographic stripping platesAR. 10. The histochemical staining (Unna-Brachet methylgreen-pyronin andHale methods) was performed according to Pearse (1960) and Immers (1954).The results of measurements were tested statistically.

Terminology and abbreviation

The terminology of sea urchin development is not well standardized. A goodrepresentation of different regions of the early pluteus larva and their designationare given in Kiihn (1955, p. 181). The dorsal side extends from the 'acron' tothe blastopore on one side; in the first pluteus stage the wall consists of asquamous epithelium and is supported and extended by the body rods. Thiswhole-region was early (see Schmidt, 1904) called the 'apical region', because ofits elongated, pointed shape. In the following the designation,' apical region ordirection' is used synonymously with 'dorsal region or direction'. The ventralside encompasses mainly the oral field, which is surrounded by the ciliary bandof which the early distinguishable acron forms a part.

The' attachment zone' is the zone of the vegetal ectoderm to which the primarymesenchyme cells first attach themselves. 'Embryos' or 'embryonic' refer toStages before the early pluteus stage. This is considered to begin when the buds ofthe anal arms appear.

RESULTS

Some morphological and histochemical data

Figures A and B (Plate 1) allow the comparison of a normal gastrula, 32 hr.after fertilization, and an embryo of the same age which was transferred to sulfate-free sea water soon after fertilization and raised in this medium. The sectionswere stained according to Unna-Brachet. The embryo shown in Fig. B. (Plate 1)has undergone a rather strong animalization. The intestine is reduced and has avery restricted lumen. On the top of the rudimentary intestine a group of ag-gregated secondary mesenchyme cells is seen; a few more detached mesenchymecells are also present. Characteristic of the mesenchyme cells is their sphericalor only slightly elongated shape. This is in contrast to the shape of the secondary


mesenchyme cells of the control embryo where these cells have formed numerouspseudo- and filopodia. They are spun out between entoderm and ectoderm andform numerous contacts with both. The primary mesenchyme cells are lessconspicuous in Fig. A. They form a well-known pattern whose establishment hasbeen thoroughly analysed by Gustafson and his co-workers (see Gustafson &Wolpert, 1961,1963). The mass of cytoplasm surrounding the nuclei is relativelygreater in the primary than in the secondary mesenchyme cells, whereas thepseudopodia of the former are correspondingly shorter, at least when the patternof cell contact has been established within the primary mesenchyme and betweenthis and the ectoderm. The atypical arrangement of the primary mesenchymein embryos which are radially symmetrical as a consequence of lack of sulfate hasbeen mentioned in the introduction. When the animalization progresses further,as, for example, to the extent found in the embryo shown in Fig. B, skeletonformation may become irregular or even be suppressed. In embryos of the typerepresented in Fig. B the staining is definitely weaker in the entoderm-mesenchymethan in the control embryo (Fig. A). In contradistinction, the nuclear staining ofthe ectoderm cells is as strong as in the control embryo of the same age. Themorphological distinction between primary and secondary mesenchyme cellsbecomes less owing to the tendency of both kinds of cells to becomespherical.

A comparison between the embryos represented in Figs. A and B shows alsothat the number of cells must be reduced by lack of sulfate, at least when its effectis as strong as in the present case. A certain delay in cell division is probablyinvolved. In the acron region, however, the number of nuclei seems to be aboutas great as in the control. Moreover, the cylindrical cells of the acron form ahigher epithelium than in the control. In the more vegetal region of the ectodermthe nuclei are fewer. There is an enrichment of cells in the direction of the animalpole as if the adhesion of the cells were stronger here.

Fig. C represents an embryo raised in sulfate-free sea water and fixed 32 hr.after fertilization and stained with the Hale reagent. The contrast between thestrongly stained ectoderm and the unstained entoderm and mesenchyme isobvious. The staining is, however, confined to the nuclei. It is difficult to decidewhether the stained region encompasses also a rim of cytoplasm. There is noHale staining of the blastocoel and of the hyaline layer. Only a small spotcorresponding to the most vegetal region of the embryo has been stained. Theregions mentioned are strongly stained in the control. The stained vegetal spot isonly a small remnant of the strongly stained material which is found in the lumenof the normal entoderm. The reader is referred to Fig. 24 in Immers (19616).

Incorporation of tritiated thymidine into DNA

Two series of Paracentrotus embryos were raised for about 27 hr. in normal(+ SO 4) and sulfate-free ( - SO^) sea water. During the late gastrulation periodbeyond 22 hr. after fertilization the embryos were exposed for 5 hr. to tritiated

J. Embryol. cxp. Morph. Vol. 14, Part 3

EXPLANATION OF PLATES

FIGS. A-K. Embryos of Paracentrotus lividus, fixed in Carnoy's fluid 32 hr. (Figs. A-C) or27 hr. (Figs. D-K) after fertilization, embedded in histowax and cut in section at 5 [x.The embryos are sectioned in Figs. A-F and G-K parallel to the animal-vegetal axis andare oriented in the Figs, with the animal pole upside. Fig. G is a transverse section.

PLATE 1

FIG. A. Normal gastrula. Staining according to Unna-Brachet. DNA appears dark in thephotograph. Frontal section, 540 x .

FIG.B. Gastrula raised in sulfate-free sea water. Staining according to Unna-Brachet. 540 x.FIG. C. Gastrula raised in sulfate-free sea water. Staining according to Hale with counter-

staining by PAS. The nuclei of the ectoderm appear dark owing to a reaction betweenunmasked phosphate groups of nucleic acids and the Fe+++ of Hale reagent. 600 x .

FIG. D. Normal gastrula. Autoradiography of incorporated 3H-thymidine. Counterstainingwith Giemsa. Median section, oral (ventral) side to the left. 600 x. For concentrationand radioactivity of thymidine, see Table 1, exp. 2.

FIGS. E and F. Gastrulae raised in sulfate-free sea water. Autoradiography of incorporated3H-thymidine; 600 x and 700 x respectively; see further legend to Fig. D.

J. IMMERS and J. RUNNSTROM (Facing page 292)

J. Embryol. exp. Morph. Vol. 14, Part 3

PLATE 2

FIG. G. Gastrula raised in sulfate-free sea water. Autoradiography of incorporated thymidine;640 x ; see further legend to Fig. D.

FIGS. H AND I. Normal gastrulae. Autoradiography of incorporated 3H-uridine; median sectionoral (ventral) side to the left (H) and more frontal section (J); 580x ; counterstainingwith Giemsa; see further Table 2, exp. 2.

FIGS. J AND K. Gastrulae raised in sulfate-free sea water; autoradiography of incorporated3H-uridine; 640 x ; counterstaining with Giemsa; see further Table 2, exp. 2.

J. IMMERS and J. R U N N S T R O M (Facing page 293)


thymidine. The data in Table I show that the incorporation of thymidine wasconsiderably lowered when the embryos had been raised in sulfate-free sea water.This is in keeping with the observation of a reduced number of cells in embryossubjected to lack of sulfate as compared with control embryos raised in normalsea water.

TABLE 1

Incorporation of 3//-thymidine into embryos o/Paracentrotus lividus raisedin normal and sulfate-free sea water

In exp. 1 the added 3H-thymidine concentration was 3-25x 10~6M with 0-63 /zC./ml. andin exp. 2 the concentration was 6-5 x 10~6 M with 1 -25 /zC./ml. n is number of parallel testsamples assayed in each exp.

Age ofembryos Means of C. P.M./J00 embryosduring

Exp. incubation Difference withNo. (in hr.) n + SO4 — SO4 standard error t P

1 21-5—26-5 4 7 - l ± 0 - 2 4-4±0-6 2-7±0-3 7-5 00052 22—27 3 138-9 + 9-5 33-2+ 1 -9 105 • 7 ± 5 • 6 18-8 <0-005

The study of the autoradiographs of 27-hr, old embryos revealed a certainlocalization of the incorporation of the 3H-thymidine in control embryos. Inthe stage examined, gastrulation has been achieved, the acron still carries a tuftof stereocilia and the dorso-ventral symmetry is manifested by a flattening of athicker oral (ventral) side and a more stretched thinner apical (dorsal) side.Fig. D (Plate 1). represents an approximately median section of a control embryowhere oral and aboral (apical) sides may be distinguished. The oral vegetal regionof the ectoderm showed a strong thymidine incorporation in several embryos.The incorporation was also rather strong in the vegetal apical region. It was aconstant feature that the thymidine incorporation was, on the whole, strongerin the oral than in the apical region. In particular the more animal part of theapical region was very poorly labelled, even at this stage, when the flattening ofthe apical cells was still moderate. It is well seen in Fig. D that the cells showingincorporation were many fewer in the animal apical region than in the oral one,Moreover, when incorporation did occur, the grains were fewer. The cells of theapical region which gradually form a thin squamous epithelium have thus aStrongly reduced frequency of mitotic division. The oral region gives rise to theciliary band and later to the stomodeum. A strong incorporation at a ratheranimal level of the oral side could be regarded as a preparation of the stomodeumformation. This region can be seen in Fig. D, but was studied preferentially incross sections where the anterior region of the archenteron was seen to approachthe ectoderm. The incorporation into the mesenchyme cells of the 27-hr, embryoswas low even compared with that in the more animal apical region.

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294 J. IMMERS and J. RUNNSTR5M

In the embryos raised in sulfate-free sea water the ventro-dorsal polarity ofthymidine incorporation is replaced by a more pronounced animal-vegetalpolarity, as Fig. E shows. The groups of densely accumulated grains correspondto the extended acron region with its tuft of stereocilia. The maximum mitoticrate is confined to this region rather than to an oral region. In many cases theincorporation of 3H-thymidine was particularly strong in the region of the acroncells adjacent to the blastocoel, see Fig. F. This is also the position of the nucleiin this region as follows from Fig. B.

An incorporation occurs also within the reduced ento-mesoderm, see Figs.E-G. The grains proved, however, to be fewer and less crowded than in the acronregion. As seen in Figs. E-G the mesenchyme cells which surround the rudi-mentary entomesoderm remained unlabelled. The pronounced bilateral sym-metry found in the control embryos is not present in the embryos raised in- SO4 sea water; see the cross-section represented in Fig. G. The polarity foundin embryos raised in — SO^ sea water was mainly an animal-vegetal one.

Incorporation of trMated uridine

Measurements of the incorporation of tritiated uridine into embryos ofParacentrotus are shown in Table 2. As in the experiments with thymidine, theembryos were 27 hr. old at the end of the incorporation period. The controlembryos raised in + SOiT sea water showed a considerably higher incorporation of

TABLE 2

Incorporation of^H-uridine into embryos of Paracentrotus lividus raised innormal and sulfate-free sea water

In exp. 1 the concentration of 3H-uridine was 6-5 x 10~6 M with 7-2 /uC./ml.; in exp. 2 theconcentrat ion was 3 - 4 x 10~6 M with 3-7 /zC./ml. n is the number of parallel test samplesassayed in each exp.

Age ofembryos Means of C.P.M.I 100 embryosduring

Exp. incubation Difference withNo. {inhr.) n +SOJ -SO^ standard error t P

1 21-5-26-5 3 2371 ±16-6 158-2±14-9 78-9±12-8 6-1 0-0252 22-27 3 251-8± 17-1 99-6± 7-2 152-2±10-7 14-2 0-005

3H-uridine than those raised in - S O ^ sea water. Uridine will enter ratherdirectly into ribonucleic acid (RNA) but after transformation to cytidine orthymine it may also enter deoxyribonucleic acid (DNA). It has been demon-strated above that deprivation of SCXr reduces the incorporation of 3H-thymidineinto the embryos. This indicates that the rate of synthesis of DNA is loweredwhen the embryos are raised in - SO4 medium. The data of Table 2 tend to showthat the rate of incorporation of 3H-uridine is reduced in embryos raised in - SO 4


medium to a level comparable with the reduced incorporation of 3H-thymidinerecorded in Table 1.

A number of autoradiographs showed that 3H-uridine is incorporated into thenuclei and, in lesser degree, also into the cytoplasm. The regional differenceswere less pronounced than in the case of incorporation of 3H-thymidine. How-ever, the ventral side of the ectoderm showed, on the whole, a stronger and moreuniform incorporation than other regions of the ectoderm; see Fig. H (Plate 2).\n the stage particularly studied (late gastrula stage), the acron is less labelledwith 3H-uridine than the more vegetal region of the ectoderm; see Figs. H and I(Plate 2). This may be an expression of the fact that the acron region has attaineda certain definite level of differentiation, whereas within the oral and apical regionnew differentiation processes are prepared. A more extensive study of these finerregional differences is, however, outside the scope of this paper.

The entoderm was not uniformly labelled. Often there seemed to be a strongertendency for incorporation in the posterior (animal) region of the entoderm thanin its anterior (vegetal) region; see Figs, H and I. The mesenchyme cells showeda certain rather weak labelling.

When control embryos of 27 hr. were compared with embryos of the same ageraised in - SO4 sea water, the difference with respect to regional incorporation of3H-uridine was striking. As shown in Figs. J and K, the ectoderm is morestrongly labelled than the entoderm. One could distinguish a tendency to reducedincorporation in the topmost part of the acron compared with that found in amore vegetal region of the ectoderm. As in control embryos, the top of the acronmay have attained a degree of differentiation which requires only a lower RNAsynthesis. In a more vegetal region of the ectoderm differentiation still takes place,which leads to the extension of the acron in a vegetal direction. The main resultis, however, the reduced incorporation in the entomesoderm, indicating alowered anabolic activity in the vegetal region of the embryo. Both DNA andRNA seems to be involved in this reduced anabolic activity.

Incorporation of Y4C-labelled amino acids into normal embryos and embryosraised in sulfate-free sea water

Beyond the measurement included in the paper by Runnstrom et al. (1964)five series of measurements of amino acid incorporation were carried out inwhich 14C-labelled amino acids in algal hydrolysate containing 14 differentamino acids were used. The agreement between the measurements was good.Text-fig. 1 illustrates one of the five experiments in which the incorporation intonormal embryos (1) and into embryos raised in sulfate-free sea water (2) werecompared. The same batch of eggs or embryos was used in both series. Theincubation with the labelled amino acids lasted each time for 30 min. The valuesin CPM/lOO embryos were plotted as a function of the time of completion ofincubation. It seems that inhibition of amino-acid incorporation prevailed as

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296 J. 1MMERS and J. RUNNSTROM

early as 12 hr. after fertilization. For the next hours the increase of incorporationof the labelled amino acids is the same in samples from the control embryos andfrom those raised in sulfate-free sea water. Later, the increase of incorporationseemed to be faster in the samples from the control than in those from embryosraised in - SOJ medium. If the values for the stages 16^-24 hr. after fertilization

250 +

5012 13-5 15 16-5 18 19-5 21 225 24

TEXT-FIG. 1. Incorporation of 14C-labelled amino acids of algal protein hydrolysate in Para-cent rot us lividus embryos raised in normal (curve 1) and in sulfate-free (curve 2) sea water.The incorporation was carried out under slow rocking in a capillary test-tube containing100 fil suspension of embryos and with radioactivity 1 /zC./ml.; incubation for 30 min •t=19°C.

are averaged, the incorporation in the embryos raised in - SO4 medium is foundto be about 70 per cent, of that in the control embryos. This is in rather goodagreement with the difference reported by Runnstrom et al (1964). Their corres-ponding value amounted to about 79 per cent.

DISCUSSION

Histochemical and incorporation data (Immers, 1959, 1961a and b) showedbeyond doubt that sulfate occurs within the sea urchin embryo mainly as a com-


ponent of sulfated polysaccharide. The absence of SO^ in the medium thusprimarily prevents the formation of sulfated polysaccharides.

Moreover, Immers (1966) has shown that acid polysaccharides prepared fromeggs and embryos of Paracentrotus Hvidus are bound to proteins; even afterdrastic treatment with proteolytic enzymes breakdown products of protein mayremain attached to the polysaccharides.

The change in morphogenesis occurring in — SO4 medium has been charac-terized as an animalization (Lindahl, 1933, 1936, 1942). Even when the ento-mesoderm does not seem to be strongly affected, an extension of the acron withits ciliary tuft occurs (see Runnstrom et ah, 1964, Figs. 2-4). This indicates adisturbance in the normal balance between animal and vegetal region. Even amoderate degree of animalization disturbs the behavior of the primary mesen-chyme cells. Their attachment to a certain zone in the vegetal region of the ecto-derm is delayed, as was described by Herbst (1904). Thus they remain locatednear to the archenteron for a prolonged period. Gradually, they become dis-placed to the attachment zone of the ectoderm, which, however, is no longer ableto induce the normal bilateral arrangement of the primary mesenchyme cells.Instead a multiple formation of triradiate skeleton rudiments occurs (Runnstromet a/., 1964, Fig. 6). These changes in normal morphogenesis may be caused bya decreased mobility of the primary mesenchyme cells, owing to the absence ofsulfated polysaccharides. At least at lower degrees of animalization the move-ments are, however, not completely suppressed. Another possibility should alsobe considered, namely, that the disturbances in arrangement of the primarymesenchyme cells are caused by changed properties of the attachment zone of theectoderm.

The structure of the attachment zone, as briefly described by Baltzer et al.(1958) for Sphaerechinus granular is, suggests a secretory tissue. Interspaces areseen between the cells. Deprivation of sulfate may change the character of thesecretion so as to delay the movements and the attachment of the primary mesen-chyme cells, even when the effect is weak.

Markman (1963) showed that treatment with actinomycin C at a concentrationof 10 /xg./ml. caused profound changes in the interaction between ectoderm andprimary mesenchyme. After early treatments, the primary mesenchyme cellsremained scattered without any order in the vegetal region of the blastocoel. Atreatment that started 10 hr. after insemination allowed the mesenchyme cells toarrange themselves in a ring although the contact with the ectoderm was impaired.There is a certain parallelism between the effect of actinomycin and that ofdeprivation of SO|\ The actinomycin may gradually block a secretion the for-mation of which is closely dependent upon transfer of genetic information. Depri-vation of SO;f, on the other hand, may block the formation of a sulfated poly-saccharide which is a necessary component of the secretion. According tounpublished observations, the attachment of primary mesenchyme cells to theectoderm may be reversed by treatment with actinomycin. A continuous secretion


seems thus to be necessary for the maintenance of the interaction between theectoderm and the primary mesenchyme cells.

The interpretations given receive additional support by observations of theautoradiographs produced in the present study. In the control embryos there isalways a zone in the vegetal ectoderm which shows a strong incorporation of3H-uridine. This may correspond to the attachment zone. The primary mesen-chyme cells, on the other hand, show hardly any labelling with 3H-uridine. Thismust mean that the production of nucleic acids is faster in the ectoderm than inthe underlying primary mesenchyme. The same holds true for the incorporationof amino acids (Markman, personal communication).

It is of great interest that the mother-cells of the primary mesenchyme, themicromeres of the 16-cell stage, show a strong incorporation of 3H-uridine aswas found by Czihak (1965). Markman (1961a) showed that at a somewhat laterstage also about 8 hours after fertilization, a small number of cells at the vegetalpole display high radioactivity after incorporation of 14C-adenine. Markmanconsidered these cells as descendants of the micromeres. The precocious for-mation of RNA may, inter alia, prepare the prospective primary mesenchymecells for their activity. The RNA formed is probably messenger RNA as, ac-cording to Wilt (1964), new ribosomal RNA appears only after the mesenchymeblastula stage.

Immers (1961a) showed earlier that the secondary mesenchyme cells lose theircapacity to form pseudo-and filopodia in embryos raised in - SO4 sea water.This has been confirmed above (see Plate 1 Figs. B & C). The secondary mesen-chyme cells thus become more spherical instead of being spun out as in normalembryos. The connections between secondary mesenchyme cells and ectodermare not, or only incompletely, established. Instead, the secondary mesenchymecells may aggregate in groups (Figs. B and C).

In the entomesoderm, only the primary invagination may take place. Thetraction of the secondary mesenchyme cells is necessary for completion of theinvagination of the entomesoderm (Gustafson & Kinnander, 1956; Dan &Okazaki, 1956). The lack of traction may explain the poor invagination of theentomesoderm, seen in Figs. B and C. The interactions between the cells may,however, also be changed.

The absence of a bilateral arrangement of the primary mesenchyme cells evenwhen attached to the ectoderm, indicates strongly that a defective state of theattachment zone may prevail even in slightly animalized larvae.

In embryos with more advanced animalization (see Runnstrom et al., 1964,Figs. 5-6) the entoderm is in varying degrees converted into ectoderm. Accordingto the concept of Runnstrom (1961a), the fan./veg. quotient' increases underthese conditions so as to correspond to a more animal 'level' than before. Therather strong incorporation of 3H-uridine in the vegetal region of the ectodermmentioned before may correspond to this shift which is also the cause of thedefective state of the attachment zone. In strongly animalized embryos the


attachment zone becomes wholly suppressed because this zone is compatibleonly with a certain range of an./veg.-values. Thus a decrease in the an./veg.quotient may likewise displace and eventually suppress the attachment zone.The same has been shown to occur in embryos vegetalized by treatment withLi+ (Herbst, 1892) or tyrosine (Fudge, 1959; Fudge-Mastrangelo, 1965).

The constriction experiments of Horstadius (1938) are of special interest in thiscontext. Only one striking case may be referred to (see I.e., Fig. 7. A1? A2). Theconstriction was almost equatorial, separating an animal region from a vegetalone. In the former an hypertrophic acron region developed; in the vegetal regionwhich contained the entomesoderm and mesenchyme the differentiation of theattachment zone failed to occur. The primary mesenchyme cells remained nearto the intestine and there formed some radially arranged triradiate skeletonpieces. No attachment zone appeared because the necessary degree of an./veg.interaction was not attained.

The constriction experiments show that even rather slight distortions of theshape of the embryo have a great influence on its differentiation. The constric-tion must primarily bring about disturbances in diffusion processes which arenecessary for the production of the gradient system of the developing embryo.It must also be kept in mind that added substances act on a graded system inwhich the different levels may have different susceptibility to or tolerance of them.Moreover, the direct effect of the added substance may be modified by inter-actions within the system.

In more strongly animalized embryos a displacement of the epithelial cellstakes place in the direction of the animal region, as is evident from a comparisonof Figs. A and B and from Figs. 3-6 of Runnstrom et al. (1964). The displace-ment results in a strong concentration of the cells within the most animalregion, a process which is probably connected with an increased adhesionof the cells. Animalization and vegetalization involve in fact a competitionfor cells. The concentration of cell material seems to be accompanied byan increased mitotic activity, as may be inferred from such pictures as Figs. EandF.

Runnstrom et al. (1964) found that animal halves raised in — SO;f mediumundergo an animalization stronger than the animalization occurring in animalhalves raised in normal sea water. The strong concentration of the cell materialin the animal direction is combined with an extension of the region carryingstereocilia. This extension may indicate that the concentration of cells alsofavors a transfer of animalizing agents from the most animal region so as toincrease the an./veg. quotient to a high value over a great part of the animalhalf.

As shown by Markman & Runnstrom (1963) the animalization of animal halvesis delayed by rather low concentrations of actinomycin C. The animalizingagents are thus without effect unless they directly or indirectly bring about atransfer of selected genetical information.


In contrast to the effects on animal halves, the absence of SO r̂ caused a vege-talization of the vegetal halves. The cells often became concentrated in thevegetal direction, with the consequence that the animal region did not get the cellsnecessary for restoring an animal centre which could give a more balanced courseto the further development. A tendency to dissociation of the cells prevailed,a tendency which decreased in the animal direction. This tendency proved,however, to be of no avail for the enhanced vegetalization (Markman, 1963;Runnstrom et al, 1964). The dissociation observed may be due to a break-up ofthe hyaline layer which is thinner at the vegetal than at the animal pole.

The sulfated mucopolysaccharides seem to act as moderators of the tendencyof the cells to concentrate in the animal or the vegetal direction. In advancedanimalization the cell concentration occurs only in the animal direction (Runn-strom et al., 1964, Figs. 5-6). In the absence of sulfated mucopolysaccharidesthe 'naked' animal cells develop an increased adhesion which is stronger thanthat of the vegetal cells.

At present it is unclear whether the moderating action is exerted by the histo-chemically demonstrated acid mucopolysaccharides of the hyaline layer or bysimilar compounds which form part of the cytoplasmic surface or by both. Thespecially formed desmosomes (see Balinsky, 1959) contribute to the adhesion ofcells. According to electron microscopic observations of these writers, thedesmosomes are conspicuous mainly in the animal region.

The sulfated polysaccharides not only participate in the production of secretionsand components of the cell surface but are also important intracellular com-ponents. This is shown by the reduction in incorporation of labelled thymidine,uridine and amino acids, when SO^ is absent in the medium. The most con-vincing evidence is the changed staining properties of the nuclei in the animalregion (see Fig. C). Markman (1957) found that treatment of sections of Carnoyfixed eggs with low concentrations of ribonuclease brought about a state in whichthe animal nuclei were stained with the Hale reagent. Immers (1956), on the otherhand, found that in embryos raised in sulfate-free sea water, the nuclei of theanimal region -were stained with the Hale reagent without previous RN-asetreatment, as was confirmed in the present work. The direct staining of the nucleimay be the consequence of a weak activation of ribonuclease in the living state.Runnstrom et al. (1964) pointed to the known capacity of sulfated polysaccharidesto serve as rather unspecific inhibitors both of nucleases and of proteases. Theirabsence would release these enzymes. The decreased incorporation of thymidine,uridine and amino acids is understandable on this basis.

Although the anabolism is decreased in the whole embryo deprived of SO^,there is nevertheless a difference between the animal and the vegetal region.According to the autoradiographic evidence, the anabolic processes are relativelyhigher in the former than in the latter region. This indicates that, on the whole,the sulfated polysaccharides are more important for cell structure and chemicalreactions in the vegetal than in the animal region of the embryo. The way in


which vegetal cells may acquire higher resistance against deprivation of SO^ isto become animalized by the mechanism discussed earlier. In a way, this resemblesthe selection of reaction patterns which Dean & Hinshelwood (1964) distin-guished in drug-treated microorganisms. According to these authors, the selectedpattern is the one in which a steady state is most rapidly attained. Evidently, theconditions which bring about an approach to a steady state are severely disturbedin the vegetal cells owing to the lack of sulfated polysaccharides, whereas in theanimal cells, an approach to a steady state is more easily brought about underthese conditions, The rate of reactions is, however, on a lower level than in thecontrol. This does not exclude, for example, components of the stereociliabeing produced to a larger extent in embryos raised in -SO4 medium than innormal ones.

Each cell in the embryo is exposed to continuous influences from other cells orcell groups. Such influences may bring about a gradual shift in established steadystates. This may lead to the enhancement of some, and suppression of other,synthetic processes. The double gradient concept is a schematic way of charac-terizing some of the more decisive influences exerted on single cells of the seaurchin embryo.

It may be inferred from experiments carried out by Horstadius (1950) that theanimalizing or vegetalizing agents remain in an active state even when the mainregions of the embryo have been delimited.

There is a close analogy between the embryos raised in — SOir medium and thosewhich have been pretreated with low doses of trypsin (Runnstrom, 1961c, 1962).Upon fertilization, the latter eggs may fail to cleave and gradually undergo a phaseseparation; a varying percentage of eggs may, however, develop with a patho-logical vegetal region, whereas the animal region may be animalized and evenextend at the expense of the vegetal one. In a few significant cases, an almostcomplete animalization was attained. It is well established that treatment withlow doses of trypsin brings about the activation of a gelating, proteolyticenzyme (Runnstrom & Kriszat, 1962). The consequences of this activation willnot be discussed in detail, but again it seems probable that the normal metabolicpattern is severely disturbed in the vegetal region. The vegetal cells are savedfrom deterioration only by being brought up to a higher an./veg. level.

Markman (1963) reported that addition of uridine to the medium had a bene-ficial effect on the development of slightly abnormal sea urchin eggs. It is ofinterest that Davidson et a/. (1960) provided evidence that uridine triphosphate,but not triphosphates of adenosine, cytosine and guanosine, stimulates thesulfatation of chondroitin sulfate B. Possibly, the presence of uridine in theexperiments of Markman favored the formation of sulfated polysaccharideswhich protect against release of various hydrolytic enzymes.

The interpretations given in this paper have admittedly a tentative character,but they are open to further experimental tests. The research will be carried onboth from its morphogenetic and its biochemical side.


SUMMARY

1. The present work forms a continuation of earlier attempts to analyse the roleof sulfate in the early development of the sea urchin. The absence of sulfate ionsin the medium means in the first place absence of sulfated mucopolysaccharides.Certain characteristic changes in morphogenesis arise as a consequence of theabsence of these compounds. In whole embryos and in animal halves an animali-zation, in vegetal halves, a vegetalization, takes place (Runnstrom et ah, 1964).According to the present paper, deprivation of sulfate ions causes a concentrationand a stronger adhesion of cells in the animal direction or, particularly in vegetalhalves, in the vegetal direction. The tendency of cells to concentrate in onedirection is moderated by the presence of sulfated mucopolysaccharides undernormal conditions.

2. Attention has been paid to the behaviour of the primary mesencheme cells.Their failure to develop the normal bilateral arrangement is attributed to loweredmotility, due to the absence of acid mucopolysaccharides. An important role isalso ascribed to a deficient differentiation of the attachment zone in the vegetalentoderm. The degree of deficiency is shown to be due to the degree of animali-zation attained in embryos raised in sulfate-free medium. Strong animalizationsuppresses completely the formation of an attachment zone. The double gradientconcept is able to account for the various changes in the attachment zone, in-cluding its eventual suppression. Moreover, the role of secretory processes inthe interaction between ectoderm and primary mesenchyme is discussed on basisof available evidence.

3. The secondary mesenchyme is also strongly affected by the absence ofsulfated polysaccharides. The cells do not disperse in the normal way but remainmore aggregated.

4. The incorporation of 3H-thymidine and 3H-uridine was shown to be con-siderably reduced in embryos raised in sulfate-free sea water. Autoradiographicstudies showed that in embryos raised in sulfate-free medium, the incorporationof these precursors is lower in the vegetal than in the animal region. The in-corporation of amino acids was studied in different stages; in confirmation ofRunnstrom et al. (1964) it was found to be lower in sulfate-free than in sulfatecontaining medium (Text-fig. 1).

5. The sum of observations indicates that lack of sulfated polysaccharides affectthe structure and anabolic pathways more strongly in the vegetal than in theanimal region of the embryo. Presumptive entomesoderm may acquire a higherresistance to lack of sulfated polysaccharides by becoming animalized, as thismeans a change in structure and reaction patterns which is compatible withdeprivation of the sulfated compounds. In extreme cases this shift may lead to acomplete animalization of the ento-mesoderm.


RESUME

Nouvelles recherches sur les effets de Vabsence de sulfates sur le developpementinitial de VOursin Paracentrotus lividus

1. Le present travail constitue la suite d'essais anterieurs sur l'analyse du rolejoue par les sulfates au debut du developpement de l'Oursin. L'absence d'ionssulfates dans le milieu entraine en premier lieu l'absence de mucopolysaccharidessulfates. Certaines modifications caracteristiques de la morphogenese sont laconsequence de l'absence de ces composes. Dans les embryons entiers et lesmoities animales survient une animalisation, dans les moities vegetatives unevegetalisation (Runnstrom et al., 1964). Selon les resultats exposes ici, la pri-vation d'ions sulfates provoque une concentration et une plus forte adhesiondes cellules dans la direction animale ou, en particulier dans les moities vegeta-tives, dans la direction vegetative. La tendance a cette concentration de cellulesdans une seule direction est moderee par la presence de mucopolysaccharidessulfates dans les conditions normales.

2. On a examine avec attention le comportement des cellules du mesenchymeprimaire. Le fait qu'elles ne peuvent prendre normalement leur dispositionbilaterale est rapporte a la diminution de leur motilite, du a l'absence de muco-polysaccharides acides.

On attribue aussi un role important a une deficience de la differentiation de lazone d'attachement dans l'endoderme vegetatif. On montre que le degre dedeficience est du au degre d'animalisation atteint chez les embryons eleves dansun milieu depourvu de sulfate. Le concept du double gradient peut rendre comptedes diverses modifications de la zone d'attachement, y compris de sa suppressioneventuelle. De plus, le role de processus secretaires dans Finteraction entrel'ectoderme et le mesenchyme primaire est discute sur la base des resultats dis-ponibles.

3. Le mesenchyme secondaire est lui aussi fortement affecte par l'absence depolysaccharides sulfates. Les cellules ne se dispersent pas normalement maisrestent plus agregees.

4. L'incorporation de thymidine-3H et d'uridine-3H est considerablementreduite dans les embryons eleves dans de l'eau de mer sans sulfate. Les recherchesautoradiographiques ont montre que dans les embryons eleves dans un milieu sanssulfate, l'incorporation de ces precurseurs est plus faible dans la region vegetativeque dans la region animale. L'incorporation des acides amines a ete etudiee adivers stades; elle est plus faible dans un milieu depourvu de sulfate que dans unmilieu en contenant (Text-fig. 1), ce qui confirme les resultats de Runnstrometal. (1964).

5. L'ensemble des observations indique que les polysaccharides sulfatesaffectent plus fortement la structure et les processus anaboliques dans la regionvegetative que dans la region animale de l'embryon. La maniere par laquellel'endoderme presomptif, par exemple, peut acquerir une plus grande resistance,

304 j . IMMERS and J. RUNNSTROM

est de devenir animalise, ce qui implique une modification de la structure et destypes reactionnels compatible avec l'absence de composes sulfates. Dans les casextremes, ce glissement peut aboutir a une animalisation complete de l'entomeso-derme.

ACKNOWLEDGEMENTS

The experimental part of this study has been made at the 'Stazione Zoologica', Naples.It is a pleasant duty to express our thanks to the Director, Dr Peter Dohrn, and his staff.Financial support from 'The Swedish Natural Sciences Research Council' and 'The SwedishCancer Society' are most gratefully acknowledged.

R E F E R E N C E S

BALINSKY, B. J. (1959). An electron microscopic investigation of the mechanism of adhesion ofcell in a sea urchin blastula and gastrula. Exptl Cell Res. 16,429-33.

BALTZER, F., CHEN, P. S. & WHITELEY, A. H. (1958). Biochemical studies on sea urchinhybrids. Exptl Cell Res. 6,192-209.

CZIHAK, G. (1965). Evidences for inductive properties of the micromere-RNA in sea urchinembryos. Naturwissenschaften, 52,141-2.

DAN, K. & OKAZAKI, K. (1956). Cyto-embryological studies of sea urchin. III. The role of thesecondary mesenchyme in the formation of the primitive gut in sea urchin larvae. Biol.

Bull. mar. biol. Lab., Woods Hole, 110, 29-42.DAVIDSON, E. A. & RILEY, J. G. (1960). Enzymatic sulfation of chondroitin B. / . Biol. Chem.

235, 3367-9.DEAN, A. C. R. & HINSHELWOOD, C. (1964). What is heredity? Nature, Lond. 202, 1046-52.FUDGE, M. W. (1959). Vegetalization of sea urchin embryos by treatment with tyrosine.

Exptl Cell Res. 18,401-4.FUDGE-MASTRANGELO, M. W. (1965). A study of the vegetalizing action of tyrosine on the

sea urchin embryo. Dissertation presented for the degree of Ph.D. Yale University.GUSTAFSON, T. & KINNANDER, H. (1956). Gastriculation in the sea urchin larva studied by aid

of time-lapse cinematography. Exptl Cell Res. 10, 733-4.GUSTAFSON, T. & WOLPERT, L. (1961). Studies on the cellular basis of morphogenesis in the

sea urchin embryo. Exptl Cell Res. 24, 64-79GUSTAFSON, T. & WOLPERT, L. (1963). The cellular basis of morphogenesis and sea urchin

development. Int. Rev. Cytol. 15,139-214.HALE, C. W. (1946). Histochemical demonstration of acid polysaccharides in animal tissues.

Nature, Lond. 157, 802.HERBST, C. (1892). Exp. Untersuchungen liber den Einfluss des veranderten chemischen

Zusammensetzung des umgebenden Medium auf die Entwicklung der Thiere. Z. wiss.Zool. 55,446-518.

HERBST, C. (1904). Uber die zur Entwicklung der Seeigellarven notwendigen anorganischenStoffe, ihre Rolle und ihre Vertretbarkeit. II Teil. Die Rolle der notwendigen anor-ganischen Stoffe. Wilhelm Roux Arch. EntwMech. Org. 17, 305-520.

HORSTADIUS, S. (1938). Schniirungsversuche an Seeigelkeimen. Wilhelm Roux Arch. Entw-Mech. Org. 138,197-258.

HORSTADIUS, S. (1950). Transplantation experiments to elucidate interactions and regulationswithin the gradient system of the developing sea urchin egg. / . exp. Zool. 153,245-76.

IMMERS, J. (1954). Chemical and histochemical demonstration of acid esters by acetic ironreagent. Exptl Cell Res. 6,127-33.

IMMERS, J. (1956). Cytological features of the development of the eggs of Paracentrotus lividusreared in artificial sea water devoid of sulfate ions. Exptl Cell Res. 10,546-8.

IMMERS, J. (1959). Autoradiographic studies on incorporation of 14C-labeled algal proteinhydrolysate in the early sea urchin development. Exptl Cell Res. 18, 585-8.


IMMERS, J. (1961a). Incorporation of 35SO4 in the sea urchin egg and larva. Ark. Zool. 13,561-4.

IMMERS, J. (19616). Comparative study of the localization of incorporated 14C-labeled aminoacids and 35SO4 in the sea urchin ovary, egg and embryo. Exptl Cell Res. 24, 356-78.

IMMERS, J. (1962). Investigation on macromolecular sulfated polysaccharides in sea urchindevelopment. Uppsala: Almquist &Wicksell.

IMMERS, J. (1966). Preparation of acid polysaccharides from sea urchin material and theirfractionation by gel filtration. Acta Chem. Scand. (under publication).

KUHN, A. (1955). Vorlesungen iiber Entwicklungsphysiologie. Berlin: Springer.LINDAHL, P. E. (1933). Uber 'animalisierte' und 'vegetativisierte' Seeigellarvan. Wilhelm

Roux Arch. EntwMech. Org. 128, 661-4.LINDAHL, P. E. (1936). Zur Kenntniss der physiologischen Grundlagen der Determination im

Seeigelkeim. Acta Zool. Stockh. 17,179-365.LINDAHL, P. E. (1942). Contribution to the physiology of form generation in the sea urchin

development. Q. Rev. Biol. 17, 213-27.MARKMAN, B. (1957). Studies on the nuclear activity in the differentiation of the sea urchin

embryo. Exptl Cell Res. 12, 424-6.MARKMAN, B. (1961a). Regional differences in isotopic labeling of nucleic acid and protein in

early sea urchin development. Exptl Cell Res. 23,118-29.MARKMAN, B. (19616). Differences in isotopic labeling of nucleic acid and protein in early sea

urchin development Exptl Cell Res. 23,197-200.MARKMAN, B. (1963). Morphogenic effects of some nucleotide metabolites and antibiotics

on early sea urchin development. Ark. Zool. (2) 16,207-17.MARKMAN, B. & RUNNSTROM, J. (1963). Animal and vegetal halves of sea urchin larvae sub-

jected to temporary treatment with actinomycin C and mitomycin C. Exptl Cell Res. 31,615-8.

PEARSE, A. G. E. (1960). Histochemistry. London.RUNNSTROM, J. (1961a). The role of nuclear metabolism in the determination of the sea urchin

egg. Path. Biol., Paris, 9, 781-5.RUNNSTROM, J. (19616). Remarks on control and structure and differentiation in cells and cell

system. In Biol. Structure and Function, Proc. 1, IUB/IUBS Symp. (ed. T. W. Goodwinand O. Lindberg) 2,465-74.

RUNNSTROM, J. (1961c). Effect of pretreatment of the sea urchin eggs with trypsin of differentdoses with respect to cortical changes, cleavage and further development. Exptl CellRes. 22, 576-608.

RUNNSTROM, J. (1962). Differential effects of pretreatment of sea urchin eggs with low dosesof trypsin. Zool. Bidr. Upps. 35, 385-95.

RUNNSTROM, J. & KRISZAT, G. (1962). The gelating effect of lower doses of trypsin on seaurchin eggs and its removal by exposure to glutathione. Exptl Cell Res. 28,192-207.

RUNNSTROM, J., HORSTADIUS, S., IMMERS, J. & FUDGE-MASTRENGELO, M. (1964). An analysisof the role of sulfate in the embryonic differentiation of the sea urchin (Paracentrotuslividus). Revue suisse Zool. T 71, 21-54.

SCHMIDT, J. (1904). Zur Kenntniss der Larvenentwicklung von Echinus microtuberculatus.Verh. phys.-med. Ges. Wu'rzb. 36, 207-336.

WILT, F. H. (1963). The synthesis of ribonucleic acid in sea urchin embryos. Biochem. biophys.Res. Commun. 11, 447-51.

{Manuscript received 1st July 1965)

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