/ . Embryol. exp. Morph., Vol. 14, Part 3, pp. 289-305, December 1965
Printed 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 . RUNNSTROM 1
From the Wenner-Gren Institute, University of Stockholm
WITH TWO PLATES
THE morphological effects of sulfate-free medium on sea urchin embryos were
described 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 sulfate
in the development of the sea urchin. Immers (1956, 1959, 1961a and b, 1962)
studied the distribution and function of acid mucopolysaccharides in early
developmental stages of sea urchins, mainly Paracentrotus lividus. A dominant
group 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 by
staining of sectioned specimens with the ferri-acetic reagent of Hale (1946). In
blastulae or gastrulae raised in sulfate-free sea water these regions are negative
with respect to Hale staining (Immers, 19616). On the other hand, the ectodermal
nuclei of the animal region of the embryos are stained with the Hale reagent
although the nuclei of the vegetal region remained unstained (I.e.).
Runnstrom et al. (1964) extended these studies and included observations on the
effect of sulfate-free medium on animal and vegetal halves. A survey and discussion of previous work were made by Runnstrom et al (1964).
The morphological effect of the lack of sulfate involves an animalization of the
embryo. The degree of animalization may vary with the batch of eggs used; it
is further dependent on the degree of removal of the sulfate from the medium in
which the embryos are raised and the stage and duration of treatment. At the
lowest degrees of animalization, the archenteron attains its normal size; nevertheless, an extension of the acron with its stereocilia takes place; the normal bilateral
symmetry of the embryo is replaced by a radial symmetry expressed both in
ectoderm and entoderm. The primary mesenchyme cells show no bilateral
arrangement. As a consequence smaller supernumerary pieces of skeleton appear
which to varying degrees manifest the radial symmetry of the embryos subjected
to sulfate-free medium. Increasing animalization leads to a stronger development
1
Authors' address: The Wenner-Gren Institute, University of Stockholm, Norrtullsgatan
16, Stockholm Va, Sweden.
290
J. IMMERS and J. RUNNSTROM
of the ectoderm with extension of the acron so that two-thirds of the embryo may
be covered by stereocilia; the entoderm becomes reduced or suppressed and the
mesenchyme loses to an increasing extent its capacity for migration and normal
distribution.
Runnstrom et ah (1964) demonstrated that lack of sulfate in the medium causes
reduced incorporation of amino acids into whole embryos. Lack of sulfate also
reduced the incorporation of amino acids into isolated animal halves (corresponding to the mesomere material). In vegetal halves (macromere + micromere
material) on the other hand, lack of sulfate did not decrease the amino acid incorporation, which even in the presence of sulfate is rather low (Markman, 1961a).
Previous studies indicated strongly that lack of sulfate may interfere with protein
synthesis within the embryos. The sulfated polysaccharides seem to be of
particular importance for the maintenance of the integrity of the vegetal region
of the sea urchin embryo. The present paper is a continuation of those of Immers
and of Runnstrom et ah referred to above. It includes a study of the incorporation
of 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 were
used in these experiments. The handling of gametes was the customary one (see
Immers, 1956). Fertilized eggs were raised under slow rocking in glass trays
covered with glass plates. The media were normal sea water (+ SO^) or sulfatefree sea water ( - SO^). The temperature was kept at 19° to 20° C.
At different times, 8-10 ml. suspensions were transferred from the trays to
smaller elongated containers to which labelled precursors were added. The
suspensions were subject to rocking during the time of exposure which lasted for
tritiated thymidine and tritiated uridine 5 hrs. and for 14C-labelled amino acids,
30 min. at different final concentrations of the labelled precursors. Further
details 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 protein
hydrolysate (14 different amino acids 100 /xC./mg.) were used. All isotopes were
supplied by the Radiochemical Centre, Amersham. The different labelled compounds were incorporated into embryos taken from one and the same egg
suspension, all embryos thus being in the same stage of development.
After incubation the embryos were fixed, washed and the radioactivity measured in an automatic 'Nucleo' counter or in a 'Tracer lab.' windowless gas flow
counter. The details of procedures were the same as those used by Markman
(1961&). The fixation in Carnoy's fluid and the subsequent washing in
96 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 treatment of the samples with 5 per cent trichloracetic acid (TCA) for 45 min. at
80° 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 influenced
the number of counts by only about 5 per cent, which is less than the error given
by the counters. Thus the method used must give a fairly accurate measure of
incorporation of the labelled amino acids into proteins. This was confirmed also
in other ways (Markman, to be published).
Moreover, lipids do not seem to interfere with the incorporation of precursors
of nucleic acids.
Autoradiographs were made with Kodak autoradiographic stripping plates
AR. 10. The histochemical staining (Unna-Brachet methylgreen-pyronin and
Hale 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 good
representation of different regions of the early pluteus larva and their designation
are given in Kiihn (1955, p. 181). The dorsal side extends from the 'acron' to
the blastopore on one side; in the first pluteus stage the wall consists of a
squamous epithelium and is supported and extended by the body rods. This
whole-region was early (see Schmidt, 1904) called the 'apical region', because of
its elongated, pointed shape. In the following the designation,' apical region or
direction' is used synonymously with 'dorsal region or direction'. The ventral
side encompasses mainly the oral field, which is surrounded by the ciliary band
of which the early distinguishable acron forms a part.
The' attachment zone' is the zone of the vegetal ectoderm to which the primary
mesenchyme cells first attach themselves. 'Embryos' or 'embryonic' refer to
Stages before the early pluteus stage. This is considered to begin when the buds of
the 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 sulfatefree sea water soon after fertilization and raised in this medium. The sections
were 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 a
very restricted lumen. On the top of the rudimentary intestine a group of aggregated secondary mesenchyme cells is seen; a few more detached mesenchyme
cells are also present. Characteristic of the mesenchyme cells is their spherical
or only slightly elongated shape. This is in contrast to the shape of the secondary
292
J. IMMERS and J. RUNNSTROM
mesenchyme cells of the control embryo where these cells have formed numerous
pseudo- and filopodia. They are spun out between entoderm and ectoderm and
form numerous contacts with both. The primary mesenchyme cells are less
conspicuous in Fig. A. They form a well-known pattern whose establishment has
been thoroughly analysed by Gustafson and his co-workers (see Gustafson &
Wolpert, 1961,1963). The mass of cytoplasm surrounding the nuclei is relatively
greater in the primary than in the secondary mesenchyme cells, whereas the
pseudopodia of the former are correspondingly shorter, at least when the pattern
of cell contact has been established within the primary mesenchyme and between
this and the ectoderm. The atypical arrangement of the primary mesenchyme
in embryos which are radially symmetrical as a consequence of lack of sulfate has
been mentioned in the introduction. When the animalization progresses further,
as, for example, to the extent found in the embryo shown in Fig. B, skeleton
formation may become irregular or even be suppressed. In embryos of the type
represented in Fig. B the staining is definitely weaker in the entoderm-mesenchyme
than in the control embryo (Fig. A). In contradistinction, the nuclear staining of
the ectoderm cells is as strong as in the control embryo of the same age. The
morphological distinction between primary and secondary mesenchyme cells
becomes less owing to the tendency of both kinds of cells to become
spherical.
A comparison between the embryos represented in Figs. A and B shows also
that the number of cells must be reduced by lack of sulfate, at least when its effect
is as strong as in the present case. A certain delay in cell division is probably
involved. In the acron region, however, the number of nuclei seems to be about
as great as in the control. Moreover, the cylindrical cells of the acron form a
higher epithelium than in the control. In the more vegetal region of the ectoderm
the nuclei are fewer. There is an enrichment of cells in the direction of the animal
pole 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 the
strongly stained ectoderm and the unstained entoderm and mesenchyme is
obvious. The staining is, however, confined to the nuclei. It is difficult to decide
whether the stained region encompasses also a rim of cytoplasm. There is no
Hale staining of the blastocoel and of the hyaline layer. Only a small spot
corresponding to the most vegetal region of the embryo has been stained. The
regions mentioned are strongly stained in the control. The stained vegetal spot is
only a small remnant of the strongly stained material which is found in the lumen
of 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 period
beyond 22 hr. after fertilization the embryos were exposed for 5 hr. to tritiated
J. Embryol. cxp. Morph.
Vol. 14, Part 3
E X P L A N A T I O N OF PLATES
FIGS. A-K. Embryos of Paracentrotus lividus, fixed in Carnoy's fluid 32 hr. (Figs. A-C) or
27 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 and
are 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 the
photograph. 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 counterstaining by PAS. The nuclei of the ectoderm appear dark owing to a reaction between
unmasked phosphate groups of nucleic acids and the Fe + + + of Hale reagent. 600 x .
FIG. D. Normal gastrula. Autoradiography of incorporated 3H-thymidine. Counterstaining
with Giemsa. Median section, oral (ventral) side to the left. 600 x . For concentration
and radioactivity of thymidine, see Table 1, exp. 2.
FIGS. E and F. Gastrulae raised in sulfate-free sea water. Autoradiography of incorporated
3
H-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 section
oral (ventral) side to the left (H) and more frontal section (J); 580x ; counterstaining
with Giemsa; see further Table 2, exp. 2.
FIGS. J AND K. Gastrulae raised in sulfate-free sea water; autoradiography of incorporated
3
H-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)
Lack ofsulfate and the development o/Paracentrotus
293
thymidine. The data in Table I show that the incorporation of thymidine was
considerably 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 embryos
subjected to lack of sulfate as compared with control embryos raised in normal
sea water.
TABLE 1
Incorporation of 3//-thymidine into embryos o/Paracentrotus lividus raised
in normal and sulfate-free sea water
In exp. 1 the added 3 H-thymidine concentration was 3-25x 10~6M with 0-63 /zC./ml. and
in exp. 2 the concentration was 6-5 x 10~6 M with 1 -25 /zC./ml. n is number of parallel test
samples assayed in each exp.
Exp.
No.
Age of
embryos
during
incubation
(in hr.)
n
+ SO4
— SO4
1
2
21-5—26-5
22—27
4
3
7-l±0-2
138-9 + 9-5
4-4±0-6
33-2+ 1 -9
Means of C. P.M./J00 embryos
Difference with
standard error
2-7±0-3
105 • 7 ± 5 • 6
t
7-5
18-8
P
0005
<0-005
The study of the autoradiographs of 27-hr, old embryos revealed a certain
localization of the incorporation of the 3H-thymidine in control embryos. In
the stage examined, gastrulation has been achieved, the acron still carries a tuft
of stereocilia and the dorso-ventral symmetry is manifested by a flattening of a
thicker oral (ventral) side and a more stretched thinner apical (dorsal) side.
Fig. D (Plate 1). represents an approximately median section of a control embryo
where oral and aboral (apical) sides may be distinguished. The oral vegetal region
of the ectoderm showed a strong thymidine incorporation in several embryos.
The incorporation was also rather strong in the vegetal apical region. It was a
constant feature that the thymidine incorporation was, on the whole, stronger
in the oral than in the apical region. In particular the more animal part of the
apical region was very poorly labelled, even at this stage, when the flattening of
the apical cells was still moderate. It is well seen in Fig. D that the cells showing
incorporation 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 the
apical region which gradually form a thin squamous epithelium have thus a
Strongly reduced frequency of mitotic division. The oral region gives rise to the
ciliary band and later to the stomodeum. A strong incorporation at a rather
animal level of the oral side could be regarded as a preparation of the stomodeum
formation. This region can be seen in Fig. D, but was studied preferentially in
cross sections where the anterior region of the archenteron was seen to approach
the ectoderm. The incorporation into the mesenchyme cells of the 27-hr, embryos
was low even compared with that in the more animal apical region.
20
294
J. IMMERS and J. R U N N S T R 5 M
In the embryos raised in sulfate-free sea water the ventro-dorsal polarity of
thymidine incorporation is replaced by a more pronounced animal-vegetal
polarity, as Fig. E shows. The groups of densely accumulated grains correspond
to the extended acron region with its tuft of stereocilia. The maximum mitotic
rate is confined to this region rather than to an oral region. In many cases the
incorporation of 3H-thymidine was particularly strong in the region of the acron
cells adjacent to the blastocoel, see Fig. F. This is also the position of the nuclei
in 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 acron
region. As seen in Figs. E-G the mesenchyme cells which surround the rudimentary entomesoderm remained unlabelled. The pronounced bilateral symmetry 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 found
in embryos raised in — SO^ sea water was mainly an animal-vegetal one.
Incorporation of trMated uridine
Measurements
Paracentrotus are
embryos were 27
embryos raised in
of the incorporation of tritiated uridine into embryos of
shown in Table 2. As in the experiments with thymidine, the
hr. old at the end of the incorporation period. The control
+ SOiT sea water showed a considerably higher incorporation of
TABLE 2
Incorporation of^H-uridine into embryos of Paracentrotus lividus raised in
normal and sulfate-free sea water
In exp. 1 the concentration of 3 H-uridine was 6-5 x 10~ 6 M with 7-2 /uC./ml.; in exp. 2 the
concentration was 3 - 4 x 10~ 6 M with 3-7 /zC./ml. n is the number of parallel test samples
assayed in each exp.
Exp.
No.
Age of
embryos
during
incubation
{inhr.)
n
1
2
21-5-26-5
22-27
3
3
3
Means of C.P.M.I 100 embryos
+SOJ
2 3 7 1 ±16-6
251-8± 17-1
-SO^
158-2±14-9
99-6± 7-2
Difference with
standard error
t
P
78-9±12-8
152-2±10-7
6-1
14-2
0-025
0-005
H-uridine than those raised in - S O ^ sea water. Uridine will enter rather
directly into ribonucleic acid (RNA) but after transformation to cytidine or
thymine it may also enter deoxyribonucleic acid (DNA). It has been demonstrated above that deprivation of SCXr reduces the incorporation of 3H-thymidine
into the embryos. This indicates that the rate of synthesis of DNA is lowered
when the embryos are raised in - SO4 medium. The data of Table 2 tend to show
that the rate of incorporation of 3H-uridine is reduced in embryos raised in - SO 4
Lack ofsulfate and the development o/Paracentrotus
295
medium to a level comparable with the reduced incorporation of 3H-thymidine
recorded in Table 1.
A number of autoradiographs showed that 3H-uridine is incorporated into the
nuclei and, in lesser degree, also into the cytoplasm. The regional differences
were less pronounced than in the case of incorporation of 3H-thymidine. However, the ventral side of the ectoderm showed, on the whole, a stronger and more
uniform incorporation than other regions of the ectoderm; see Fig. H (Plate 2).
\n the stage particularly studied (late gastrula stage), the acron is less labelled
with 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 attained
a certain definite level of differentiation, whereas within the oral and apical region
new differentiation processes are prepared. A more extensive study of these finer
regional differences is, however, outside the scope of this paper.
The entoderm was not uniformly labelled. Often there seemed to be a stronger
tendency for incorporation in the posterior (animal) region of the entoderm than
in its anterior (vegetal) region; see Figs, H and I. The mesenchyme cells showed
a certain rather weak labelling.
When control embryos of 27 hr. were compared with embryos of the same age
raised in - SO4 sea water, the difference with respect to regional incorporation of
3
H-uridine was striking. As shown in Figs. J and K, the ectoderm is more
strongly labelled than the entoderm. One could distinguish a tendency to reduced
incorporation in the topmost part of the acron compared with that found in a
more vegetal region of the ectoderm. As in control embryos, the top of the acron
may have attained a degree of differentiation which requires only a lower RNA
synthesis. 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 result
is, however, the reduced incorporation in the entomesoderm, indicating a
lowered anabolic activity in the vegetal region of the embryo. Both DNA and
RNA seems to be involved in this reduced anabolic activity.
Y4
C-labelled amino acids into normal embryos and embryos
raised 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 in
which 14C-labelled amino acids in algal hydrolysate containing 14 different
amino acids were used. The agreement between the measurements was good.
Text-fig. 1 illustrates one of the five experiments in which the incorporation into
normal embryos (1) and into embryos raised in sulfate-free sea water (2) were
compared. The same batch of eggs or embryos was used in both series. The
incubation with the labelled amino acids lasted each time for 30 min. The values
in CPM/lOO embryos were plotted as a function of the time of completion of
incubation. It seems that inhibition of amino-acid incorporation prevailed as
Incorporation of
20*
296
J. 1MMERS and J. RUNNSTROM
early as 12 hr. after fertilization. For the next hours the increase of incorporation
of the labelled amino acids is the same in samples from the control embryos and
from those raised in sulfate-free sea water. Later, the increase of incorporation
seemed to be faster in the samples from the control than in those from embryos
raised in - SOJ medium. If the values for the stages 16^-24 hr. after fertilization
250 +
50
12 13-5
15 16-5
18 19-5 21 225
24
1. Incorporation of 14C-labelled amino acids of algal protein hydrolysate in Paracent 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 containing
100 fil suspension of embryos and with radioactivity 1 /zC./ml.; incubation for 30 min •
TEXT-FIG.
t=19°C.
are averaged, the incorporation in the embryos raised in - SO4 medium is found
to be about 70 per cent, of that in the control embryos. This is in rather good
agreement with the difference reported by Runnstrom et al (1964). Their corresponding value amounted to about 79 per cent.
DISCUSSION
Histochemical and incorporation data (Immers, 1959, 1961a and b) showed
beyond doubt that sulfate occurs within the sea urchin embryo mainly as a com-
Lack ofsulfate and the development o/Paracentrotus
297
ponent of sulfated polysaccharide. The absence of SO^ in the medium thus
primarily prevents the formation of sulfated polysaccharides.
Moreover, Immers (1966) has shown that acid polysaccharides prepared from
eggs and embryos of Paracentrotus Hvidus are bound to proteins; even after
drastic treatment with proteolytic enzymes breakdown products of protein may
remain attached to the polysaccharides.
The change in morphogenesis occurring in — SO4 medium has been characterized as an animalization (Lindahl, 1933, 1936, 1942). Even when the entomesoderm does not seem to be strongly affected, an extension of the acron with
its ciliary tuft occurs (see Runnstrom et ah, 1964, Figs. 2-4). This indicates a
disturbance in the normal balance between animal and vegetal region. Even a
moderate degree of animalization disturbs the behavior of the primary mesenchyme cells. Their attachment to a certain zone in the vegetal region of the ectoderm is delayed, as was described by Herbst (1904). Thus they remain located
near to the archenteron for a prolonged period. Gradually, they become displaced to the attachment zone of the ectoderm, which, however, is no longer able
to induce the normal bilateral arrangement of the primary mesenchyme cells.
Instead a multiple formation of triradiate skeleton rudiments occurs (Runnstrom
et a/., 1964, Fig. 6). These changes in normal morphogenesis may be caused by
a decreased mobility of the primary mesenchyme cells, owing to the absence of
sulfated polysaccharides. At least at lower degrees of animalization the movements are, however, not completely suppressed. Another possibility should also
be considered, namely, that the disturbances in arrangement of the primary
mesenchyme cells are caused by changed properties of the attachment zone of the
ectoderm.
The structure of the attachment zone, as briefly described by Baltzer et al.
(1958) for Sphaerechinus granular is, suggests a secretory tissue. Interspaces are
seen between the cells. Deprivation of sulfate may change the character of the
secretion so as to delay the movements and the attachment of the primary mesenchyme cells, even when the effect is weak.
Markman (1963) showed that treatment with actinomycin C at a concentration
of 10 /xg./ml. caused profound changes in the interaction between ectoderm and
primary mesenchyme. After early treatments, the primary mesenchyme cells
remained scattered without any order in the vegetal region of the blastocoel. A
treatment that started 10 hr. after insemination allowed the mesenchyme cells to
arrange themselves in a ring although the contact with the ectoderm was impaired.
There is a certain parallelism between the effect of actinomycin and that of
deprivation of SO|\ The actinomycin may gradually block a secretion the formation of which is closely dependent upon transfer of genetic information. Deprivation of SO;f, on the other hand, may block the formation of a sulfated polysaccharide which is a necessary component of the secretion. According to
unpublished observations, the attachment of primary mesenchyme cells to the
ectoderm may be reversed by treatment with actinomycin. A continuous secretion
298
J. IMMERS and J. RUNNSTROM
seems thus to be necessary for the maintenance of the interaction between the
ectoderm and the primary mesenchyme cells.
The interpretations given receive additional support by observations of the
autoradiographs produced in the present study. In the control embryos there is
always a zone in the vegetal ectoderm which shows a strong incorporation of
3
H-uridine. This may correspond to the attachment zone. The primary mesenchyme cells, on the other hand, show hardly any labelling with 3H-uridine. This
must mean that the production of nucleic acids is faster in the ectoderm than in
the underlying primary mesenchyme. The same holds true for the incorporation
of amino acids (Markman, personal communication).
It is of great interest that the mother-cells of the primary mesenchyme, the
micromeres of the 16-cell stage, show a strong incorporation of 3H-uridine as
was found by Czihak (1965). Markman (1961a) showed that at a somewhat later
stage also about 8 hours after fertilization, a small number of cells at the vegetal
pole display high radioactivity after incorporation of 14C-adenine. Markman
considered these cells as descendants of the micromeres. The precocious formation of RNA may, inter alia, prepare the prospective primary mesenchyme
cells for their activity. The RNA formed is probably messenger RNA as, according to Wilt (1964), new ribosomal RNA appears only after the mesenchyme
blastula stage.
Immers (1961a) showed earlier that the secondary mesenchyme cells lose their
capacity 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 mesenchyme cells thus become more spherical instead of being spun out as in normal
embryos. The connections between secondary mesenchyme cells and ectoderm
are not, or only incompletely, established. Instead, the secondary mesenchyme
cells may aggregate in groups (Figs. B and C).
In the entomesoderm, only the primary invagination may take place. The
traction of the secondary mesenchyme cells is necessary for completion of the
invagination of the entomesoderm (Gustafson & Kinnander, 1956; Dan &
Okazaki, 1956). The lack of traction may explain the poor invagination of the
entomesoderm, 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 even
when attached to the ectoderm, indicates strongly that a defective state of the
attachment 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. According
to the concept of Runnstrom (1961a), the fan./veg. quotient' increases under
these conditions so as to correspond to a more animal 'level' than before. The
rather strong incorporation of 3H-uridine in the vegetal region of the ectoderm
mentioned before may correspond to this shift which is also the cause of the
defective state of the attachment zone. In strongly animalized embryos the
Lack ofsulfate and the development o/Paracentrotus
299
attachment zone becomes wholly suppressed because this zone is compatible
only 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 with
Li + (Herbst, 1892) or tyrosine (Fudge, 1959; Fudge-Mastrangelo, 1965).
The constriction experiments of Horstadius (1938) are of special interest in this
context. Only one striking case may be referred to (see I.e., Fig. 7. A1? A2). The
constriction was almost equatorial, separating an animal region from a vegetal
one. In the former an hypertrophic acron region developed; in the vegetal region
which contained the entomesoderm and mesenchyme the differentiation of the
attachment zone failed to occur. The primary mesenchyme cells remained near
to the intestine and there formed some radially arranged triradiate skeleton
pieces. 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 the
shape of the embryo have a great influence on its differentiation. The constriction must primarily bring about disturbances in diffusion processes which are
necessary 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 in
which the different levels may have different susceptibility to or tolerance of them.
Moreover, the direct effect of the added substance may be modified by interactions within the system.
In more strongly animalized embryos a displacement of the epithelial cells
takes place in the direction of the animal region, as is evident from a comparison
of Figs. A and B and from Figs. 3-6 of Runnstrom et al. (1964). The displacement results in a strong concentration of the cells within the most animal
region, a process which is probably connected with an increased adhesion
of the cells. Animalization and vegetalization involve in fact a competition
for cells. The concentration of cell material seems to be accompanied by
an increased mitotic activity, as may be inferred from such pictures as Figs. E
andF.
Runnstrom et al. (1964) found that animal halves raised in — SO;f medium
undergo an animalization stronger than the animalization occurring in animal
halves raised in normal sea water. The strong concentration of the cell material
in the animal direction is combined with an extension of the region carrying
stereocilia. This extension may indicate that the concentration of cells also
favors a transfer of animalizing agents from the most animal region so as to
increase the an./veg. quotient to a high value over a great part of the animal
half.
As shown by Markman & Runnstrom (1963) the animalization of animal halves
is delayed by rather low concentrations of actinomycin C. The animalizing
agents are thus without effect unless they directly or indirectly bring about a
transfer of selected genetical information.
300
J. IMMERS and J. RUNNSTROM
In contrast to the effects on animal halves, the absence of SO^r caused a vegetalization of the vegetal halves. The cells often became concentrated in the
vegetal direction, with the consequence that the animal region did not get the cells
necessary for restoring an animal centre which could give a more balanced course
to 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 of
the hyaline layer which is thinner at the vegetal than at the animal pole.
The sulfated mucopolysaccharides seem to act as moderators of the tendency
of the cells to concentrate in the animal or the vegetal direction. In advanced
animalization the cell concentration occurs only in the animal direction (Runnstrom et al., 1964, Figs. 5-6). In the absence of sulfated mucopolysaccharides
the 'naked' animal cells develop an increased adhesion which is stronger than
that of the vegetal cells.
At present it is unclear whether the moderating action is exerted by the histochemically demonstrated acid mucopolysaccharides of the hyaline layer or by
similar compounds which form part of the cytoplasmic surface or by both. The
specially formed desmosomes (see Balinsky, 1959) contribute to the adhesion of
cells. According to electron microscopic observations of these writers, the
desmosomes are conspicuous mainly in the animal region.
The sulfated polysaccharides not only participate in the production of secretions
and components of the cell surface but are also important intracellular components. This is shown by the reduction in incorporation of labelled thymidine,
uridine and amino acids, when SO^ is absent in the medium. The most convincing evidence is the changed staining properties of the nuclei in the animal
region (see Fig. C). Markman (1957) found that treatment of sections of Carnoy
fixed eggs with low concentrations of ribonuclease brought about a state in which
the animal nuclei were stained with the Hale reagent. Immers (1956), on the other
hand, found that in embryos raised in sulfate-free sea water, the nuclei of the
animal region -were stained with the Hale reagent without previous RN-ase
treatment, as was confirmed in the present work. The direct staining of the nuclei
may be the consequence of a weak activation of ribonuclease in the living state.
Runnstrom et al. (1964) pointed to the known capacity of sulfated polysaccharides
to serve as rather unspecific inhibitors both of nucleases and of proteases. Their
absence 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 relatively
higher in the former than in the latter region. This indicates that, on the whole,
the sulfated polysaccharides are more important for cell structure and chemical
reactions in the vegetal than in the animal region of the embryo. The way in
Lack ofsulfate and the development o/Paracentrotus
301
which vegetal cells may acquire higher resistance against deprivation of SO^ is
to become animalized by the mechanism discussed earlier. In a way, this resembles
the selection of reaction patterns which Dean & Hinshelwood (1964) distinguished in drug-treated microorganisms. According to these authors, the selected
pattern is the one in which a steady state is most rapidly attained. Evidently, the
conditions which bring about an approach to a steady state are severely disturbed
in the vegetal cells owing to the lack of sulfated polysaccharides, whereas in the
animal cells, an approach to a steady state is more easily brought about under
these conditions, The rate of reactions is, however, on a lower level than in the
control. This does not exclude, for example, components of the stereocilia
being produced to a larger extent in embryos raised in -SO4 medium than in
normal ones.
Each cell in the embryo is exposed to continuous influences from other cells or
cell groups. Such influences may bring about a gradual shift in established steady
states. This may lead to the enhancement of some, and suppression of other,
synthetic processes. The double gradient concept is a schematic way of characterizing some of the more decisive influences exerted on single cells of the sea
urchin embryo.
It may be inferred from experiments carried out by Horstadius (1950) that the
animalizing or vegetalizing agents remain in an active state even when the main
regions of the embryo have been delimited.
There is a close analogy between the embryos raised in — SOir medium and those
which have been pretreated with low doses of trypsin (Runnstrom, 1961c, 1962).
Upon fertilization, the latter eggs may fail to cleave and gradually undergo a phase
separation; a varying percentage of eggs may, however, develop with a pathological vegetal region, whereas the animal region may be animalized and even
extend at the expense of the vegetal one. In a few significant cases, an almost
complete animalization was attained. It is well established that treatment with
low doses of trypsin brings about the activation of a gelating, proteolytic
enzyme (Runnstrom & Kriszat, 1962). The consequences of this activation will
not be discussed in detail, but again it seems probable that the normal metabolic
pattern is severely disturbed in the vegetal region. The vegetal cells are saved
from deterioration only by being brought up to a higher an./veg. level.
Markman (1963) reported that addition of uridine to the medium had a beneficial effect on the development of slightly abnormal sea urchin eggs. It is of
interest that Davidson et a/. (1960) provided evidence that uridine triphosphate,
but not triphosphates of adenosine, cytosine and guanosine, stimulates the
sulfatation of chondroitin sulfate B. Possibly, the presence of uridine in the
experiments of Markman favored the formation of sulfated polysaccharides
which 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 on
both from its morphogenetic and its biochemical side.
302
J. IMMERS and J. RUNNSTROM
SUMMARY
1. The present work forms a continuation of earlier attempts to analyse the role
of sulfate in the early development of the sea urchin. The absence of sulfate ions
in the medium means in the first place absence of sulfated mucopolysaccharides.
Certain characteristic changes in morphogenesis arise as a consequence of the
absence of these compounds. In whole embryos and in animal halves an animalization, in vegetal halves, a vegetalization, takes place (Runnstrom et ah, 1964).
According to the present paper, deprivation of sulfate ions causes a concentration
and a stronger adhesion of cells in the animal direction or, particularly in vegetal
halves, in the vegetal direction. The tendency of cells to concentrate in one
direction is moderated by the presence of sulfated mucopolysaccharides under
normal 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 lowered
motility, due to the absence of acid mucopolysaccharides. An important role is
also ascribed to a deficient differentiation of the attachment zone in the vegetal
entoderm. The degree of deficiency is shown to be due to the degree of animalization attained in embryos raised in sulfate-free medium. Strong animalization
suppresses completely the formation of an attachment zone. The double gradient
concept is able to account for the various changes in the attachment zone, including its eventual suppression. Moreover, the role of secretory processes in
the interaction between ectoderm and primary mesenchyme is discussed on basis
of available evidence.
3. The secondary mesenchyme is also strongly affected by the absence of
sulfated polysaccharides. The cells do not disperse in the normal way but remain
more aggregated.
4. The incorporation of 3H-thymidine and 3H-uridine was shown to be considerably reduced in embryos raised in sulfate-free sea water. Autoradiographic
studies showed that in embryos raised in sulfate-free medium, the incorporation
of these precursors is lower in the vegetal than in the animal region. The incorporation of amino acids was studied in different stages; in confirmation of
Runnstrom et al. (1964) it was found to be lower in sulfate-free than in sulfate
containing medium (Text-fig. 1).
5. The sum of observations indicates that lack of sulfated polysaccharides affect
the structure and anabolic pathways more strongly in the vegetal than in the
animal region of the embryo. Presumptive entomesoderm may acquire a higher
resistance to lack of sulfated polysaccharides by becoming animalized, as this
means a change in structure and reaction patterns which is compatible with
deprivation of the sulfated compounds. In extreme cases this shift may lead to a
complete animalization of the ento-mesoderm.
Lack ofsulfate and the development o/Paracentrotus
303
RESUME
Nouvelles recherches sur les effets de Vabsence de sulfates sur le developpement
initial de VOursin Paracentrotus lividus
1. Le present travail constitue la suite d'essais anterieurs sur l'analyse du role
joue par les sulfates au debut du developpement de l'Oursin. L'absence d'ions
sulfates dans le milieu entraine en premier lieu l'absence de mucopolysaccharides
sulfates. Certaines modifications caracteristiques de la morphogenese sont la
consequence de l'absence de ces composes. Dans les embryons entiers et les
moities animales survient une animalisation, dans les moities vegetatives une
vegetalisation (Runnstrom et al., 1964). Selon les resultats exposes ici, la privation d'ions sulfates provoque une concentration et une plus forte adhesion
des cellules dans la direction animale ou, en particulier dans les moities vegetatives, dans la direction vegetative. La tendance a cette concentration de cellules
dans une seule direction est moderee par la presence de mucopolysaccharides
sulfates dans les conditions normales.
2. On a examine avec attention le comportement des cellules du mesenchyme
primaire. Le fait qu'elles ne peuvent prendre normalement leur disposition
bilaterale est rapporte a la diminution de leur motilite, du a l'absence de mucopolysaccharides acides.
On attribue aussi un role important a une deficience de la differentiation de la
zone d'attachement dans l'endoderme vegetatif. On montre que le degre de
deficience est du au degre d'animalisation atteint chez les embryons eleves dans
un milieu depourvu de sulfate. Le concept du double gradient peut rendre compte
des diverses modifications de la zone d'attachement, y compris de sa suppression
eventuelle. De plus, le role de processus secretaires dans Finteraction entre
l'ectoderme et le mesenchyme primaire est discute sur la base des resultats disponibles.
3. Le mesenchyme secondaire est lui aussi fortement affecte par l'absence de
polysaccharides sulfates. Les cellules ne se dispersent pas normalement mais
restent plus agregees.
4. L'incorporation de thymidine-3H et d'uridine-3H est considerablement
reduite dans les embryons eleves dans de l'eau de mer sans sulfate. Les recherches
autoradiographiques ont montre que dans les embryons eleves dans un milieu sans
sulfate, l'incorporation de ces precurseurs est plus faible dans la region vegetative
que dans la region animale. L'incorporation des acides amines a ete etudiee a
divers stades; elle est plus faible dans un milieu depourvu de sulfate que dans un
milieu en contenant (Text-fig. 1), ce qui confirme les resultats de Runnstrom
etal. (1964).
5. L'ensemble des observations indique que les polysaccharides sulfates
affectent plus fortement la structure et les processus anaboliques dans la region
vegetative que dans la region animale de l'embryon. La maniere par laquelle
l'endoderme presomptif, par exemple, peut acquerir une plus grande resistance,
304
j . IMMERS and
J. R U N N S T R O M
est de devenir animalise, ce qui implique une modification de la structure et des
types reactionnels compatible avec l'absence de composes sulfates. Dans les cas
extremes, ce glissement peut aboutir a une animalisation complete de l'entomesoderme.
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 Swedish
Cancer Society' are most gratefully acknowledged.
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