J. Embryol. exp. Morph. 74, 69-77 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
69
Restriction of developmental potential and
trochoblast ciliation in Patella embryos
By C. JANSSEN-DOMMERHOLT 1 , R. VAN WIJK 2 AND
W. L. M. GEILENKIRCHEN1
From the Zoological Laboratory, Utrecht University and the Laboratoire
Arago, Banyuls sur Mer, France
SUMMARY
At the 64-cell-stage embryos of Patella develop a prototroch consisting of four groups of
four cilia-bearing cells. Ciliogenesis of isolated blastomeres and trochoblasts was studied, as
well as the effect on it of cleavage arrest caused by cytochalasin B treatment. Isolation of
blastomeres or trochoblast cells has no influence on ciliogenesis; neither has arrest of cleavage
in whole embryos after the third cleavage. However, cleavage arrest before third cleavage
completely prevents ciliogenesis. Thus, third cleavage is decisive for the expression of the
developmental potential of the primary trochoblasts. Impairment of DNA synthesis by aphidicolin in the S-phase preceding third cleavage also prevents ciliogenesis. It is concluded that
a determinant for ciliogenesis as well as certain nuclear factors must be segregated into the
micromeres at third cleavage for ciliogenesis to occur in the prototroch cells.
INTRODUCTION
Ciliated prototroch cells of Molluscs appear early in the development. The
early development of Patella has been described by Wilson (1904). The first
quartet of micromeres gives rise to sixteen primary trochoblasts (Figs 1 and 2).
These cells do not divide further and develop cilia within 1 h after their last
cleavage. The primary trochoblasts derive from the micromere daughter cells
Ia 2 -ld 2 , which each divide twice. In this way four interradial groups of four
primary trochoblasts each are formed (Fig. 2). Cilia are also produced by descendants of the apical rosette cells l a ^ l d 1 (Fig. 2), which form ciliated accessory
prototroch cells and apical tuft cells. The second quartet of micromeres produces
secondary trochoblasts.
Wilson (1904) showed that isolated primary trochoblasts differentiate in exactly the same way as they would have done in the intact embryo. It is generally
accepted that the capacity for self-differentiation of cells in early Molluscan
development depends on localized determinants which are generated during
1
Author's address: Zoological Laboratory, University of Utrecht, Transitorium III,
Padualaan
8, 3508TB Utrecht, The Netherlands.
2
Author's address: Laboratory of Molecular Celbiology, University of Utrecht, Transitorium III, Padualaan 8, 3508TB Utrecht, The Netherlands.
70
C. JANSSEN-DOMMERHOLT AND OTHERS
4 1 1 1 - .111
1a -1d
1
4 11 4_i 1
1a -1d
,112
1a -
-•
4 121 4 J21
1a -1d
4 12 4 J 2
1a -Id
1a'22-1d122
1a-1d
1 21 * ,21
1a -1d
1a2-1d2
A-D
2
cleavage
4
I
la
1
211
D
-
Pr
4 212 - 2 1 2
. 221 . ,221
4 22
4
,22
1a -1d
5th
,1a
1a -1d
Pr
l i a 2 2 2 - Id222Pr
1A-1D
3rd
4th
Fig. 1. Cell lineage of the first micromere quartet of Patella. Pr = prototroch cell.
The end-cells in the lines terminating in—»go on dividing.
Fig. 2. 58-cell Patella embryo seen from the upper pole, after division of the rosette
cells ( l a n - l d n ) and the basal cells (Ia 12 -ld 12 ). Primary trochoblasts stippled (after
Wilson, 1904).
oogenesis. Localized determinants seem to be basic to cell specification in many
developing systems, but despite many efforts an exact understanding of their
nature, distribution and functional significance is still lacking (Davidson, 1976;
Freeman, 1979; Whittaker, 1979).
Determination of ciliogenesis in Patella embryos
71
In this paper we show at which time in development the determinants for
ciliogenesis in the primary trochoblasts are segregated and that their capacity to
evoke ciliation depends on their allocation to the first quartet of micromeres.
MATERIALS AND METHODS
Patella vulgata collected at the coast of Normandy were kept in the laboratory
in circulating sea water (16 °C). To obtain gametes the animals are opened
laterally along the shell (van den Biggelaar, 1977). Parts of the testis or ovary are
removed and placed in a dish containing filtered sea water. The eggs and sperm
then separate naturally into the water. Eggs exposed to sea water round off and
lose their chorion. After washing the eggs twice with sea water they are fertilized
by adding a few drops of a highly diluted sperm solution. Eggs are about 160 /im
in diameter. The embryos were reared at 18 °C in a constant temperature room.
Synchronously cleaving eggs were obtained by selecting eggs which started
first cleavage within lmin. Embryos were dissociated at the 2-, 4-, 8-, 16- or
32-cell stage by treatment with Ca ++ -free sea water starting before cleavage.
Cytochalasin B (CCB) solutions were made by diluting a standard solution of
10 fig CCB/ml and 1 % DMSO with sea water. Aphidicolin solutions used were
dilutions with sea water of a standard solution of 50 /ig/ml plus 1 % DMSO in
distilled water. For observation of nuclei whole mounts of Feulgen-stained embryos (van den Biggelaar, 1971) were made.
For scanning electron microscopy embryos were fixed for 30min in 2-5 %
glutaraldehyde in 0-1 M-cacodylate buffer (pH 7-3) and postfixed for 1 h in a 1 %
OsO4 solution in the same buffer (temperature 20 °C). After dehydration in
acetone and cyclohexane the embryos were dried over P2O5. After mounting the
eggs on aluminium stubs they were sputter-coated with gold and examined in a
Cambridge 600M scanning electron microscope.
RESULTS
Development of isolated blastomeres
Cleavages in Patella embryos are synchronous up to the 32-cell stage. According to our measurements the four cleavage cycles took 27, 27, 31 and 41min,
respectively, at a constant temperature of 18 °C. Primary trochoblasts isolated
after fifth cleavage went through their final divisions 75 min later. About 1 h after
this final cleavage moving cilia could be observed.
Blastomeres were isolated by embryo dissociation as described under
Materials and Methods. The isolation procedure had a retarding effect on cell
cycle duration. The mean intercleavage time was 10-15 % longer than the
intercleavage time of the respective cleavage cycles in intact embryos.
All blastomeres isolated at the 2- or 4-cell stage produced ciliated trochoblasts;
so did all the micromeres isolated at the 8-cell stage. At fourth cleavage each
72
C. JANSSEN-DOMMERHOLT AND OTHERS
micromere divides into a smaller I 1 cell (apical rosette cell) and a larger I 2 cell
(primary trochoblast). Each primary trochoblast divides twice and forms four
cilia-bearing cells (Fig. 2). Isolated primary trochoblasts divided and formed cilia
as well (Fig. 3). The I 1 , so-called apical rosette cells go on cleaving beyond the
sixth cleavage round (Fig. 1). Ciliogenesis-determining factors are also present
in these apical rosette cells. A number of their descendants differentiate into
ciliated accessory prototroch cells and into apical tuft cells. Moreover, the
second quartet of micromeres produces ciliated secondary trochoblasts (Wilson,
1904). The secondary and accessory prototroch cells become ciliated several
hours after the primary trochoblasts have developed cilia.
.Effects of cytochalasin B on cleavage and ciliogenesis
We used the lowest concentration of cytochalasin B (0-1/ig/ml sea water)
which inhibited cleavage in Patella but had no visible effect on mitosis. Mitosis
was not impaired in eggs continuously incubated from before first cleavage till
after the normal time of sixth cleavage. Treated embryos and controls were
compared with respect to number and synchrony of mitoses up to 2 h after sixth
cleavage in the controls. The duration of the mitotic cycles in treated embryos
was slightly shorter than in controls; the treated embryos were one mitosis ahead
at the normal time of sixth cleavage, but we were unable to express this in terms
of cell-cycle duration. The nuclei in the treated embryos were localized near the
egg surface and all went through mitosis at the same time, thus failing to show
the mitotic desynchronization that occurs in normal embryos.
Isolated trochoblasts (Ia 2 -ld 2 ) and 16-cell embryos were incubated with
cytochalasin B continuously from lOmin after fourth cleavage. Divisions were
no longer observed. In both groups cilia developed on the undivided I 2 cells in
synchrony with untreated controls.
Intact 8-cell embryos were incubated continuously from lOmin after third
cleavage. Divisions were no longer observed; cilia developed on all four
Fig. 3. Four prototroch cells reared from a I2 trochoblast isolated after fourth
cleavage. All four cells have produced cilia. Scanning electron micrograph. Magn.
xlOOO.
Figs 4-7. Scanning electron micrographs of cleavage inhibited embryos.
Fig. 4. Cleavage inhibition at the 8-cell stage with cytochalasin B. Embryo shown
2 h after 64-cell stage in control embryos was reached. The four cleavage-inhibited
micromeres produced cilia at approximately the same time as in the controls. Magn.
X500.
Fig. 5. Third cleavage partially inhibited with cytochalasin B. Three micromeres
were formed, which then produced cilia. Magn. x500.
Fig. 6. Third cleavage partially inhibited with cytochalasin B. Two micromeres were
formed, which then produced cilia. Magn. x500.
Fig. 7. Third cleavage partially inhibited with cytochalasin B. Only one micromere
was formed, which then produced cilia. Magn. X500.
Determination of ciliogenesis in Patella embryos
Figs 3-7
73
74
C. JANSSEN-DOMMERHOLT AND OTHERS
Table 1. Effect of continuous incubation with cytochalasin B on cilia formation
Start of incubation
uncleaved egg
2-cell stage
4-cell stage
8-cell stage
16-cell stage
32-cell stage
ca. 64-cell stage
isolated trochoblasts
la 2 -Id 2
Formation of cilia
—
+
+
+
+
+
micromeres (Fig. 4). At the time of cilia formation the uncleaved micromeres
contained the number of nuclei expected if mitotic activity was unimpaired.
Continuous incubation with cytochalasin B of 1-cell, 2-cell and 4-cell embryos
again prevented cleavage, mitosis being unimpaired. However, cilia never
developed on these embryos (Table 1).
In order to study the exact stage from which cytochalasin B prevents
ciliogenesis, we treated eggs just before third cleavage, at third cleavage, 5 min
after the start of third cleavage, and 10 min after third cleavage. When treatment
started before or at third cleavage, no cleavage furrow was formed or the furrow
was retracted, respectively; no cilia developed. Treatment starting at 10 min
after third cleavage inhibited further cleavage but had no effect on ciliogenesis;
as expected ciliation was observed on all four micromeres (Fig. 4).
Treatment starting at 5 min after the onset of third cleavage gave variable
results, even within one and the same embryo. Sometimes the cleavage furrow
was retracted, sometimes it was not. Thus embryos were obtained consisting of
8, 7, 6, 5 or 4 cells with 4, 3, 2,1 or 0 micromeres, respectively. Figs 4 , 5 , 6 and
7 show such embryos. Cilia only formed on micromeres that were present as
such. It is clear from these data that determinants for ciliation will only be
expressed if they are segregated into micromeres at third cleavage. Thus third
cleavage is decisive for the expression of the developmental potential of the
primary trochoblasts. In this respect it is truly asymmetrical.
In another series of experiments the effect of cytochalasin B treatment during
one cell cycle only was studied. Embryos were treated from 10 min after second
cleavage to 10 min after third cleavage and then washed in sea water. The embryos resumed cleavage, but in not one case did the embryos form cilia at the
time appropriate for primary trochoblasts, nor during the 3h to follow. Overnight the embryos formed an apical tuft and cells with short cilia typical of
accessory and secondary trochoblasts. Apparently suppression of ciliogenesis is
irreversible for primary trochoblasts, whereas in the other cells ciliogenesis is not
suppressed. In conclusion it can be said that the asymmetry of third cleavage with
Determination of ciliogenesis in Patella embryos
75
respect to irreversible effects on ciliogenesis only pertains to the primary
trochoblasts.
Effects of aphidicolin on ciliogenesis
Aphidicolin, a known inhibitor of DNA polymerase-a activity, was used to
study the role of DNA replication in ciliogenesis. Aphidicolin concentrations of
1 and 0-5/ig/ml sea water were tested in continuous incubation experiments
starting at stages shortly before second, third, fourth, fifth and sixth cleavages.
One or two cleavages occurred after the start of treatment. After some hours,
the embryos disaggregated into single cells. These never developed cilia. Feulgen staining of whole embryos showed fragmented nuclear material. These experiments show that aphidicolin, in addition to its effect on the nucleus, affects
cleavage and cell adhesion.
Embryos continuously incubated in 0-1 /ig aphidicolin/ml went on cleaving. If
treatment started shortly before fifth or sixth cleavage no effect on development
was observed; normal trochophores developed in 24h. A slight effect on cell
adhesion was noted, however. This effect was much stronger if treatment began
one cleavage cycle earlier; treatment starting shortly before the fourth cleavage
caused the embryos to dissociate into single cells. Ciliation of the dissociated
cells was not impaired, however.
Embryos treated from before third cleavage disaggregated and the cells
showed a marked retardation of ciliation. With treatment starting before second
cleavage the embryos again disaggregated but no ciliation of the cells was observed at all.
In conclusion it can be said that aphidicolin affects cell adhesion in early
embryos of Patella and may inhibit ciliation, possibly by impairment of DNA
synthesis following second cleavage.
Effects of actinomycin D
In order to study the possible role of nuclear activity in the ciliation of the
prototroch cells we used treatment with actinomycin D, a known inhibitor of
RNA synthesis. Actinomycin D at a concentration of 5/ig/ml suppressed 90 %
or more of uridine incorporation into trochoblast RNA during cilia formation (to
be published). This concentration did not inhibit ciliogenesis. Even a tenfold
higher concentration had no effect whatsoever on cilia formation, even if the
eggs were continuously treated from 1 h before first cleavage.
DISCUSSION
The differentiation of primary trochoblasts of Patella into prototroch cells
comprises a limited number of S-phases anddeavages, and the formation of cilia.
The Patella egg provides for this differentiation process in two steps. The first
step is at third cleavage, when the micromeres are separated from the
76
C. JANSSEN-DOMMERHOLT AND OTHERS
macromeres. The second step is the first division of the micromeres, which
separates the primary trochoblasts from the apical rosette cells.
With respect to the differentiation of trochoblasts into cilia-bearing cells, the
experiments with cytochalasin B clearly show that the determination of
ciliogenesis occurs between second and third cleavage. Third cleavage as such
makes the determination definitive as it separates the nuclei, cytoplasm and
cortex of the micromeres from those of the macromeres.
As shown by a strong increase in tubulin synthesis in the primary trochoblasts
shortly before cilia formation (results to be published), factors promoting ciliation override other synthetic activities during the final step in the differentiation
process.
One could think that the specialization of the primary trochoblasts is entirely
regulated by the localization of cytoplasmic or cortical factors in the future
prototroch cells. However, localization cannot be the only factor involved.
Aphidicolin in very low concentrations inhibits cilia formation if applied at the
time of second cleavage. Application after the S-phase following second
cleavage no longer has an effect on ciliation. This strongly suggests the participation in ciliogenesis of nuclear activity at the 4-cell stage. We argue that during
the S-phase in question the nuclei are being prepared for specific activities, so
that upon segregation into the correct cytoplasmic environment, they will be able
to support cellular activities leading to ciliogenesis. The latter activities are
apparently limited in time, because, if the separation of the micromeres is postponed for the duration of one cell cycle, cilia no longer develop. Clearly a nuclear
change is obligatory and as important as the localization phenomenon. After the
nuclear change the segregation of certain localized factors into a micromere must
occur within a definite time span.
Experimental data favouring this interpretation have been obtained by Cather
(1973), who observed that in Ilyanassa polar lobe material inhibits ciliation of
cells in the pretrochal region of the larva. Brachet, de Petrocelis & Alexandre
(1981) found that ciliogenesis could be suppressed in Chaetopterus embryos by
aphidicolin. The explanatory description by Freeman (1979) of the dynamics of
the localization process in Cerebratulus and Mnemiopsis fits the observations in
Patella. It cannot, however, explain the results of our experiments in which 1, 2
or 3 ciliated micromeres were formed, while blastomeres in which the third
cleavage furrow retracted never produced cilia.
Cytochalasin-arrested Ciona embryos develop tyrosinase activity in pairs of
blastomeres if cleavages are inhibited after the 8-cell stage, but not at earlier
stages (Whittaker, 1979). These results are clearly similar to the present findings
in Patella.
We wish to thank the members of the staff of the Laboratoire Arago for their hospitality and
for the opportunity to use their facilities, and particularly Dr J. Marthy for his inspiring
presence. We thank Dr J. Faber for his critical comments. Thanks are due to Dr W. Berendsen
for his help with the scanning electron microscopy.
Determination of ciliogenesis in Patella embryos
11
REFERENCES
J., DE PETROCELIS, B. & ALEXANDRE, H. (1981). Studies on differentiation without
cleavage in Chaetopterus. Effects of inhibition of DNA synthesis with aphidicolin. Differentiation 19, 47-54.
CATHER, J. N. (1973). Regulation of apical cilia development by the polar lobe of llyanassa
(Gastropoda, Nassaridae). Malacologia 12, 213-223.
DAVIDSON, E. H. (1976). Gene Activity in Early Development. New York: Academic Press.
FREEMAN, G. (1979). In Determinants of Spatial Organization (eds S. Subtelny & I. R. Konigsberg). New York: Academic Press.
VAN DEN BIGGELAAR, J. A. M. (1971). Timing of the phases of the cell cycle with tritiated
thymidine and Feulgen cytophotometry during the period of synchronous division in Lymnaea. J. Embryol. exp. Morph. 26, 351-366.
VAN DEN BIGGELAAR, J. A. M. (1977). Development of dorsoventral polarity and
mesentoblast determination in Patella vulgata. J. Morph. 154, 157-171.
WHITTAKER, J. R. (1979). In Determinants of Spatial Organization (eds S. Subtelny & I. R.
Konigsberg). New York: Academic Press.
WILSON, E. B. (1904). Experimental studies in germinal localization./, exp. Zool. 1,197-268.
BRACHET,
{Accepted 26 November 1982)
EMB74