/. Embryol. exp. Morph. Vol. 42, pp. 237-260, 1977
Printed in Great Britain © Company of Biologists Limited 1977
237
The establishment of the oral-aboral axis
in the ctenophore embryo
BY GARY FREEMAN 1
From the Friday Harbor Laboratories, University of Washington, and the
Department of Zoology, University of Texas at Austin
SUMMARY
In a small percentage of normal embryos and in a higher percentage of embryos centrifuged
prior to the first cleavage the positions of the polar bodies and the site of the first cleavage
furrow do not coincide. These cases have been used to establish whether polar body formation
sites or first cleavage initiation sites correlate best with the oral-aboral axis of the embryo.
In all cases when the first cleavage is initiated at a site different from the site where the polar
bodies were given off, the pattern of the first four cleavages is normal, the segregation of
comb plate potential at these stages is normal, and the larvae that form are normal. The
extent to which comb plate potential is localized along the oral-aboral axis of the embryo
prior to the first cleavage, during the first cleavage and at the 2-cell stage was also examined.
These experiments demonstrate that the oral-aboral axis is established at the time of the first
cleavage, that cleavage plays a causal role in setting up the axis, and that comb plate-forming
potential begins to be localized in the aboral region of the embryo at this time.
INTRODUCTION
Developmental biologists traditionally argue that many aspects of the pattern
of cleavage and the localization of cytoplasmic factors which play a role in
specifying different types of cell differentiation are determined by a 'promorphological scaffold' which is laid down during oogenesis (Wilson, 1925).
This scaffold is generally thought of from a formal standpoint as one or more
axial coordinates with the property of polarity; however, at present its structural
basis is obscure.
Most of the evidence for a promorphological scaffold is based upon studies
which have demonstrated a correlation between one or more structural features
of the oocyte and a symmetry property of the embryo. There have been only a
few cases in which the significance of this kind of correlation has been tested
experimentally. One example frequently cited in support of this notion, on both
correlative and experimental grounds, is based upon studies of embryogenesis
in ctenophores (Wilson, 1925; Schliep, 1929).
Figure 1 outlines the pattern of early cleavages in ctenophore embryos and
1
Author's address: Department of Zoology, University of Texas at Austin, Austin, Texas
78712, USA.
l6
EMB 42
238
G. FREEMAN
Fig. 1. Diagrams of thefirstcleavage, the 2-, 4-, 8-, and 16-cell stage embryos and the
larvae. All stages are shown from the side in the tentacular plane; the oral pole is
marked by a polar body. The first cleavage is initiated at the future oral pole and
passes through what will be the sagittal plane of the larvae. The second cleavage
also begins at the future oral pole of the embryo, and passes through what will be the
future tentacular plane of the larvae. The third cleavage begins in the oral region;
it divides each of the four blastomeres along a plane oblique to the first division
plane. The cells at the ends of the tentacular axis are referred to as ' E ' blastomeres
while the more oral cells are referred to as ' M ' blastomeres. During the fourth
cleavage each blastomere gives off an ' e ' or an 'm' micromere toward the aboral
pole.
indicates how this pattern is related to the symmetry properties of the larva. The
early cleavages in these animals are unipolar; the furrow is initiated at a circumscribed site and spreads out from there passing through the cell. There are
several reports which indicate that the first cleavage originates at or near the site
where the polar bodies are given off. Marking experiments show that this site
corresponds to the future oral region of the larva; the apical organ will form
opposite this site (Reverberi & Ortolani, 1963). These two loci define the oralaboral axis of the larva.
There are several environmental factors that could act upon the developing
oocyte to set up an oral-aboral axis in it. Oogenesis in ctenophores is mediated
by an oocyte-nurse cell system, a syncytial complex containing one central
oocyte connected by intercellular bridges with three nurse cell clusters (Dunlap,
1966; Pianka, 1974). During oocyte maturation polar bodies are released from
the end of the oocyte where the nucleus has been located throughout oogenesis.
This region bears a consistent relationship with the three intercellular bridges
connecting the nurse cell clusters to the oocyte. There is also a consistent
spatial arrangement between the oocyte-nurse cell complex and the tissues
surrounding it during oogenesis.
Several investigators have claimed, on the basis of experimental studies, that
factors specifying comb plate cilia formation are localized in the ctenophore egg
Oral-aboral axis in the ctenophore embryo
239
prior to the 2-cell stage (Driesch & Morgan, 1895; Fischel, 1903; Yatsu, 1912a).
This claim implies some kind of promorphological organization. Unfortunately
these studies are difficult to evaluate because certain key pieces of information
are missing (see Discussion).
The purpose of this study is to test the validity of the notion that the uncleaved
ctenophore egg has a promorphological scaffold in the form of an oral-aboral
axis. This problem has been approached in two ways: (1) The correlation
between the position of the polar bodies and the site where the first cleavage is
initiated was examined. In a small percentage of normal embryos and in a
higher percentage of experimentally manipulated embryos the positions of the
polar bodies and the site of origin of the first cleavage furrow do not coincide.
These cases have been used to establish whether polar body sites or first
cleavage initiation sites correlate best with the oral-aboral axis of the embryo.
(2) The extent to which comb plate-forming potential is localized along the
'oral-aboral' axis of the embryo prior to the first cleavage, during the first
cleavage, and at the 2-cell stage was examined.
These experiments demonstrate that the oral-aboral axis is established at the
time of first cleavage, that cleavage plays a causal role in setting up the axis,
and that comb plate-forming potential begins to be localized in the aboral
region of the embryo at this time.
MATERIALS AND METHODS
Three species were used in this study: Bolinopsis microptera, Pleurobrachia
pileus and Pleurobrachia bachei. The descriptions of Dunlap (1966) and Kozloff
(1974) have been used as a basis for identification. Fertile eggs were obtained
through natural spawnings. These species are in reproductive condition at
Friday Harbor from the last part of April to the first part of June. During this
season only about half of the adults of each species will contain eggs. Adults
were generally collected the day before they were to be used to obtain gametes.
Individual animals were placed in finger bowls. During the evening hours the
finger bowls were placed in the dark in an incubator at 10-11 °C. The next
morning they were removed from the dark and examined at frequent intervals
in order to detect animals preparing to spawn; the eggs of these animals will
be visible in their spawning sinuses. There is a time interval when individuals
of each species are most apt to spawn after being brought into the light; these
time intervals are 1-1-5 h for P. pileus, 1-5-2 h for Bolinopsis and 4-5 h for
P. bachei. Individuals of a given species which do not spawn at the expected
time will frequently spawn at some other time. Spawning also occurs with a
high frequency when the animals are in the dark. Individual Bolinopsis will
sometimes spawn more than once during the 12 h period after they have been
removed from the dark. During spawning sperm are released first and eggs a few
minutes later (Dunlap, 1966). In P. bachei large numbers of sperm are
16-2
240
G. FREEMAN
frequently released; under these conditions fertilization is invariably polyspermic and the eggs develop abnormally. This condition can be mitigated by
transferring an animal that has begun to release sperm to a large volume of sea
water and stirring the sea water so that the sperm will disperse.
The distances between different sites on the egg surface were measured using
a dissecting microscope at 150 x with a goniometer eye piece. Eggs were orientated
with the two sites to be measured at the periphery, the egg centered with respect
to the eye piece, and the angle between the two sites was then measured. Two
sources of error put limits on the accuracy of these measurements: (1) If a series
of measurements are made between two sites on a given egg the range over
which the measurements will vary is about 5°. This error is probably related to
slight differences in the orientation of the egg from measurement to measurement. (2) When an egg is in the process of dividing it flattens perpendicular to
the plane of cleavage. If these eggs are oriented in certain ways the angle
between two sites being measured will be slightly different from what it would
be if the egg was a perfect sphere. These two sources of error place the accuracy
of these goniometric measurements in the range of + 5-10°.
The procedures used to remove egg envelopes, for marking selected regions
of the egg surface with carbon particles, for cutting uncleaved eggs or blastomeres into parts, and for isolating blastomeres have been described previously
(Freeman, 1976; Freeman & Reynolds, 1973). One new experimental procedure
involved centrifugation of uncleaved eggs. A sucrose cushion (0-5 ml of 1-1 molal
sucrose) was placed in a conical centrifuge tube. About 20 eggs were layered on
top of the cushion in 0-2-0-3 ml of sea water and centrifuged for 5 min at ca.
5000 g in an IEC table top clinical centrifuge with a horizontal rotor. The eggs
were then washed several times to remove the sucrose. Intact embryos in their
envelopes were reared in millipore filtered pasteurized sea water in the wells
(1-5 ml vol.) of glass spot plate dishes. Isolated blastomeres and eggs with their
envelopes removed were reared in the same way, except that the wells were
lined with a coat of 2 % agar.
The embryos and isolated blastomeres were raised at 10-12 °C. Under these
conditions the time interval from spawning to initiation of the first cleavage
is about 60 min for P. bachei, 80 min for P. pileus, and 130 min for Bolinopsis.
The time interval between the two-cell stage and the first appearance of comb
plate cilia in these embryos is 13-14 h for P. bachei, 15-16 h for P. pileus, and
19-20 h for Bolinopsis. In some of the experiments reported here the presence
or absence of comb plate cilia was monitored; these observations were always
made 3-6 h after these cilia first appeared (see Freeman, 1976 for procedure).
In other experiments the normality of larvae was assayed; these observations
were usually made 12-24 h after comb plate cilia first appeared.
The cytological methods and procedures used to make reconstructions of eggs
from serial sections have been described previously (Freeman, 1976; Freeman &
Reynolds, 1973).
Oral-aboral axis in the ctenophore embryo
241
RESULTS
I. The correlation between the site of polar body formation and the
origin of the first cleavage furrow
Normal development. During the process of oocyte maturation in ctenophores,
the oocyte moves into a spawning sinus and then to the exterior of the animal
via a gonopore (Dunlap, 1966). The oocyte gives off the first polar body in the
spawning sinus (Dunlap, 1966). The second polar body is either present at the
time the oocyte emerges from the gonopore, or is given off within a few minutes
after emergence. The release of the second polar body occurs on schedule, even
in unfertilized eggs. The region of the oocyte where the polar bodies are given
off is the first region that passes through the gonopore. Fertilization probably
takes place while the oocyte passes through the gonopore or very shortly after
it is liberated into the sea water. As the oocyte passes through the gonopore
a fluid filled space forms between it and the vitelline membrane which surrounds
it.
The polar bodies can adhere to the surface of the egg, float in the space
between the egg and the vitelline membrane, or adhere only to the vitelline
membrane. Twenty per cent of the P. bachei eggs, 75 % of the P. pileus eggs
and 45 % of the Bolinopsis eggs have one or both polar bodies attached. This
average figure is somewhat misleading, since for a given species in three out of
four spawnings only a small percentage of eggs may have one or more polar
bodies attached, while in the fourth spawning over 75 % of the eggs may have
at least one polar body attached. This variability in the percentage of eggs from
a given spawning with at least one polar body attached is probably related to
the timing of polar body formation relative to the raising of the vitelline
membrane. An animal that has spawned eggs in which a high percentage of
cases have at least one polar body attached may subsequently spawn eggs in
which only a low percentage of the eggs have at least one polar body attached.
If only one polar body adheres to the egg it is always the second one. The first
polar body can almost always be distinguished from the second one because the
former usually divides to form two polocytes which remain attached to each
other. There are a number of cases, especially for P. pileus, in which both polar
bodies are attached to the egg. Since they are always within 10° of each other,
they are probably given off at the same site. There is no indication that the polar
bodies change their relative distance from each other.
The relationship between the region where the polar bodies are given off and
the site where the first cleavage is initiated was examined by measuring the
distance between these two sites before the first cleavage furrow was half way
across the egg. These measurements (Table 1) show that the first cleavage
furrow can originate in any quadrant of the egg with reference to the site of
polar body formation. Figure 2 shows a case for each species in which the site
of the first cleavage furrow does not correspond to the region where the polar
242
G. FREEMAN
B -
Fig. 2. Photographs of eggs in which the site of polar body formation does not
correlate with the origin of the first cleavage furrow: (A) Bolinopsis, (B) P. pileus,
(C) P. bachei. All photographs are at the same magnification. The bar indicates
50 /tm. The arrow indicates the polar body.
Table 1. The relationship between the site ofpolar body formation and the site
of origin of the first cleavage furrow
Species
P. bachei
P. pileus
Bolinopsis
Angle between polar body and origin
of cleavage furrow
Number
of eggs
examined
0-45°
45-90°
90-135°
135-180°
77
94
93
87%
53%
46%
8%
25%
29%
4%
15%
17%
1%
7%
8%
A
bodies were given off. Nevertheless the most probable site of furrow formation is
in the quadrant nearest the site of polar body formation; as the distance from
this site increases the probability of furrow formation decreases. Table 1 also
indicates that there are differences among the species in the degree of correlation between the site of polar body formation and the site where the first
cleavage is initiated. This correlation is quite strong in P. bachei but only of
moderate strength in Bolinopsis and P. pileus. In those cases in which the first
cleavage furrow originated between 45° and 135° from the site of polar body
formation, the plane of the first cleavage does not correlate with the site of the
polar body formation.
The suitability of using attached polar bodies as surface landmarks has been
studied by applying a carbon mark to the surface of the egg within 10° of the
site where the polar body is and measuring the distance between the mark and
the polar body at frequent intervals up until the third cleavage. Five Bolinopsis,
eight P. pileus and three P. bachei eggs were marked within 10 min after spawning. There were no cases in which the distance between the carbon mark and
Oral-aboral axis in the ctenophore embryo
243
Table 2. The positions of the pronuclei or zygote nucleus relative to the site of
polar body formation at different times after fertilization in P. pileus
Time after
(min)
Angle between polar bodies and the nucleus
Nuclear type
0-45°
45-90°
90-135C
135-180°
5
0
0
Female pronucleus
16
0
0
1
Male pronucleus
10
5
15
0
0
Female pronucleus
16
2
2
2
Male pronucleus
9
5
25
1
0
0
Zygote nucleus
14
5
3
Pronuclei*
2
4
* It is difficult to distinguish between male and female pronuclei at 25 min.
The eggs off. pileus were always monospermic. Yatsu (19126) has suggested that fertilization is normally polyspermic in Beroe ovata; however, these eggs are much larger than those
of P. pileus.
Fig. 3. Reconstruction of three P. pileus eggs fixed 15 min after spawning. In each case
the female pronucleus has moved from the site where the polar bodies were given off
toward the male pronucleus.
the polar body changed prior to the first cleavage; if the mark or the polar body
was not near a cleavage furrow the distance between the mark and the polar
body was not changed during cleavage (see Freeman, 1976 for observations on
the behavior of carbon marks near cleavage furrows). In two of these Bolinopsis
and five of the P. pileus eggs the origin of the first cleavage furrow was over
45° from the polar body and the carbon mark. Observations that have been
made on these eggs in the course of cutting experiments indicate that the
polar bodies adhere firmly to the egg. This evidence suggests that the polar
bodies remain at the site where they originate and indicates that they are a stable
landmark.
The role of nuclear events in establishing the site of the first cleavage has been
studied by measuring the positions of male and female pronuclei relative to the
site of polar body formation at various time intervals prior to the first cleavage.
Even though the ctenophore egg is translucent it is difficult to make judgements about the movement of the male and female pronuclei in living eggs. The
244
G. FREEMAN
Fig. 4. Photographs showing eggs approximately 15 min after centrifugation. Note
the polar bodies and the stratification of the egg cytoplasm: (A) Bolinopsis, (B)
P.pileus, (C) P. bachei. All photographs are at the same magnification. The bar
indicates 50 /«n. The arrow indicates the polar body.
Table 3. The relationship between the site of polar body formation and the
centripetal pole of centrifuged eggs
Species
Number
of eggs
examined
P. bachei
P. pileus
Bolinopsis
93
105
155
Angle between polar body and the centripetal pole
0-45°
45-90'
90-135
135-180°
23%
15%
30%
38%
30%
27%
13%
35%
21%
26%
20%
21%
description presented here is based upon P. pileus eggs which have been fixed
and sectioned for cytological study. For each spawning a sample of 20 eggs was
monitored in order to establish the percentage that cleaved and the synchrony
of the first cleavage. The eggs from a given spawning were used for cytological
analysis if at least 75 % of the eggs cleaved and at least 75 % of the cleaving eggs initiated their first cleavage within 10 min of the same event in the
first egg.
The ctenophore egg has a centrolecithal organization: it is composed of an
inner endoplasmic zone of yolk spheres and a thin outer layer of basophilic
cortical cytoplasm which surrounds the endoplasm. The male and female
pronuclei always reside in the cortical cytoplasm just under the cell membrane.
Observations on the relative positions of the male and female pronuclei in eggs
as a function of time after spawning are summarized in Table 2. In eggs fixed
5 min after spawning the male pronucleus is highly condensed (average diameter ca. 2 fim) while the female pronucleus is not condensed (average diameter
ca. 6-1 yM-m). The female pronucleus is almost always directly under the region
of the egg surface where the polar bodies are. The male pronucleus can be found
245
Oral-aboral axis in the ctenophore embryo
' B
Fig. 5. Photographs showing the origin of the first cleavage in centrifuged eggs.
Note the axis along which the egg is stratified and the polar bodies: (A) Bolinopsis,
(B) P.pileus, (C) P. bachei. All photographs are at the same magnification. The bar
indicates 50/*m. The arrow indicates the polar body.
Table 4. The relationship between the origin of the first cleavage furrow and the
centripetal pole of centrifuged eggs
Species
P. bachei
P. pileus
Bolinopsis
Angle between
centripetal Number of
pole and
eggs
polar bodies examined
0-45°
45-90°
90-135°
135-180°
Total
0-45°
45-90°
90-135°
135-180°
Total
0-45°
45-90°
90-135°
135-180°
Total
16
27
9
18
70
12
22
28
15
77
34
29
23
22
108
Angle between the centripetal pole and the
origin of the furrows
0-45°
45-90°
90-135°
135-180°
75%
22%
56%
39%
43%
75%
41%
M%
20%
25%
74%
44%
56%
54%
25%
54%
71 %
53%
56%
12%
48%
43%
32%
32%
0%
4%
0%
0%
1-5%
0%
0%
11%
20%
0%
0%
0%
5%
1-5%
0%
5%
7%
7%
5%
0%
0%
0%
9%
2%
31 %
88%
48%
48%
50%
61 %
8%
0%
4%
9%
9%
5%
in any quadrant of the egg but is found most frequently in the quadrant where
the polar bodies are. This observation suggests that the sperm most often fuses
with the egg near the site where the polar bodies are given off. At 15 min after
fertilization the male pronucleus is less condensed (average diameter ca. 4 /«n).
There is no change in the morphology of the female pronucleus. The distribution
of the male pronuclei is roughly the same as it was 5 min after spawning; the
distribution of female pronuclei is much less uniform. In two cases the female
246
G. FREEMAN
Table 5. The relationship between the origin of the first cleavage
furrow and the polar bodies of centrifuged eggs
Angle between
Number of
centripetal
pole and
eggs
Species polar bodies examined
P. bachei
P.pileus
Bolinopsis
Angle between the polar bodies and the origin
of the furrow
0-45°
45-90°
90-135°
135-180°
0-45°
45-90°
90-135°
135-180°
Total
16
27
9
18
100%
70%
44%
6%
0%
30%
55%
50%
0%
0%
0%
28%
0%
0%
0%
6%
70
57%
31%
10%
2%
0-45°
45-90°
90-135°
135-180°
Total
12
22
28
15
92%
45%
61%
13%
0%
41%
21%
40%
77
52%
27%
0%
5%
11%
27%
10-5%
8%
9%
7%
20%
10-5%
0-45°
45-90°
90-135°
135-180°
Total
34
29
23
22
85%
72%
35%
14%
12%
17%
13%
27%
0%
7%
26%
36%
3%
4%
26%
23%
108
56%
17%
15%
12%
pronuclei moved more than 45° from the site of the polar body emission. In
most cases the female pronucleus appears to have moved toward the site where
the male pronucleus is located. Figure 3 shows three egg reconstructions which
document this point. At 25 min the pronuclei have fused in the majority of
cases. In those cases where the pronuclei have fused the zygote nucleus is
generally in the quadrant between 0° and 45° from the site where the polar
bodies have been given off. If the pronuclei have not fused they are generally
in close proximity to each other, but some distance from the site where the
polar bodies have been given off. The results of similar observations on Bolinopsis eggs indicate that the female pronucleus behaves in essentially the same
way in this species. These cytological studies suggest that in undisturbed eggs,
the female pronucleus is initially at the site where the polar bodies were given
off, but that it will migrate some distance in order to fuse with the male
pronucleus if the latter is not in the immediate vicinity.
Centrifugation experiments. While in some eggs the region where the first
cleavage furrow originates bears no relationship to the site of polar body
formation, in many cases these sites closely coincide. However, one can alter
this correlation by centrifuging eggs prior to the first cleavage. In each centrifugation experiment about 20 eggs were set aside as a control. These eggs were
treated in the same way as the experi mentals were excepf that they were not
centrifuged. They were used to establish the per cent cleavage and the synchrony
247
Oral-aboral axis in the ctenophore embryo
Table 6. The segregation of comb plate-forming potential in eggs where the origin
of the first cleavage furrow corresponds to the site of polar body formation
and at different distances from this site
Species
Angle between
polar body and
origin of
the furrow
Blastomere
pair
isolated
Number
of eggs
studied
Number of blastomeres in
a pair which form comb
plate cilia
i
Both
A
One*
Neither
\
0-20°
3
0
1
E and M
11
1
0
2
0
E and e
2
0
7
1
80-100°
E and M
—
—
—
E and e
1
1
2
1
E and M
160-180°
1
0
4
0
E and e
0-20°
E and M
3
0
14
0
P. pileus
0
3
10
2
E and e
5
2
14
1
E and M
80°-100°
1
4
7
1
E and e
5
0
17
0
E and M
160-180°
0
0
10
3
E and e
0
12
3
0-20°
Eand M
0
Bolinopsis
2
0
7
0
E and e
0
1
3
10
80-100°
E and M
2
0
4
0
E and e
0
4
14
0
160-180°
E and M
0
3
1
9
E and e
* In every case where only one blastomere formed comb plate cilia that blastomere was the
E macromere when the E and M blastomere pair was assayed and the e micromere when the
e and E blastomere pair was assayed
P. bachei
of the first cleavage in each spawning. Centrifugation always took place at least
20 min prior to initiation of the first cleavage in controls. In most cases the eggs
were probably centrifuged after pronuclear fusion. In eggs centrifuged with
sufficient force the cortical cytoplasm takes up a centripetal position while the
endoplasm takes up a centrifugal position (Fig. 4). Examination of these eggs
with phase optics indicates that the zygote nucleus ends up in the cortical cytoplasmic mass in the centripetal region of the egg. In certain batches of eggs from
all three species, especially for P. pileus, some centrifuged eggs formed blebs
on their surfaces; these eggs were always discarded. After centrifugation the
cortical cytoplasm slowly envelops the endoplasm re-establishing the original
centrolecithal organization. The time this process takes depends both upon the
centrifugal force used and the species. Within 30-45 min it is frequently difficult
to identify the centripetal pole of P. pileus eggs, while this pole can be identified
for 90-120 min in uncleaved eggs of Bolinopsis and P. bachei.
Table 3 summarizes observations on the relationship between the centripetal
pole of the centrifuged eggs and the site where the polar bodies formed. These
248
G. FREEMAN
z_\
Fig. 6. The early developmental history of embryos in which there was an inappropriate segregation of comb plate-forming potential during the 8- or 16-cell stage. The
top part of each figure indicates the site of origin of the first cleavage furrow and the
site where the polar bodies were given off. A hypothetical distribution for the factors
which specify comb plates, as defined by the position of the polar bodies is indicated
by x 's. In those cases in which the egg has been centrifuged the stratification of
cortical cytoplasm is indicated by shading. The bottom part of each figure shows the
location of the blastomeres from these embryos in which there was not an appropriate
segregation of comb plate-forming potential at the 8- or 16-cell stage. (A) A P. bachei
embryo, all four of the E, M blastomere pairs were assayed. An E, M blastomere pair
in which neither blastomere formed comb plates is on the same side of the sagittal
plane as the E, M pair in which both blastomeres formed comb plates. (B) A
P.pileus embryo, three of the E, M blastomere pairs were assayed. One of the E,
M pairs for which there is a normal segregation of comb plate potential is on the
same side of the sagittal plane as the E, M pair in which both blastomeres formed
comb plates. (C) A P. pilots embryo, all four of the E, M blastomere pairs were
assayed. Three of these pairs showed a normal segregation of developmental
potential. (D) A P.pileus embryo, only the blastomere pair indicated was assayed.
results indicate that the centripetal end of the centrifuged egg bears no relationship to the region of polar body extrusion. Eight P.pileus eggs were marked
with carbon within 10° of the polar body prior to centrifugation. Centrifugation
did not change the distance between the mark and the polar body. These
centrifugation experiments provide another way of demonstrating that the polar
bodies are a stable marker.
Figure 5 shows a set of centrifuged eggs with an attached polar body in which
the first cleavage is beginning. Table 4 examines the site of origin of the first
cleavage furrow as a function of distance from the centripetal end of the egg for
subpopulations of eggs in which the site of polar body formation is at different
distances from the centripetal region. This table defines the most centripetal end
of the egg as 0° while the most centrifugal end is 180°; 0-45° is entirely within
the cortical cytoplasmic region of the centrifuged egg. The boundary between
the cortical and the endoplasmic region of the egg is somewhere between 45°
Oral-aboral axis in the ctenophore embryo
249
Table 7. The degree of normality for larvae where the origin of the first cleavage
furrow corresponds to or is different from the site of polar body formation
Normalcy of larvae
Species
Treatment
prior to
cleavage
Angle between
polar bodies
and origin of
the furrow
Normal
larvae
Larvae in which
1-3 quadrants Abnormal
are normal*
larvaef
0-45°
23
3
0
1
45-180°
4
0
2
7
1
0-45°
Centrifuged
6
45-180°
4
1
23
None
P. pile us
0
0-45°
0
0
24
45-180°
0
17
16
0-45°
2
Centrifuged
45-180°
13
1
17
None
0-45°
18
0
Bolinopsis
0
1
26
45-180°
1
20
0-45°
14
7
Centrifuged
17
9
45-180°
5
* A larva with an abnormal quadrant has either a greatly reduced number of comb plates
or no comb plates in that quadrant; these larvae frequently are missing one tentacle pouch.
The ctenophore larvae is composed of four identical quadrants which are defined by the
sagittal and tentacular planes.
t Abnormal larvae either have no comb plate cilia or a greatly reduced number of comb
plate cilia; most of these cases also have an abnormal apical organ and abnormal tentacle
pouches. These cases frequently exhibited a cleavage delay or cleaved abnormally during
early embryogenesis. Almost all of the abnormal larvae are derived from centrifuged eggs.
P. bachei
None
and 90°, while the region between 90° and 180° contains endoplasm. In almost
every case the site of origin of the first cleavage furrow occurs within or just
at the edge of the cortical cytoplasm. If the first cleavage furrow originated near
the interface between the cortical and endoplasmic layers the plane of cleavage
was always parallel to this interface. Table 5 examines the site of origin of the
first cleavage furrow as a function of distance from the site of polar body formation for subpopulations of eggs in which the site of polar body formation is
different distances from the centripetal pole. A comparison of Tables 5 and 1
indicates that the site of origin of the first cleavage furrow, with reference to the
site where the polar bodies are given off, can be altered substantially by centrifugation especially when the polar bodies are some distance from the centripetal
pole.
The observations made on normal eggs and the centrifugation experiments
reported here make it clear that the first cleavage furrow does not have to
originate at the site of polar body formation. At this point the developmental
consequences of having the first cleavage originate at a site other than the region
where the polar bodies have been given off will be examined. One way that this
250
G. FREEMAN
problem has been approached is by comparing the cleavage pattern in embryos
in which the site of the origin of the first cleavage furrow corresponds to the
site of polar body emission with embryos in which it does not. The other approach
to this problem has examined the way in which developmental potential is
segregated during cleavage and the normality of the larvae that develop from
these two classes of embryos.
In those ctenophore eggs where the site of origin of the first cleavage differs
from that of polar body formation there may be a change in the sites where one
or more subsequent cleavage furrows originate. Such an effect has been demonstrated in other kinds of embryos (e.g. Morgan & Spooner, 1909). The first
four cleavages (see Fig. 1) were carefully monitored for at least 15 eggs from
each species in which the first cleavage furrow originated between 90° and 180°
from the region where the polar bodies were given off. These cases were selected
either from batches of untreated eggs or from eggs that had been centrifuged.
A carbon mark was placed on each egg at the site where the first cleavage was
initiated and the origin of the subsequent cleavages was examined with reference
to this mark and to the site where the polar bodies were given off. While in
some cases a given cleavage was blocked or delayed, especially after centrifugation, there was no indication of any other changes in cleavage pattern. In every
case the micromeres were given off during the fourth cleavage at a region which
corresponded to the pole of the uncleaved egg directly opposite the site of origin
of the first cleavage furrow.
The segregation of comb plate-forming potential in embryos where the origin
of the first cleavage furrow differs from the site of polar body formation has
been studied by isolating blastomeres at the 8- and 16-cell stages and monitoring
their ability to differentiate. Blastomere isolation experiments delimit the following sequence for the segregation of comb plate-forming potential during cleavage
in normal embryos. At the 4-cell stage each blastomere has the ability to give
rise to an isolate which subsequently differentiates comb plate cilia cells, at the
8-cell stage only the E macromere inherits this capability, while only the
e micromere inherits it at the 16-cell stage (see Reverberi, 1971 for a review). If
one supposes that the factors specifying comb plate cilia formation are localized
at the aboral pole of the uncleaved egg and that this pole can be identified by
using the site of polar body formation as a marker, a disturbance in the segregation of developmental potential might result if the sites of origin of the first
cleavage furrow and polar body formation differ. In each species a number of
eggs were selected in which (1) the origin of the first cleavage furrow was within
20° of the site of polar body emission, (2) the origin of the first cleavage furrow
was within 80-100° of this region, or (3) the origin of the first cleavage furrow
was within 160-180° of this site. These cases were selected from batches of both
untreated and centrifuged eggs. When eggs were operated on at the 8-cell stage
each blastomere that made up an E and M macromere pair derived from
the same progenitor cell at the 4-cell stage was isolated and its ability to
Oral-aboral axis in the ctenophore embryo
251
differentiate comb plate cilia was monitored. When the eggs were operated on at
the 16-cell stage each blastomere that made up an e micromere E macromere pair
derived from an E macromere progenitor cell at the 8-cell stage was isolated
and its ability to differentiated comb plate cilia was monitored. These results
are summarized in Table 6. Only normally developing isolates are reported in
this table; if an isolate lost more than 10 % of its cells, or if there was evidence
that a cleavage block had occurred it was not included. Twelve per cent of the
isolates were discarded for these reasons; almost all of these cases originated
from centrifuged eggs. If one isolate from any pair was not suitable for analysis
the other member of the pair was also discarded. The results show that, regardless of the site of origin of the first cleavage furrow with reference to the region
of polar body formation, the segregation of comb plate cilia-forming potential
was nearly always normal. However, both the blastomeres that make up a pair
formed comb plates in four cases; Fig. 6 documents the history of each case.
This figure shows that the region of cytoplasm sampled in these cases did not
bear a consistent relationship to the hypothetical oral-aboral axis marked by
the region where the polar bodies were given off.
In those centrifuged eggs in which the plane of the first cleavage parallels the
boundary between the endoplasm and the cortical cytoplasm two blastomere
lineages result in which there are quantitative differences in cytoplasmic composition of the cells. One of these lineages is rich in endoplasm but poor in cortical cytoplasm while the other lineage is cortical cytoplasm rich but endoplasm poor.
In most of these cases the segregation of comb plate-forming potential is normal
regardless of the cytoplasmic composition of the blastomere pair (see Freeman
& Reynolds, 1973 for a description of the behavior of egg fragments made up
exclusively of cortical cytoplasm).
Larvae were also allowed to develop from intact embryos selected from untreated and centrifuged eggs in which the first cleavage originated at varying
distances from the site of polar body formation. These cases were carefully
examined after the mouth, the apical organ, comb rows and tentacle pouches
could be clearly identified. If one discounts the cases in which cleavage was
abnormal the embryos from all three species developed into normal larvae
regardless of the relationship between the site of origin of the first cleavage
furrow and the site of the polar body formation (Table 7).
These observations on normal and centrifuged eggs have made it clear that
the site of origin of the first cleavage furrow does not have to correspond to the
region where the polar bodies were given off. In those cases where the origin of
the first cleavage does not correspond to the region of polar body formation, the
oral-aboral axis of the embryo is clearly related to the site of origin of the first
cleavage furrow.
252
G. FREEMAN
II. The localization of comb plate-forming potential in the unc/eaved
egg, during the first cleavage, and at the 2-cell stage
At some point in development prior to the 16-cell stage the factors which
specify comb plate differentiation must become localized at the aboral pole of
the ctenophore embryo. Studies which have mapped the distribution of comb
plate-forming potential in Mnemiopsis have indicated that there is a moderate
localization of these factors at the aboral pole of the blastomeres in the 2-cell
FIGURE 7
Fig. 7. Description of the operations on uncleaved eggs, eggs undergoing their first
cleavage and the blastomere of the 2-cell embryo. (A) Operation in which a cut is
made along the presumptive equatorial plane of the uncleaved egg separating the
presumptive oral region from the presumptive aboral region. (B) Operation in which
a cut is made along the presumptive oral-aboral axis of the uncleaved egg generating
two fragments which contain presumptive oral and aboral cytoplasmic regions. After
operation 'A' or ' B ' the diameter of the blastomere fragments were measured.
(Note: In each case the diameter of the fragment that did not cleave was measured;
in addition, in several cases the diameter of the cleaving fragment was measured
before cleavage began. In those cases in which only the diameter of the uncleaved
fragment was measured, this value was used to estimate the volume of the cleaving
fragment by subtracting the volume of the uncleaved fragment from the average
volume of the eggs for the species used. The diameter of the P. pileus egg is 171 ± S.D.
7-6 /tm; the diameter of the P. bachei egg is 140 ± S.D. 7-4 fim). Usually only one
fragment will cleave following an operation. This fragment is allowed to generate
four blastomeres, the blastomeres are then separated and each is raised in isolation.
Operations 'A' and ' B ' were done prior to pronuclear fusion in some cases and
after fusion in other cases. The time span between the operation and the initiation
of cleavage did not influence the results.
(C) Operation in which a cut is made along the equatorial plane during the first
cleavage separating the nucleated oral region from the enucleated aboral region of
the egg. In effecting this operation the cut was always made through the uncleaved
portion of the egg prior to the advance of the furrow through that area. After this
operation the enucleated fragment was measured. The cleaving fragment was
allowed to generate four blastomeres, the blastomeres were then separated and
raised in isolation. (D) Operation in which a cut is made along the developing first
cleavage furrow to give two nucleated fragments each containing presumptive oral
and aboral cytoplasmic regions. Each 'blastomere' was allowed to divide once
more, the four blastomeres were separated and each raised in isolation. (Note: this
operation is not strictly comparable to operation ' C in that the volume of the
blastomeres isolated at the 4-cell stage is about twice the volume of the blastomeres from the experimentals).
(E) Operation in which a cut is made along the equatorial plane separating a
nucleated oral region from an enucleated aboral region of one blastomere at the
2-cell stage. These eggs were orientated prior to the operation by marking the site
where the first cleavage furrow originated. (F) Operation in which a cut is made along
the oral-aboral axis in the tentacular plane of one blastomere at the 2-cell stage to
give a nucleated fragment containing oral and aboral cytoplasmic regions and an
enucleated fragment of similar composition. These eggs were orientated prior to the
operation by marking one end of the oral-aboral axis and the tentacular end of the
blastomere. The blastomeres of the embryo were separated and the marked blastomere was cut. After operations ' E ' and ' F ' the enucleated fragment was measured,
the nucleated fragment was allowed to cleave once and the two blastomeres were
separated and raised in isolation.
Oral-aboral axis in the ctenophore embryo
253
Fig. 7
stage embryo (Freeman, 1976). In this study similar procedures have been used
to establish whether comb plate potential is localized in the uncleaved egg.
A known volume of cytoplasm was excised from the 'aboral' region of either
the uncleaved egg, an egg in which the first cleavage is occurring, or a blastomere
of a 2-cell stage embryo. The ability of the remaining nucleated fragment to
differentiate comb plate cilia was then assayed. If the factors responsible for
specifying comb plate cilia differentiation are not localized in the future aboral
region of the embryo when the operation is done, one would expect the remaining isolate to form comb plate cilia. If these factors are localized exclusively in
the aboral region at the time of the operation, the isolates derived from the
nucleated fragment remaining after the operation would not form comb plate
cilia. As a control another set of operations was done in which cytoplasmic
regions of comparable volume were removed, but where the fragment that
EMB 42
254
G. FREEMAN
remained after the operation contained cytoplasm of both presumptive oral and
aboral regions of the embryo.
Most of these operations were performed on the eggs and embryos of
P. pileus; these cases are supplemented by a smaller group of operations that
were performed on P. bachei. In uncleaved eggs the polar bodies were used to
mark the oral pole. They are a reliable marker in P. bachei but are less reliable
in P. pileus (Table 1). The poor correlation between the site of polar body
formation and the oral-aboral axis for P. pileus was mitigated to a certain
extent by analyzing separately those cases where the origin of the first cleavage
furrow corresponded to the site of polar body formation. When an egg is cut
into an oral and an aboral half, usually only the oral half cleaves; however,
there are some cases in which the aboral half cleaves. By way of analogy with
other eggs that have been cut into two parts, it is reasonable to suppose that the
division center of the male pronucleus is necessary for cleavage and that the
fragment which cleaves is either haploid or diploid. For the operations on eggs
that were going through their first cleavage, and the 2-cell stage embryos, the
origin of the first cleavage furrow was used to mark the oral-aboral axis. Only
the oral half of embryos cut at these stages will continue to cleave. These
operations are outlined in Fig. 7. When the developmental stage was reached
which was equivalent to the 4-cell stage, the blastomeres were separated and
raised in isolation in order to assay their ability to differentiate comb plate cilia.
These results are summarized in Table 8.
If the presumptive aboral region was removed from a 2-cell stage blastomere
of P. pileus or P. bachei, 33-37 % of the isolates did not differentiate comb plate
cilia. When control operations were done at this stage that gave blastomere
fragments in which both the presumptive oral and aboral regions were present,
only 12-21 % of the isolates failed to differentiate comb plate cilia. Figures 8
and 9 show the sizes of the nucleated fragments obtained after these operations
and the distribution of cases that did not form comb plate cilia. As the size of
the aboral cytoplasmic region removed gets larger, the ability of the nucleated
fragment that remains to support comb plate cilia differentiation declines
(Figs. 8 A and 9 A). When both oral and aboral cytoplasmic regions are present
(Figs. 8 B and 9B) the failure to form comb plate cilia is not related to the volume
of the nucleated fragment. This comparison between nucleated fragments of
comparable size which either have or do not have an aboral region indicates
that comb plate-forming potential is partially localized in the aboral region of
these 2-cell stage embryos.
If the presumptive aboral region was removed from an uncleaved egg or an
egg that was undergoing its first cleavage, almost all of the isolates derived
from the nucleated fragment will develop comb plate cilia. In those cases in
which comb plate cilia did not develop, there was a less pronounced relationship
between the size of the aboral region removed from the egg and its ability to
differentiate (Figs. 8C, D and 9C, D). A comparison of these operations with
Oral-aboral axis in the ctenophore embryo
255
Table 8. The effects of removing defined cytoplasmic regions from uncleaved eggs,
cleaving eggs, and 2-cell blastomeres on the differentiation of comb plate cilia
by their isolated EM blastomere derivatives*
Species
Stage
Operation!
Number
of
eggs
Comb plate
cilia
Number
differof
entiation
isolates
(%)
Equatorial-isolating
37
10
92
presumptive oral region
(7 A)
82
3
11
Equatorial-isolating presumptive aboral region
(7 A)
17
94
5
Axial-isolating presumptive oral and aboral
regions (7B)
63
First cleav- Equatorial-isolating pre87
18
age
sumptive oral region (7C)
47
96
12
Axial-isolating presumptive oral and aboral
regions (7D)
Equatorial-isolating
pre30
15
63
2-Cell
sumptive oral region (7E)
16
8
88
Axial-isolating presumptive oral and aboral
regions (7F)
59
16
Equatorial-isolating pre86
P. pileus
Uncleaved
egg
sumptive oral regionselected (7 A)
53
14
91
Equatorial-isolating presumptive oral regionsunselected (7 A)
39
10
92
Equatorial-isolating presumptive aboral region
(7 A)
88
24
97
Axial-isolating presumptive oral and aboral
regions (7B)
121
32
First cleav- Equatorial-isolating pre89
age
sumptive oral region
(7C)
131
34
99
Axial-isolating presumptive oral and aboral
regions (7D)
Equatorial-isolating pre83
42
67
2-Cell
sumptive oral region (7E)
48
27
Axial-isolating presump79
tive oral and aboral region
(7F)
* Only normally developing isolates are reported in this table; if an isolate lost more than
10% of its cells, or if there was evidence that a cleavage block had occurred it was not ineluded. About 8 % of the isolates were discarded for these reasons: smaller isolates tend to
develop abnormally more frequently than larger isolates do.
t These operations are shown in Fig. 7.
P. bachei
Uncleaved
egg
17-2
256
G. FREEMAN
18 r
16 r
14 _
12
10
J
8
6
4
-i—i—i—i—i—r
Percentage of normal egg size
Percentage of normal blastomere size
Fig. 8
10 8
6
4
i
i
i r
oooooooo
I
I I I
I
^4
o o ot oooroooo
o
i
i
oooooo oo
i i i T i i °? i
Fig. 9
Figs. 8 and 9. The size distribution of the nucleated fragments produced following
operations on the uncleaved egg, the cleaving egg, and the 2-cell embryo of P. pi/eus
(Fig. 8) and P. bachei (Fig. 9). Each isolate that developed from a blastomere fragment was assigned the relative volume of the fragment. The size distribution of the
fragments which produced comb plates are white and those that did not are colored
black. (A) The size distribution of fragments produced after an equatorial cut
removed the presumptive aboral region from a blastomere of a 2-cell stage embryo
(Fig. 7E). Note the size distribution of the isolates that did not form comb plate
cilia. (B) The size distribution of the fragments produced after a cut was made along
the tentacular plane of a blastomere from a 2-cell embryo giving a blastomere fragment with both oral and aboral cytoplasmic regions (Fig. 7F). (C) The size distribution of the fragments produced after an equatorial cut removed the presumptive
aboral region from the cleaving egg (Fig. 7C). (D) The size distribution of the
fragments produced after an equatorial cut removed the presumptive aboral region
from the uncleaved egg (Fig. 7 A).
Oral-aboral axis in the ctenophore embryo
257
the control operations and with the operations done on uncleaved eggs in which
the presumptive aboral region cleaved shows that a comparable percentage of
isolates differentiated comb plate cilia in all cases. Of special interest are two
uncleaved P. bachei eggs which were cut into presumptive oral and aboral
halves; in each of these cases both halves cleaved. Yatsu (1912a) has described
a similar case for Beroe; presumably these eggs were fertilized by two sperm.
All eight isolates from both halves of one egg were successfully raised in isolation. Only one of these isolates, from the aboral half, did not differentiate comb
plate cilia. In the other case only three isolates from the oral half and three
isolates from the aboral half were raised successfully in isolation. All six of
these isolates developed comb plate cilia. These comparisons suggest that the
factors which specify comb plates are not yet localized exclusively in the presumptive 'aboral' region of the uncleaved egg or in the uncleaved portion of
the egg which is undergoing its first cleavage.
DISCUSSION
This study demonstrates that the oral-aboral axis of the ctenophore embryo
is set up as a consequence of the first cleavage. This demonstration is based upon
the observation that the location of this axis is determined by the site where the
first cleavage furrow originated for eggs in which this site is different from the
site of polar body formation. The observations made here which indicate that
there is not always a one to one correspondence between the site of polar body
formation and the origin of the first cleavage furrow in normal eggs do not agree
with the published reviews on ctenophore development which imply that there
is a strict one to one correspondence (Wilson, 1925; Schliep, 1929; Korschelt,
1936; Reverberi, 1971; Pianka, 1974). If one looks beyond these reviews to their
primary sources one sees that there is almost no data on the relationship between
the site of polar body formation and the origin of the first cleavage furrow
(Yatsu, 19126; Komai, 1922; Reverberi & Ortalani, 1963). In fact, Komai (1922)
shows a 2-cell embryo in which the first cleavage furrow clearly originated more
than 45° from the site of polar body formation.
The fact that the first cleavage can originate some distance from the point of
polar body extrusion in normal eggs indicates that the frequently observed
coincidence of these two sites is probably due only to chance. I suspect that the
site of origin of the first cleavage furrow corresponds to the site where the sperm
fuses with the egg for this group of animals. Further studies on the fertilization
process and measurements of any male pronuclear migration are needed to
validate this suggestion. Several other studies have demonstrated that there can
be a close correlation between the site of fertilization and the positioning of
cleavage furrows (see Morgan, 1927 and Guerrier, 1971 for reviews). During
the process of spawning in ctenophores the first part of the oocyte surface
exposed to the sea water is the pole where the polar bodies are given off (Dunlap,
258
G. FREEMAN
1966). Consequently, the probability of sperm-egg fusion will be highest at this
pole which would then be translated into a high probability of furrow initiation
at the same site. While the site of fertilization or zygote nucleus formation may
determine the placement of the oral-aboral axis, the actual establishment of the
axis appears to be related to the act of cytokinesis. The centrifugation experiments presented here show that if the zygote nucleus is moved to a new location,
the cleavage furrow which forms at this new site corresponds to the oral-aboral
axis of the embryo.
This study has also examined the extent to which the potential for comb plate
cilia differentiation is localized in the presumptive aboral region of uncleaved
eggs, cleaving eggs and 2-cell stage embryos. These experiments suggest that the
potential for comb plate differentiation begins to become localized in the
aboral region of the embryo as a consequence of the first cleavage. This judgement does not agree with the conclusions reached by Driesch & Morgan (1895),
Fischel (1903) and Yatsu (1912a) who all argued that there was some localization of comb plate-forming potential prior to the 2-cell stage.
Experiments analogous to those described here in which eggs were cut into
parts prior to the initiation of the first cleavage have been done by Driesch &
Morgan (1895) and Yatsu (1912a) on the egg of Beroe ovata. In Yatsu's experiments eggs were cut either before or after polar body formation. Beroe ovata
seems to differ from other ctenophores in that both polar bodies are reportedly
given off after fertilization. The eggs cut prior to polar body formation could
not be oriented; the size of the fragments produced was not indicated. Thirteen
cases developed to the point where they could be analyzed; 11 formed eight
rows of comb plates while two cases formed seven rows. In the experiments
which were performed after polar body formation 12 fragments were produced.
Three developed normally, two cases were missing from two to six rows of
comb plates, while the other seven cases were classified as 'more or less defective
as Fischel, 1903 has found out' (almost all of Fischel's defective cases had eight
rows of comb plates, but had a smaller number of plates in some rows). In his
1911 paper Yatsu indicates how these eggs were cut and presents diagrams
showing the relative size of the fragments; however, he does not relate the
origins of the egg fragments to the quality of the resulting larvae. Driesch &
Morgan (1895) studied 16 eggs cut at random; the time of the operation with
reference to the initiation of the first cleavage was not recorded. Six of their
cases developed into normal larvae; eight had four normal rows on one side,
and only one or two comb rows which were frequently rudimentary on the other
side. In the last two cases, the egg was cut into a large and a small fragment and
the small fragment cleaved; these did not form comb plate cilia. These experiments suggest that a change occurs in the Beroe egg during polar body formation
which makes it more difficult for an egg fragment to develop normally. However, it is not clear that the change involves the localization of comb plateforming potential at a particular site.
Oral-aboral axis in the ctenophore embryo
259
Eggs have also been cut into parts at the beginning of the first cleavage by
Fischel (1903) and Zeigler (1898) working on Bero'e and by Freeman & Reynolds
(1973) working on Mnemiopsis. Fischel removed variable amounts of egg
cytoplasm from the aboral region (six cases), while in six similar operations
Zeigler, and Freeman & Reynolds removed up to 50 % of the egg cytoplasm.
All of these cases developed into normal larvae. The experiments reported here
confirm these findings. Fischel also removed variable amounts of cytoplasm
from the oral region to one side of the developing furrow. He reports that this
operation causes defects in comb plate differentiation. I have tried to repeat
this experiment on P. pileus (Freeman, unpublished results). While I don't feel
that I have done enough operations to make a definitive statement, the results
at hand do not indicate that the operation causes a decrease in the number of
comb plates.
The fact that there is a moderate localization of comb plate-forming potential
in the aboral region of the 2-cell stage blastomeres of P. pileus and P. bachei
confirms the results of similar experiments which have been done on Mnemiopsis
(Freeman, 1976). The failure to detect this localization prior to the 2-cell stage
provides another line of evidence which, is consistent with the argument that an
oral-aboral axis does not exist in the embryo prior to this stage. The increase
in comb plate-forming potential in the aboral region of the embryo after the
first cleavage furrow has passed through that region, suggest that the process of
cytokinesis itself, or the local cytoplasmic movements which accompany it may
play a role in setting up this localization of developmental potential.
I feel that there has been a tendency on the part of developmental biologists
to try to explain too many features of early development on the basis of some
kind of hypothetical spatial organization which is supposed to be laid down
during oogenesis. Clearly, there are many kinds of animal eggs in which some
kind of promorphological organization exists. However, this study and published
work on spiralians (Tadano, 1962; Guerrier, 1968) show that eggs from some
groups of animals have essentially no promorphological organization at the end
of oogenesis and acquire this organization only as a consequence of the initiation of embryonic development. The Fucus egg has been one of the main models
used in analyzing the intracellular events that accompany the development of
polarity in an unstructured system (Quatrano, 1973). The eggs of ctenophores
and certain spiralians may be excellent material for similar studies on animal
cells.
This work was supported by Grant GM 20024-03 from the National Institutes of Health.
I want to thank Dr Dennis Willows, the director of the Friday Harbor Laboratories, for
facilitating my work there, and Drs Antone Jacobson and Helen Pianka for reading this
manuscript.
260
G. FREEMAN
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1
A verbatim English translation of this paper is available on request.
(Received 18 April 1977, revised 17 June 1977)