/. Embryol. exp. Morph. 75, 87-100 (1983)
Printed in Great Britain © The Company of Biologists Limited 1983
Establishment of embryonic axes in larvae of the
starfish, Asterina pectinifera
By TETSUYA KOMINAMI 1
From the Department of Zoology, Faculty of Science, Kyoto University
SUMMARY
In order to clarify the relationships between the first cleavage plane and the embryonic axes,
early cleavage pattern of the fertilized eggs of the starfish, Asterina pectinifera was reexamined . It was ascertained that the polar bodies were formed at the site to which the germinal
vesicle had closely located before the initiation of the meiotic division, and that the first
cleavage plane passed near this site of polar body formation. While some of the early embryos
of this starfish were observed to show various cleavage patterns during early cleavage stage,
more than 70 % of the embryos developed according to, so to say, the 'typical' cleavage pattern.
Next, horseradish peroxidase (HRP) was injected into one of the blastomeres of the 2-cellor 8-cell-stage embryos. The embryos were allowed to develop up to either the early gastrula
or the early bipinnaria stage and stained to detect the descendants of the blastomere injected
with HRP. In early gastrulae still retaining radial symmetry, the activity of HRP injected at
the 2-cell stage was found only in one side of the embryo partitioned by one of the symmetrical
planes. When one of the four blastomeres lying nearer to the polar bodies at the 8-cell stage
was marked with HRP, its descendants constituted one quarter of the anterior part of the
gastrula, and descendants of a blastomere opposite the polar bodies were found in the posterior region of the embryo. It was concluded that the animal-vegetal (AV) axis was preexisting in the fertilized egg and that the first cleavage plane contained this primary axis.
In early bipinnariae with their dorsoventral (DV) axes already established, the region of
activity of the HRP injected at the 2-cell stage was still demarcated by a plane which passed
through the AV axis, but the plane of the boundary had nofixedrelation to the DV axis. The
results indicate that the first cleavage plane does not necessarily correspond to the median
plane of the starfish larva, unlike the case in sea-urchin eggs (Horstadius & Wolsky, 1936). In
other words, the DV axis of the starfish embryo is not predetermined in the fertilized egg, and
might be established in the course of development through cell-to-cell interactions, while the
AV axis is established mainly according to the pre-existing egg polarity.
INTRODUCTION
Establishment of the embryonic axes in early development of echinoderm eggs
has been studied by several workers (reviewed by Horstadius, 1973). Using a
surgical technique and the famous pigment band as a natural marker, Horstadius
(1927) provided evidence that the animal-vegetal (AV) axis pre-exists before
fertilization in Paracentrotus eggs. Recently Schroeder (1980) extended this preexistence of the AV axis to other species of sea urchins by employing the jelly
canal as another marker of the egg.
1
Author's address: Department of Zoology, Faculty of Science, Kyoto University, Kyoto,
606,Japan.
88
T. KOMINAMI
Few experimental analyses have been made, however, of the establishment of
the secondary axis, that is, the dorsoventral (DV) axis. Difficulty in analysing the
properties of the DV axis is due partly to lack of a proper natural marker, but
mostly to the highly 'regulative' properties of echinoderm eggs: those experiments that involve the separation or removal of some part of the embryo, which
has been a successful means for examining 'mosaic' type eggs, do not provide
definite answers. The crucial experiment would be to observe intact whole embryos, preferably with some sort of artificial markers.
In recent years, it has been demonstrated that the use of horseradish peroxidase
(HRP) is an excellent means for tracing cell lineages (Weisblat, Sawyer & Stent,
1978; Hirose & Jacobson, 1979; Batakier & Pedersen, 1982). By employing HRP
as a marker, the author could follow the descendants of the HRP-injected blastomeres in starfish embryos. To my knowledge, no systematic experimental study
has been made with starfishes to determine either the AV or DV axis of the egg.
Since starfish eggs do not form micromeres, the embryonic axis is not expressed
until the late blastula stage. This disadvantage of starfish egg as material seems to
have been overcome by the use of HRP, as reported in the present paper.
MATERIALS AND METHODS
Animals
Asterina pectinifera were collected along the shore of Tokyo Bay in April and
Wakasa Bay in September, and kept in aquaria supplied with circulating cold sea
water (15 °C).
Oocyte maturation
Excised ovaries were rinsed three times with artificial sea water (A.S.W.,
Jamarin U, Jamarin Lab., Osaka), and immersed in A.S.W. containing 2 x
10~6M-l-methyladenine (Kanatani, Shirai, Nakanishi & Kurokawa, 1969) for
20min at 20°C to induce reinitiation of meiosis. After this treatment, oocytes
that had undergone germinal vesicle breakdown were shed from the ovaries.
These were rinsed three times with A.S.W.
Insemination
Oocytes were inseminated before first polar body formation with a suspension
of dry sperm. By inseminating the oocytes during this period, the time of the first
cleavage was scheduled according to the time of 1-methyladenine treatment
(Kominami & Satoh, 1980), so the time of the initiation of treatment was employed to describe the developmental time. Embryos were cultured in A.S.W.
at20°C.
Injection of HRP into blastomeres
Injection pipettes were prepared by pulling glass tubes ( 0 = 1 mm) over a
Establishment of embryonic axes in starfish
89
micro gas burner. The tips were broken with watchmaker's forceps to make the
final tip diameter 3-5 jum. HRP (type VI; Sigma Chemical Co., St. Louis, MO.)
was dissolved at a concentration of 10 % in Ca++ -free artificial sea water. This
solution was introduced into an injection pipette, and sealed with liquid paraffin.
The micropipette was held by a micromanipulator (Model MK-2, Narishige Sci.
Inst. Lab., Tokyo) and the solution was injected into the blastomeres, using an
injection syringe (Injecting device, Ernst Leitz Ltd., Midland, Ontario) under
a microscope. Embryos at the 2-cell or 8-cell stage with an intact fertilization
membrane could be penetrated by the injection pipette. An intact fertilization
membrane was indispensable for preserving the organization of the cleaved
blastomeres (Dan-Sohkawa, 1976). The quantities of solution injected were not
precisely determined, but were about 4-10pi for the 2-cell- and 1-2pi for the
8-cell-stage embryos.
Enzyme reaction
Early gastrulae (24-28 h after the induction of maturation, 20 °C) and early
bipinnariae (48-52h) were fixed with 1-5% glutaraldehyde in A.S.W. for l h
then rinsed in 0-1 M-phosphate buffer (pH 6-4) for 30 min at 20 °C. These samples
were preincubated for 20 min in 0-3 % 3,3'-diaminobenzidine dissolved in 0-1 Mphosphate buffer then an adequate quantity of 1 % peroxide was added (the final
concentration was about 0-01 %). The reaction was continued for 5-10 min and
was stopped by washing the samples twice with 0-1 M-phosphate buffer.
RESULTS
Polar body formation
In immature oocytes the germinal vesicle is located eccentrically, closely apposing the oocyte membrane. The polar bodies are subsequently formed at this
point after the reinitiation Of meiosis induced by 1-methyladenine (Shirai &
Kanatani, 1980, Fig. 1, A-C). This spatial relationship was not modified by
fertilization, as judged from the time-lapse videofilms taken from the initiation
of germinal vesicle breakdown through fertilization to the successive formation
of the two polar bodies (Fig. 1, D-F).
First cleavage
First cleavage took place about 170 min after the initiation of 1-methyladenine
treatment at 20 °C. The plane of the first cleavage is commonly said to pass
through the site of polar body formation. To make sure that such a spatial
relationship exists, the distance of the site of polar body formation from the first
cleavage plane was measured on photographs after the two blastomeres were
completely separated by the cleavage furrow (Fig. 2). This distance was less than
20/im in most cases, and even in extreme cases did not exceed 50/mi. Since the
90
T. KOMINAMI
B
1A
\
Fig. 1. Polar body formation. (A) and (D) Germinal vesicles located eccentrically,
closely apposing the oocyte membrane. Arrows indicate the presumed sites of polar
body formation. (B) After germinal vesicle breakdown. (C) At the nearest point of
the germinal vesicle to the oocyte surface two polar bodies have been extruded
(arrowheads). Oocytes were not inseminated. (E) The presumed sites of polar body
formation was not modified by fertilization. (F) The first and second cleavage furrows passed near the site of polar body formation. Bar: 100 jum.
shorter diameter (perpendicular to the first cleavage plane) of blastomeres is
about 100/Am, it can be said that the first cleavage furrow passes near the polar
body formation site.
Early cleavages
Second cleavage took place perpendicular to the first cleavage plane, and also
passed near the polar body formation site. After two divisions, the polar bodies
were observed on either one of the four blastomeres. The third cleavage plane
was perpendicular to both the first and the second cleavage planes. Frequently
the third cleavage was slightly unequal. In this case the blastomeres nearer to the
site of polar body formation were smaller than their sister blastomeres. After the
fourth cleavage, two layers of blastomeres, each containing eight blastomeres in
a circle, were formed (Fig. 3, IA-D). In the fifth cleavage each layer divided into
two layers, resulting in four layers of eight blastomeres each. Thereafter the
cleavage direction became non-uniform, so it is difficult to describe the organization of the blastulae. This cleavage pattern (Fig. 31) occurred in about 70 % of
the embryos, and will hereafter be called the 'typical' pattern. In the rest of the
embryos, the 'typical' pattern was altered as early as the second cleavage. In
Establishment of embryonic axes in starfish
91
100 urn
150
100
50
0*-
10
20
30
40
50 (pan)
Distance of polar-body-formation site
Fig. 2. Distance of the polar-body-formation site from the first cleavage plane.
Ordinate; Number of embryos. Abscissa; Distance of polar bodies from the first
cleavage plane (,um). In more than 90 % of the embryos, this distance was less than
20,um. Blastomeres of the 2-cell-stage embryos were about 100fim in shorter
diameter.
some embryos the two cleavage planes were oblique with each other (Fig. 311).
In extreme cases, the two cleavage axes were perpendicular (Fig. 3III), although
the 8-cell-stage embryos apparently had the same arrangement of blastomeres
as the 'typical' embryos.
Distribution of HRP-labelled cells in early gastrulae
a) Injection at the 2-cell stage
Early gastrulae with an archenteron at the vegetal pole were cylindrical in
shape, and radially symmetrical around the AV axis. HRP was injected into
either blastomere of the 2-cell-stage embryos. Samples were fixed 24-28 h after
the reinitiation of meiosis and observed for the distribution of HRP-labelled
cells. Some results are shown in Fig. 4. After the enzyme reaction, labelled cells
were stained reddish brown, and could be easily distinguished from the nonlabelled cells. At a glance it is clear that labelled cells and non-labelled cells did
not extensively intermingle, and that the boundary between the two groups of
cells was rather clear. The distribution of labelled cells in the gastrula shown in
92
T.
JIB
^^x
KOMINAMI
nc
Fig. 3. Various cleavage patterns during the early cleavage stages (from 2-cell to
16-cell stage). IA-ID; The 'typical' cleavage pattern occurring in about 70 % of the
embryos. The 16-cell-stage embryos consist of two layers of eight blastomeres each.
IIA-IID; The second cleavage plane of one blastomere is oblique to that of the other
blastomere, so the arrangement at the 8-cell or 16-cell stage is disturbed. About 20 %
of the embryos had this cleavage pattern. IIIA-IIID; The second cleavage plane of
one blastomere is almost perpendicular to that of the other blastomere; this case
represents about 10 % of the embryos. An embryo of this type appears 'typical' at
the 8-cell, but at the 16-cell stage the arrangement of blastomeres is again disturbed.
Arrows indicate the sites of polar body formation. Bar: lOO/xm.
Fig. 4A is just the right half of the embryo. Clearly, labelled and non-labelled
regions are separated by a plane including the AV axis. Figure 4B shows a
gastrula in which the labelled ectodermal region was somewhat smaller than the
non-labelled region, whereas the labelled endodermal region was somewhat
larger. This indicates a slight shift in the plane of partition from the AV axis. In
Fig. 4C, the labelled ectodermal region was somewhat larger than the nonlabelled ectoderm, and the labelled archenteron was somewhat smaller compared with the non-labelled part, the reverse of Fig. 4B. In total, about half the
body of the embryo seemed to be labelled. This situation is more clearly seen in
Fig. 4D, which is a top view of the same specimen as in Fig. 4C. In most embryos,
labelled cells were found only on one side of the embryo with respect to the
Establishment of embryonic axes in starfish
93
symmetrical planes of the early gastrula. As the boundary can be thought to
coincide with the first cleavage plane, it is concluded that one of the blastomeres
of the 2-cell-stage embryo gave rise to half of both the ectoderm and endoderm
of the early gastrula. This may also indicate that the first cleavage plane contained the AV axis of the future embryo.
b) Injection at the 8-cell stage
Next, HRP was injected into one of the blastomeres at the 8-cell stage. Unlike
those described above (Fig. 31), the cleavage patterns were not so uniform
among embryos. Only the embryos that had shown the 'typical' pattern at the
4-cell stage were used in this experiment. The half containing the polar-bodyformation site with respect to the third cleavage plane was named the 'upper' half
and the other half named the 'lower' half. HRP was injected into one of the
blastomeres of the 'upper' half at the 8-cell stage, and the distribution of labelled
cells in the early gastrula was observed. Figures 5A and 5B illustrate one of the
4A
B
Fig. 4. Early gastrulae (24-28 h) following injection of HRP into one of the blastomeres at the 2-cell stage. (A) HRP activity is found only in the right half of this early
gastrula. Arrows indicate the boundary between labelled and non-labelled cells. The
boundary contains the AV axis of the embryos. (B) Another example in which the
boundary has shifted a little to the right of the anterior head region and to the left
of the archenteron tip. (C) HRP activity is detected in the left half. (D) Top view of
the same embryo shown in C. Bar: 100 jitm.
EMB75
94
T. KOMINAMI
results. Labelled cells were localized in one quadrant of the embryos distributed
from the animal pole down to two thirds of the ectoderm in the AV direction,
as could be seen by viewing the embryo from the animal pole. When HRP was
injected into one of blastomeres of the 'lower' half at the 8-cell stage, labelled
cells were found both in the ectodermal region near the vegetal pole and in some
part of the archenteron (Figs 5C and 5D). These results indicate that blastomeres
in the 'upper' half of the 8-cell-stage embryo constituted only the ectoderm of the
animal region, and that blastomeres of the 'lower' half differentiated into both
ectodermal cells near the vegetal pole and endodermal cells that gave rise to the
archenteron of the early gastrula. Therefore, the site of polar body formation
corresponds to the animal pole (head region, or the most anterior part) of the
early gastrula, and its antipode corresponds to the region where gastrulation
begins.
\
5A
B
...A
C
D
Fig. 5. Early gastrulae (24-28 h) following injection of HRP into one of the blastomeres at the 8-cell stage. (A) HRP was injected into one of the 'upper' blastomeres.
Labelled cells are distributed from the anterior (animal) to the posterior pole in two
thirds of the ectoderm. (B) The same embryo shown in A. The anterior ectoderm of
about one fourth the circumference is labelled. (C) HRP was injected into one of the
'lower' blastomeres. HRP activity is found in the posterior ectoderm and in the
archenteron. (D) Another example of the same type of injection with the embryo
squashed slightly to more easily reveal the distribution of labelled cells. Arrows
indicate the boundaries. Bar: 100 jum.
Establishment of embryonic axes in starfish
95
Distribution of HRP-labelled cells in early bipinnariae
a) Injection at the 2-cell stage
HRP-injected embryos developed to the early bipinnaria stage (48-52 h after
the initiation of development) were fixed and stained for HRP. At this stage of
development, the DV axis of the embryo could be easily discerned by the
presence of the oral opening on the ventral side. The archenteron had differentiated into oesophagus, stomach, intestine and mesodermal tissues (coeloms and
mesenchyme cells). Some samples are shown in Fig. 6 and in Fig. 7 with
diagrammatic representations. In the example shown in Fig. 6B and in Fig. 7B,
labelled cells were found only in the right half of the embryo, and the boundary
between the two groups of cells (labelled and non-labelled cells) coincided with
the median plane of the early bipinnaria. Each blastomere of the 2-cell-stage
embryo thus constituted just the right or the left half, respectively, of the early
bipinnaria. However, this situation was observed in only a few cases. In the
example shown in Fig. 6B and in Fig. 7B, labelled cells were found only in the
ventral half of the embryo. The DV axis of the embryo, in this case, was determined in a direction perpendicular to the first cleavage plane. Directly opposite
to the example in Fig. 6B and Fig. 7B, the dorsal half of the embryo (both
ectoderm and endoderm) was labelled in the example in Fig. 6C and Fig. 7C.
Separation of labelled and non-labelled cells was in many other cases neither
left-right nor dorsal-ventral. As shown in Figs 6D, E, F and Figs 7D, E, F,
labelled cells were found both in part of the ventral half and in part of the dorsal
half. In these cases, the plane dividing the labelled parts from the non-labelled
parts of the embryo did not correspond to the median plane. Instead the planes
took various angles to the median planes. As the plane containing the boundary
between the two groups of cells (labelled and non-labelled) was thought to
correspond with the first cleavage plane, it was concluded that the first cleavage
plane did not necessarily coincide with the median plane of the early bipinnariae.
In other words, the DV axis was determined independently of the first cleavage
plane in the course of development.
b) Injection at the 8-cell stage
HRP was injected into one of the blastomeres at the 8-cell stage, and embryos
were allowed to develop to the early bipinnaria stage. It was expected that only
one fourth of the anterior ectoderm would be labelled if HRP were injected into
one of the blastomeres of the 'upper' half (animal hemisphere), taking into
account the results described above. One of these embryos is shown in Figs 8A
and 8B. Only the ectoderm of the anterior right half of the ventral side was
labelled. It was also expected that about one fourth of the posterior region of
the ectoderm and the digestive tract would be labelled if HRP were injected into
one of the blastomeres of the 'lower' half (vegetal hemisphere). The result
96
T. KOMINAMI
B
D
Fig. 6. Early bipinnariae (48-52 h) following injection of HRP into one of blastomeres at the 2-cell stage. (A) Upper half (right half as to the median plane of the
embryo) is labelled. Thefirstcleavage plane in this case corresponds to the median
plane of the early bipinnaria. (B) Almost all of the ventral half is labelled and the
dorsal half is not labelled. The first cleavage plane in this embryo separates the
ventral half from the dorsal half. (C) An embryo which is opposite the embryo shown
in B. The dorsal half is labelled. (D), (E), (F) In these embryos, the first cleavage
planes are oblique to the median plane of the early bipinnariae. Portions of both the
ventral and dorsal halves are labelled, but the labelled area is still about half of each
embryo. Arrows indicate the boundaries. Notice the labelled cells, especially at the
anus. Bar: 100jum.
represented by Figs 8C and 8D fulfilled this expectation. In addition, the cells of
the stomodaeum were labelled in the example shown in Figs 8A and 8B, whereas
they were not labelled in the example shown in Figs 8C and 8D. This indicates
that the cells of the stomodaeum of the early bipinnaria originate from
Establishment of embryonic axes in starfish
7A
V
B
V
97
v
v
Fig. 7. Diagrammatic representations of the embryos shown in Fig. 6. Embryos are
viewed from the posterior pole with the ventral side upward. Fig. 7A corresponds to
Fig. 6A. Fig. 7B corresponds to Fig. 6B, and so on. Dots indicate the distribution of
the marker enzyme. D; Dorsal. V; Ventral.
a blastomere of the 'upper' half of the 8-cell-stage embryo, i.e., they originate
from the ectoderm of the early gastrula.
DISCUSSION
Several papers have reported the usefulness of HRP to trace the fate of blastomeres (Weisblat et al. 1978; Hirose & Jacobson, 1979; Baiakier & Pedersen,
1982). HRP turned out to be useful for marking blastomeres in starfish embryos
also. As shown in Fig. 4, for example, HRP injected into one of the blastomeres
98
8A
T. KOMINAMI
B
Fig. 8. Early bipinnariae following injection of HRP into one of the blastomeres at
the 8-cell stage. (A) HRP was injected into an 'upper' blastomere. Ventral view. The
anterior ectoderm is labelled. A double arrow indicates the labelled stomodaeum.
(B) Same embryo shown in A. Side view. Arrows indicate the boundary between
labelled and non-labelled cells. About two thirds of the ectoderm from anterior to
posterior and about one fourth the circumference are labelled. (C) HRP was injected
into one of the 'lower' blastomeres at the 8-cell stage. Ventral view. The posterior
ectoderm and the archenteron are labelled. A double arrow indicates the nonlabelled stomodaeum. (D) Side view of the same embryo shown in C. Bar: 100/im.
at the 2-cell stage was localized on either side of a symmetrical plane of the early
gastrula. This indicates that HRP molecules have not moved from the descendent cells of the HRP-injected blastomere to descendants of another blastomere
(Tupper & Saunders, Jr, 1972). Blastomeres of the embryo did not intermingle
much during early embryogenesis at least up to the early gastrula and in fact these
conditions were not modified up to the early bipinnaria stage except for the
migration of mesenchyme cells.
The data presented here clearly show that the first cleavage plane contains the
AV axis, which becomes the anteroposterior axis in the early gastrula. It was also
ascertained that the 'upper' blastomeres in reference to the site of the polar
bodies formed the anterior part of the gastrula ectoderm and that the 'lower'
blastomeres formed the posterior ectodermal region and the archenteron (Figs
5 and 8).
Establishment of embryonic axes in starfish
99
HRP-injected, 'upper' blastomeres of the 8-cell-stage embryos formed
approximately the anterior two thirds of the ectoderm in the early gastrula
(24-28 h), or in early bipinnaria (48-52 h). The 'lower' blastomeres of the 8-cellstage embryos formed the posterior third of the ectoderm and the archenteron.
The 32-cell-stage embryo consists of four layers of eight blastomeres each. Of
these four layers, the 'lower' two are derived from the 'lower' blastomeres of the
16-cell-stage embryo. The present results indicate that only the lowest layer of
blastomeres at the 32-cell stage may form the digestive tract.
The most significant result obtained in these experiments is that the first
cleavage plane does not necessarily contain the DV axis of the starfish larva (Figs
6 and 7). In amphibian eggs, the first cleavage plane, which becomes the future
median plane of the embryo, passes through the sperm entrance point and the
grey crescent that is formed opposite the sperm entrance point. Therefore, the
entrance of a spermatozoon into an egg is the critical event in establishment of
the DV axis before the first cleavage (Ancel & Vintemberger, 1948). Horstadius
& Wolsky (1936) concluded that the DV axis pre-exists at the 1-cell stage of the
fertilized eggs of the sea urchin, on the basis of their experiments involving
isolation and recombination of vitally stained blastomeres. The observation of
Horstadius (1925) on Asterias gibbosa is the sole prior information on the origin
of the DV axis in starfish eggs. Some of this species are reportedly oval in shape.
Taking advantage of this irregularity, he found that the first cleavage furrow
separates dorsal from ventral side in the larvae of that starfish. However, no
similar study has ever been reported using spherical eggs. HRP-injection
methods as adopted in the present work seem to have offered a clear answer. The
DV axis of the starfish larva is probably established during early embryogenesis
by means of cell-to-cell interactions.
Determination of the AV axis, on the other hand, may be traced to the stage
of polar body formation, since the present study revealed the fixed relationship
between the site of polar body formation and the eccentric position of the germinal vesicle, a relationship which is not affected by fertilization. Possibly, the
AV axis could be determined even in immature oocytes. A similar conclusion
was reached using vital staining methods (Shirai & Kanatani, 1980). Each blastomere isolated from the 16-cell-stage embryo is known to be totipotent, that is,
to be able to construct a dwarf but morphologically normal bipinnaria (DanSohkawa & Satoh, 1978). The problem of the embryonic axis in relation to such
regulative development would be whether each blastomere develops by newly
establishing an AV axis or develops according to its own polarity possibly laid
down along the AV axis of the whole embryo before isolation.
Though it turns out to be clear that there is no fixed correlation between the
first cleavage plane and the median plane of the larva, the role of sperm entrance
in establishing the DV axis remains to be resolved. The entrance of a spermatozoon may affect the embryonic axes determination just as in amphibian embryos (Ancel & Vintemberger, 1948). This is a debated subject for starfishes.
100
T. KOMINAMI
I wish to thank Associate Prof. Takeshi Yanase, Osaka Kyoiku Univ., for affording us
opportunities to utilize glass tube puller and also thank Prof. Mitsuki Yoneda for his technical
advice during the course of this study and critical reading of the manuscript.
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ANCEL,
(Accepted 10 August 1982)