395
Development 120, 395-404 (1994)
Printed in Great Britain © The Company of Biologists Limited 1994
The establishment of bilateral asymmetry in sea urchin embryos
Elizabeth R. McCain* and David R. McClay
Zoology Department, 243 Biological Science Building, Duke University, Box 90325, Durham, North Carolina 27708-0325, USA
*Author for correspondence
SUMMARY
Although much is known about the specification and determination of the two primary axes (animal/vegetal and
dorsoventral or oral/aboral) in a number of embryos, little
is understood about bilaterality. In the sea urchin, left/right
asymmetry is crucial to normal development as the echinus
or adult rudiment is positioned on the left side of the larva.
We examined the establishment of bilateral asymmetry in
Lytechnis variegatus embryos by determining the relationship of the first cleavage planes to the left/right axis.
Embryos were bisected at different times to determine
when the bilateral axis is committed. These lineage tracing
and cell separation experiments demonstrated that the first
cleavage plane divides the embryo into left and right
halves, although this is conditional until after late blastula
stage. The relationship between the specification of the
dorsoventral axis and the bilateral axis was examined
experimentally. In other species when the dorsal and
ventral halves of early echinoderm embryos (preblastula)
are separated, the dorsal half often reverses (180°) its
dorsoventral axis. We asked whether those larvae with an
inverted dorsoventral axis would shift the position of the
echinus rudiment from the original left side to the new left
side. If so, it would demonstrate that the larval asymmetry
is dependent upon specification of the dorsoventral axis.
Using a combination of lineage tracing and cell separation
techniques, we show that the left/right asymmetry is
specified with respect to the dorsoventral axis.
INTRODUCTION
the larva metamorphoses into an adult. Although the specification of the left/right asymmetry and, correspondingly, the
echinus rudiment have not been examined in any species of sea
urchins, a few starfish have been looked at with respect to that
issue. When starfish embryos were separated into left and right
halves at an early stage (between blastula and gastrula), the
larvae developed differentially. The left halves, which had
higher viability, formed a hydropore on the left side, while the
right halves were less viable and had a hydropore on the left
or right side, or on both sides. From these data, it was
concluded that, in starfish embryos, the position of the
hydropore is fixed and stable in the left half, but not in the right
half (Runnström, 1920; Hörstadius, 1928, 1973).
Similar experiments investigating the determination of the
dorsoventral axis in echinoderm embryos demonstrated that
the echinoderm embryo is capable of reversing its dorsoventral axis. Hörstadius (Hörstadius and Wolsky, 1936; Hörstadius, 1973) discovered that, by cutting a 16-cell stage Paracentrotus lividus embryo into dorsal and ventral halves, the
dorsal half reversed its dorsoventral polarity so that what was
originally dorsal became ventral, and ventral became dorsal
(experiment reviewed in Fig. 2A). Unfortunately, the experiment did not reveal the effect of this reversal upon the orientation of the hydropore and echinus rudiment. If the dorsal
halves had survived longer, there would have been two
possible outcomes with respect to the reversal of the left-right
asymmetry (reviewed in Fig. 2B): (1) the echinus rudiment
could have been on the left side, indicating that its location was
Bilateral animals have three axes: anteroposterior, dorsoventral and left/right. Although a considerable amount is known
of the establishment of the anteroposterior and dorsoventral
axes, very little is understood about the developmental mechanisms distinguishing left from right. One outstanding question
concerning the specification and determination of the bilateral
axis is: when and how is it established with respect to the other
axes? In other words, are the spatial relationships between the
three axes dependent upon one another so that, if the orientation of one axis changes, the other axes are also affected, or
can the specification of any of the three axes be uncoupled from
the others? There are few systems in which this can be experimentally tested. The echinoderm embryo, however, can be
used to examine the interdependency of axial specification
since the dorsoventral axis can be artificially reversed at an
early stage of development. We used this experimental system
to examine the relationship between the specification of the
dorsoventral axis and left/right asymmetry.
Unlike vertebrates, which have multiple asymmetries on
both sides of their bilateral bodies, most echinoderm larvae
have only one readily observed asymmetrical structure; a group
of coelomic cells on the left side of the gut develops into the
echinus rudiment which produces most of the future adult. The
first sign of this asymmetry is the formation of the hydropore,
a portion of the adult water vascular system (Fig. 1A). Eventually, when the echinus rudiment is fully developed (Fig. 1B),
Key words: bilateral asymmetry, left/right axis, dorsoventral axis,
embryo, cleavage pattern, sea urchin
396
E. R. McCain and D. R. McClay
dependent upon the (re)specification of the dorsoventral axis,
or (2) the echinus rudiment could have been on the right side,
indicating that the location of the specified asymmetrical
structure had not switched when the dorsoventral axis reversed
(Fig. 2B). Reversal of the dorsoventral axis in dorsal half
embryos was also observed with Heliocidaris erythrogramma,
a direct developing sea urchin, but the effect on left/right asymmetries was not noted (Raff, 1992; Henry and Raff, 1993). We
used this same basic experimental manipulation, of cutting
embryos into dorsal and ventral halves, in order to learn
whether bilaterality is established with respect to the dorsoventral axis, or whether bilateral pattern information is specified
by a mechanism distinct from that of the dorsoventral axis.
We address several specific questions in an attempt to
answer when and how bilateral asymmetry is established in the
sea urchin, Lytechinus variegatus. (1) What
is the relationship of the left/right axis to the
first cleavage planes? (2) When is the difference between the left and right sides, or
the position of the echinus rudiment,
committed? (3) Is the left/right asymmetry
dependent upon the dorsoventral axis such
that if the dorsoventral axis is reversed,
asymmetric bilaterality will also be
reversed?
whose tip had been pulled to a tip diameter of approximately 2 µm,
was attached to the end of the tubing and the DiI solution was frontfilled into the needle. The needle was mounted onto a Narishege
micromanipulator and positioned adjacent to a Leitz Dialux 12 microscope. The DiI solution, in the form of a dye/oil droplet, was microinjected into one cell of the 2-cell stage embryos, which were held in a
Kiehart chamber (Kiehart, 1982). DiI rapidly diffused throughout the
cell, but the oil droplet and some DiI were retained throughout development. For the lineage tracing experiment, one cell at the 2-cell stage
was injected with one DiI droplet. Another experiment, designed to
test for axis reversal, required that two DiI droplets be injected into
one cell at the 2-cell stage. Under this condition, the DiI droplets were
positioned so that the second cleavage would partition one droplet into
each of the two daughter cells.
Following microinjection, the embryos were removed from the
Kiehart chamber and washed with ASW. They were cultured in the
MATERIALS AND METHODS
Gamete and embryo preparation
Lytechinus variegatus adults were acquired from
North Carolina and Florida. Gametes were
obtained by intracoelomic injection of 0.5 M
KCl. The eggs were fertilized with dilute sperm
in artificial sea water (ASW) containing 8 mM paminobenzoic acid (Sigma, Inc). Before first
cleavage, the embryos were washed, poured
through a 0.75 µm nylon mesh to remove the fertilization envelope, then washed again. Embryos
were maintained in glass dishes at 23°C or in a
15°C water bath until the 2-cell stage, when they
were prepared for micromanipulation.
Lineage tracing techniques
To determine the position of the first cleavage
plane relative to the larval axis, one cell of a 2cell stage embryo was labeled with either Nile
Blue or 1,1′-dihexadecyl-3,3,3′3′-tetramethylindocarbocyanine perchlorate {DiI C16(3)} (D-384,
Molecular Probes, Eugene, Oregon), a fluorescent lipophilic dye.
DiI C16(3)
In order to label cells for several days, DiI C16(3)
was injected into the cell. It was first diluted to a
10% stock solution in 100% ethanol. From this,
a 0.3% DiI solution was made in soybean oil
(Wesson cooking oil). Polyethylene tubing (76
mm inner diameter) attached to a 0.2 ml Gilmont
syringe was backfilled filled with approximately
5 µl of mercury and then 50 µl of the 0.3% DiI
solution. A siliconized glass needle (0.85 mm
outer diameter, 0.55 mm inner diameter,
Drummond Scientific Company, Broomall, PA),
Fig. 1. L. variegatus larvae. (A) 7-day-old pluteus larvae, from the dorsal (aboral) view.
The hydropore (arrow) is on the left side of the esophagus (e), anterior to the stomach (s).
The inset is a magnification of the hydropore (arrow). (B) 24-day-old echinopluteus with
well-developed echinus rudiment (r) on the left side of the stomach. The folded tube feet
are visible (arrows). Bar, 13.1 µm.
Sea urchin axis of bilateral asymmetry
397
Fig. 2. Description of a manipulation experiment and the predicted results. (A) Hörstadius (1973) cut P. lividus embryos into dorsal and ventral
halves at the 16-cell stage, then marked the cut edge with Nile Blue. The position of the dye on the dorsal side of both halves indicated that the
dorsal half had reversed its dorsoventral axis. (B) The two possible outcomes of the experiment with respect to the position of the echinus
rudiment in the dorsal halves that had reversed their dorsoventral axis. If the polarity of the left/right asymmetry reversed in those particular
dorsal halves, the echinus rudiment would be located on the left side. If the left/right asymmetry did not invert its original polarity in the dorsal
halves then the echinus rudiment would be on the new right side. The goal of the studies reported in this paper will resolve this issue.
dark until prism stage (24 hours old) or pluteus stage (48 hours old),
when they were monitored with a fluorescent microscope. The
position of the dye and the droplet were noted.
Nile blue
For short-term cell surface labeling, a solution of 2% low melting
point agarose and 0.5% Nile Blue was backfilled into a pulled
capillary tube (tip diameter approximately 30 µm). After the agarose
had cooled, the tip of the capillary tube was applied, freehand, to the
cell membrane of a 2-cell embryo. A dissecting microscope facilitated
the process. Only those embryos that were half-labeled and continued
to develop normally were set aside for cell separation experiments.
Cell separation
Embryos were cut in half at the 2-cell, 4-cell or blastula stages. Individual embryos were placed in Ca2+/Mg2+-free ASW for one to three
minutes and a glass needle was gently forced down between the cells
that were to be separated. Once the two halves were apart, they were
briefly flushed with ASW and then moved to a 2% agarose-coated
dish which contained Millipore-filtered ASW. The half embryos from
a single separation experiment were raised as a pair in a dish. After
3-24 hours, each pair of half embryos was transferred to an agarosefree dish. The embryos were examined every other day with a
compound microscope for the position of the hydropore (on about day
7) and the echinus rudiment (on about day 20), as well as overall larval
characteristics.
Embryos were cut in half along the first or second cleavage plane.
To test for the fixation of the left/right axis, embryos were cut into
left and right halves (along the first cleavage plane) at the 2-cell stage,
early blastula (4-5 hours postfertilization) stage and the late blastula,
premesenchyme blastula stage (6.5-7.5 hours postfertilization).
Embryos were also separated into dorsal and ventral halves (along the
second cleavage plane) at the 4-cell stage, after having been marked
with Nile Blue or DiI at the 2-cell stage.
Maintenance of embryos and larvae
All embryos were raised at 23°C in 35 mm Falcon plastic dishes filled
with Millipore-filtered ASW. Every other day, larvae were transferred
to clean dishes filled with Millipore-filtered ASW. The larvae were
fed Dunaliella tertiolecta (8,000/larva) every other day after the
fourth day of development.
Axis nomenclature
Discussions related to defining the positions of the dorsal, ventral, left
and right sides of an organism can be confusing. This stems from the
fact that, although the animal/vegetal and dorsoventral axes are
defined by their specific properties, the left and right sides are characterized not by traits unique to them, but are defined geometrically
by their position relative to the other two axes. In sea urchins, the
animal/vegetal axis is fixed during oogenesis and, thus, can not be
manipulated. In contrast, the dorsoventral axis can be reversed experimentally. By definition, the left and right sides would also switch in
such embryos. But this poses a problem. If the dorsoventral axis
reversed but the property that specified the left/right asymmetry did
not, then the organs that were once on the left side would now be on
the newly defined right side (Fig. 2B). Thus, left/right characteristics
would be fixed independent of the determination or commitment of
the dorsoventral axis. If the specification of bilateral asymmetry was
inextricably bound to the dorsoventral axis, however, the reversal of
the dorsoventral axis would require that the position of the bilateral
organs also reverse. It is this relationship of axis specification that we
are examining.
RESULTS
The relationship between the left/right axis and the
first two cleavage planes of L. variegatus
DiI, in an oil-based media, was injected into one cell at the 2cell stage of 105 embryos (Fig. 3A). The location of the dye
relative to the larval axis was examined at the prism or pluteus
stages, when the dorsal, ventral, left, right, anterior and
posterior regions were distinguishable. Three patterns were
observed; labeling was on the right side, the left side, or all
over. In 75 (75/105) larvae, the dye was distributed bilaterally,
with 30 (30/75) marked on the right side and 45 (45/75) marked
on the left (Table 1). In most of the embryos, the division
between labeled and unlabeled tissue equally bisected the larva
and was parallel to the plane of bilaterality (Fig. 3B-D). The
angle of the division was sometimes tilted 5-10° to the left or
the right of the bilateral plane. In the 30 embryos where the
Table 1. Results from the DiI injection experiment
Total number injected
105
Position of DiI in larvae
Left half
Right half
All over
30
45
30
One cell at the 2-cell stage was injected and the resulting larvae were
marked on the left half, right half or all over.
398
E. R. McCain and D. R. McClay
dye was spread uniformly, it was likely
that the original dye injections were made
prior to completion of the first cleavage.
From these data, it was concluded that
the first cleavage plane divided the
embryo into left and right halves. The
second cleavage plane, therefore,
bisected the future dorsoventral axis.
This axial information was used in the
following cell separation experiments.
The timing of the commitment of
left/right asymmetry
At various stages, embryos were cut into
left and right halves along the plane of the
first cleavage. To orient embryos that
were bisected after the 2-cell stage, one
cell at the 2-cell stage was marked with
Nile Blue. Later, the marked embryos
were cut in half along the dye line.
Left/right asymmetry was assessed by
examining the overall symmetry of the
larva and, when the larva was older, the
position of the hydropore and the echinus
rudiment was noted. Pairs of larvae were
followed.
Each member of the twelve surviving
pairs derived from a bisected 2-cell stage
embryo had a left hydropore and was
normal in all other respects (Table 2A).
Six of the pairs survived long enough as
a pair to produce an echinus rudiment and
this was on the left side in all cases. Only
one individual of each of the other 6 pairs
survived to the time of rudiment
formation; all had the echinus rudiment
on the left side. There were 18 pairs in
which only one of the pair survived long
enough to observe the position of the
hydropore. Of these, all but two had a left
hydropore (Table 2A). Nine of the 16
with a left hydropore survived to produce
a left echinus rudiment. One single larva
formed a hydropore on both the left and
right sides (Table 2A) and eventually had
an echinus rudiment on the right side.
The other larva did not form a definitive
hydropore (Table 2A) or rudiment.
Of 17 pairs of early blastula halves that
survived one week, 15 produced two
larvae each of which had left hydropores.
The other two pairs consisted of one
normal larva with a left hydropore and
one that lacked coelom development
(Table 2B). One complete pair and four
single larvae survived to produce left
echinus rudiments. All 16 of the single
half blastula produced left hydropores.
Eight of those larvae survived long
enough to produce left echinus
rudiments.
Fig. 3. Lineage tracing of 2-cell L. variegatus. The left-hand column is bright field and the
right-hand column is fluorescence. (A) 2-cell stage embryo after injection of DiI/oil droplet
(arrow). (B) Prism stage embryo (ventral or oral view) with fluorescent label on its right side.
The anus (a) is in focus. (C-D) Two plutei (ventral or oral view) with fluorescent label on
their right side. The DiI/oil droplet (arrow) is visible in both the bright-field and fluorescent
light. The fluorescent images (C and D) show several labeled mesenchyme cells on the
unlabeled side of the larvae (arrowhead). Thus, pigment or skeletogenic cells migrate all
over the larvae, regardless of which side they originated from. Bar, 20.0 µm.
Sea urchin axis of bilateral asymmetry
399
Fig. 4. A pair of larvae, which were produced by cutting one late blastula (6.5 hours old) into a left half and a right half: (A-B) one half and (CD) the other half. (A) One half larva at 12 days old with a left hydropore (arrow). (B) By 28 days this half larvae develops an echinus rudiment
(arrows) on the left side. (C) At 12 days of age the larva is less developed than its counterpart (in Fig. 4A). The inset shows that the larva has
left and right hydropores (arrow), which are joined by a continuous hydroporic canal (arrowhead). (D) The larva at 28 days old with two
bilateral early echinus rudiments. The hydrocoel has formed on both sides (arrow). Bar, 21.3 µm.
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E. R. McCain and D. R. McClay
Table 2. Results from cell separation along the first
cleavage plane
(A) Cell separation at the 2-cell stage
Total number
of pairs
12
Position of hydropore for each pair
Left/Left
Right/Left
Left/None
12
0
0
Total number
of singles
18
Position of hydropore for each single
Left
Right and Left
None
16
1
1
(B) Cell separation along the first cleavage plane at the early blastula stage
Total number
of pairs
17
Position of hydropore for each pair
Left/Left
Right/Left
Left/None
15
0
2
Total number
of singles
16
Position of hydropore for each single
Left
Right
None
16
0
0
(C) Cell separation along the first cleavage plane at the late blastula stage
Total number
of pairs
14
Position of hydropore for each pair
Left/Left
Right/Left Left/Right and Left
9
2
3
Total number
of singles
13
Position of hydropore for each single
Left
Right
Right and Left
9
2
2
Embryos were cut in half along the first cleavage plane at the (A) 2-cell
stage, (B) early, non-swimming blastula stage (4-5 hours), or (C) late blastula,
premesenchyme blastula stage (6.5-7.5 hours). The larvae either survived as
pairs, or only one of a pair survived. The position of the hydropore of each
larva was noted. The / mark denotes the characteristic of each member of a
pair.
14 pairs of late blastula half embryos and 13 single late
blastula half embryos survived to the age when the hydropore
could be discerned. Both larvae in nine of the pairs had a left
hydropore. For two pairs (2/14), the hydropore was on the left
of one half of a pair and in the right half of the other. The
remaining three pairs (3/14) had one larva with a left hydropore
and the other with both left and right hydropores. Of the 13
single larvae, the majority had left hydropores, but two had a
right hydropore, and two had a left and right one (Table 2C).
In general, when one half had a left hydropore, it would eventually produce a left echinus rudiment (Fig. 4A,B). If the other
half had a left and a right hydropore, two rudiments would
result (Fig. 4C,D). From these experiments, we observed that,
prior to the late blastula stage, both halves of a bisected embryo
place the hydropore on the left side. Beginning at the late
blastula stage, bisection sometimes resulted in embryos with
hydropores on the right side or with both a left and a right
hydropore.
Dorsoventral axis reversal in the dorsal half
embryos: what happens to bilaterality?
Two separate experiments tested the hypothesis that, when the
dorsoventral axis is reversed, the position of bilaterally asymmetric structures is also respecified. First, we observed the
location of the hydropore and echinus rudiment that developed
in dorsal half L. variegatus embryos (See Fig. 2B). One cell at
the 2-cell stage was marked with Nile Blue and at the 4-cell
stage the embryos were cut along the second cleavage plane
into dorsal and ventral halves. The Nile Blue was simply used
to mark the orientation of the first cleavage plane. The pairs
Table 3. Results from cell separation along the second
cleavage plane
Total number
of pairs
10
Position of hydropore for each pair
Left/Left
Right/Left
Left/None
10
0
0
Total number
of singles
5
Position of hydropore for each single
Left
Right
None
5
0
0
Embryos were cut in half along the second cleavage plane at the 2-cell
stage. The larvae either survived as pairs, or only one of a pair survived. The
position of the hydropore of each larva was noted. The / mark denotes the
characteristic of each member of a pair.
were followed and examined for the position of the hydropore
and echinus rudiment. Since previous experiments have shown
that ventral halves do not invert the dorsoventral axis (Fig. 2A),
we anticipated that at least one member of a dorsoventral pair
would have a hydropore on the left side. Since the other larva
of the pair (the dorsal half) could have a hydropore on the left
or the right side, there are three possible interpretations for
each pair of dorsal and ventral larvae (outlined in Fig. 5). If
the dorsal half larva has a hydropore on the left side, either no
axis reversal occurred or both the dorsoventral axis and the
position of the hydropore reversed 180°. If, on the other hand,
the hydropore were on the right side of the dorsal half larva,
then only the dorsoventral axis reversed and the position of the
hydropore was not respecified. (Fig. 5).
Each member of the 10 pairs derived from a 4-cell stage
embryo had a left hydropore and was normal (Table 3). Of
those that survived longer, the echinus rudiment was also on
the left side. Of the 5 single embryos that came from the 4-cell
stage separation, i.e. the other half of the pair did not survive,
all had a left hydropore (Table 3). These results indicate that
the dorsoventral axis did not reverse by itself (Fig. 5). If we
assume that the dorsoventral axis reversed in the dorsal half,
as it does in other species, then left/right asymmetry also
reversed. However, the same results could occur if neither axis
reversed (Fig. 5). Therefore, in order to know whether or not
the dorsoventral axis did reverse, the following experiment was
done.
The second experiment directly addressed the question: does
the left/right axis reverse when the dorsoventral axis reverses?
Two small DiI/Wesson Oil droplets were injected into one cell
at the 2-cell stage. At the 4-cell stage, only those embryos with
one DiI droplet in two adjacent cells were cut into dorsal and
ventral halves, along the second cleavage plane. There are only
two possible outcomes to this experiment since the previous
experiment showed that left/right asymmetry does not reverse
unless the dorsoventral axis does too. If neither the dorsoventral or left/right axes reversed, then the DiI droplet would be
on the same bilateral side in both half embryos. However, if
both of these axes reversed, then the oil droplet would be on
the right side of one larva and on the left side of the other (Fig.
5). Fig. 6 shows two pairs of embryos in which this double axis
reversal occurred. One larva for each pair is labeled on the left
(Fig. 6A,C) and the other on the right (Fig. 6B,D). Out of ten
embryo pairs that appeared to develop normally, eight had the
left/right pattern of dye labeling, demonstrating that the dorsal
half larvae usually inverts both the dorsoventral axis and the
position of the hydropore. The other two pairs of dorsal and
Sea urchin axis of bilateral asymmetry
401
Fig. 5. The two outlined experiments demonstrate which axis or axes reversed when 4-cell stage embryos were cut into dorsal (D) and ventral
(V) halves. The experiment described on the left required that one cell at the 2-cell stage be marked with Nile Blue so that a cut could
consistently be made along the second cleavage plane at the 4-cell stage. For these half embryo pairs, the position of the hydropore was noted.
Since it is recognized that the ventral half does not reverse its dorsoventral axis (Hörstadius and Wolsky, 1936; Hörstadius, 1973; Raff, 1992;
Henry and Raff, 1993), the hydropore would always be on the left side of at least one half embryo, the ventral half. This embryo is described at
the top of the figure. If the hydropore was on the left side of both the dorsal and ventral halves then there were two possible interpretations. (1)
Neither the dorsoventral axis or the left/right asymmetry associated with the position of the hydropore reversed, or (2) both axes inverted. This
was what was experimentally observed, indicated by the arrows. If the hydropore had been on the right side of one half embryo (dorsal half) of
a pair, a single axis reversal occurred. This was not observed, as indicated by the stop. The other cell separation experiment, described in the
right of the figure, required that one cell at the 2-cell stage, left (L) or right (R), be injected with two DiI droplets. (This example has the dye on
the left side.) At the 4-cell stage the embryo was separated along the second cleavage plane so that each half, ventral and dorsal, contained one
DiI droplet. The position of the DiI droplet was noted in the resulting pair of larvae. If the DiI droplet were on the same side in both halves (the
left side in this example), then no axes reversed. This occurred in a small number of cases (indicated by the smaller arrow). If the DiI droplet
were on different sides of each half, then either the dorsoventral axis reversed and the position of the echinus rudiment polarity of the left/right
axis) did not invert, or both axes reversed. Although the DiI injection/separation data support both of these interpretations, the Nile Blue
experiment described above indicated that the former situation could not occur (indicated by the stop). Therefore, we can conclude that both the
dorsoventral axis and the left/right asymmetry inverted in the dorsal half embryos (indicated by the larger arrow). Information from these two
experiments allowed us to conclude that when the dorsoventral axis reversed in the dorsal half embryo, the position of the echinus rudiment
would also change relative to its original location; i.e. the dorsoventral axis could not reverse without affecting the polarity of the left/right axis.
The plutei are shown from the dorsal (aboral) view so that left and right are in the same position relative to the cleavage stage embryos from
which they were derived.
ventral halves had dye on the same side in each half; one pair
had a DiI droplet on the right side of both larvae and another
pair were both labeled on the left side (Table 4). These two
pairs of larvae clearly indicate that the neither the dorsoventral
axis or the position of the hydropore reverse in some dorsal
halves.
DISCUSSION
Axis specification and determination in a variety of organisms
Table 4. Results from cell marking and cell separation
experiment
Total number
of pairs
12
POSITION OF DiI DROPLET FOR EACH PAIR:
Right/Left
Right/Right
Left/Left
10
1
1
One cell at the 2-cell stage was injected with two DiI droplets and at the 4cell stage the marked embryos were cut in half along the second cleavage
plane. Only when both members of a pair survived was the position of the DiI
droplet noted for each dorsal and ventral half plutei.
402
E. R. McCain and D. R. McClay
has been well studied, particularly with reference
to the dorsoventral and anteroposterior
(animal/vegetal) axes. Nevertheless, the process
by which the third, bilateral, axis is established
has been a ‘deep and neglected problem’ (Brown
and Wolpert, 1990). This is partly due to the fact
that, unlike the two other axes, the left/right axis
often superficially results in a mirror image and
there are no molecular markers for molecules
found on only one side of the embryo. Since a
bilaterally symmetric animal does not require any
differential left-right positional information (only
information on the distance from the midline), the
differentiation of bilaterality often is considered
uninteresting. However, bilateral animals are
rarely completely symmetric; thus, there must be
mechanisms by which left-right asymmetries are
aligned with respect to the other two axes. Only
recently have researchers begun to investigate
these mechanisms of bilateral asymmetry specification (for reviews, see Berg, 1991; Brown et
al., 1991; Brueckner et al., 1991; Morgan, 1991;
Wood and Kershaw, 1991; Yost, 1991). We
chose the sea urchin embryo as a model system
because the position of the echinus rudiment is
bilaterally asymmetric and the embryos can be
experimentally manipulated so that left/right
specification and determination can be examined.
The study of left-right pattern formation
requires knowledge of when the bilateral axis is
first specified. In nine species of sea urchins, the
relationship between the first cleavage plane and
the dorsoventral or left/right axes has been characterized with lineage tracing markers. Four
different spatial relationships were found
(Hörstadius and Wolsky, 1936; Kominami, 1988;
Cameron et al., 1989; Henry et al., 1990, 1992).
One of these patterns, found in Lytechinus pictus,
Strongylocentrotus droebachiensis and Heliocidaris tuberculata, is that the first cleavage plane
marks the dorsoventral (aboral-oral) axis, thus
bisecting the embryo into left and right halves.
This was true for Lytechinus variegatus, as
demonstrated with a novel lineage tracing
technique. DiI, mixed with oil to create a longer
lasting intracellular microdroplet, was followed
along with the fluorescent dye which diffused
throughout the daughter cells. It was found that
the initial cleavage conditionally divides the
embryo into right and left halves. The term ‘conditional’ is used since experimental manipulations show that this normal fate is not yet fixed.
Since a switch in the dorsoventral axis also
switches the bilateral location of the left/right
structures, their position is not fixed by cytoplasmic determinants present in the egg. An unanticipated observation reinforces this conclusion. In
our lineage experiment there was a statistically
significant (Chi square P<0.001) bias towards
embryos being labeled on the right side. An
asymmetric distribution of lineage tracer was also
Fig. 6. 2-day old dorsal and ventral half larvae. These larvae were produced by
injecting one cell at the 2-cell stage with two DiI/oil droplets and then cutting the
embryo along the second cleavage plane at the 4-cell stage, so that each half (dorsal
and ventral) retained an oil droplet. The two half larvae were raised as a pair and
when they were 2 days old, they were examined for the position of the DiI/oil
droplet (arrow). All of the photographs were taken from the ventral (oral) view. The
bright-field images are in the left-hand column while the fluorescent images are in
the right-hand column. (A,B) One dorsal and ventral pair. (A) The larva is labeled
on the left side. (B) The larva is labeled on the right side. (C,D) Another dorsal and
ventral pair. (C) The larva is labeled on the left side. (D) The larva, which is tilted
slightly onto its right side, is labeled on the right side. Bar, 21.3 µm.
Sea urchin axis of bilateral asymmetry
observed in S. droebachiensis when Nile Blue was the lineage
tracer, in L. pictus when a fluorescent dye was used (Henry et
al., 1992), and in phoronids who were marked with either Nile
Blue or a fluorescent dye (Freeman, 1991). Thus, since the
marking of a cell at the 2-cell stage can somehow bias this cell
towards becoming the right side, this reinforces the conclusion
that neither the bilateral nor the dorsoventral axes are prespecified via cytoplasmic determinants.
Only a few incomplete studies examined the left-right
asymmetry in sea urchins prior to this study. When P. lividus
embryos were separated between the swimming blastula and
mesenchyme blastula stages, it was observed that the left and
right pairs had asymmetric arm deficiencies, narrowing the
time frame of bilateral determination (Hörstadius and Wolsky,
1936; Hörstadius, 1973). Unfortunately, the left/right pairs
were not reared to an age where echinus rudiment differentiation could be detected, so it was not learned whether this
singular bilaterally asymmetric feature was fixed at the time of
the operation. In our study, when L. variegatus embryos were
cut into left and right halves at different times after fertilization and raised as pairs, the resulting larvae suggest that the
left/right asymmetry still is not fixed by the time of the early
blastula stage (5.5-6.5 hours), since all pairs resulted in normal
larvae. If the left/right halves were separated during late
blastula (6.5-7.5 hours old), one half (presumably the left half)
retained normal left coelom development but the other half
sometimes had left or right hydropores, or a pair of hydropores.
This is similar to what was observed in starfish embryos
(Runnström, 1920; Hörstadius, 1928, 1973). Since abnormalities in left/right patterning arise at this time in these experimental half larvae, the late blastula stage appears to be the
period during which the left/right axis becomes committed.
This time frame also corresponds to when the dorsoventral axis
is fixed in sea urchins and starfish (Runnström, 1920; Hörstadius, 1936, 1973, Hardin et al., 1992), indicating that the commitment of both of these axes is temporally coincidental.
Earlier, it was demonstrated that when 2-cell or 16-cell stage
embryos were separated into dorsal and ventral halves, the
dorsal half could invert its dorsoventral axis (Hörstadius and
Wolsky, 1936; Hörstadius, 1973; Raff, 1992; Henry and Raff,
1993). We took advantage of this observation and manipulation technique to study how the left-right asymmetry is established with respect to the dorsoventral axis. By simply cutting
Lytechinus embryos into dorsal and ventral halves at the 4-cell
stage and raising them to an advanced larval stage, it was found
that, when the dorsoventral axis reversed, so did the left-right
polarity. This was confirmed by performing the double DiI
injection and cell separation experiment. Also, when the
dorsoventral axis did not reverse in these dorsal half embryos
(in two cases) the left/right axis did not reverse either. This
supports the hypothesis that the specification of these two axes
is intimately coupled.
The marking and cell separation experiments indicate that
in every case the sea urchin embryo specifies left/right
asymmetry coincident with the dorsoventral specification.
Although this type of pattern formation is suspected to be true
for all bilaterally asymmetric organisms (see discussion section
in Wood and Kershaw, 1991), this is the first time that it was
tested with lineage tracing and under experimental conditions
that allowed for reversal of the dorsoventral axis. One other
animal has been examined after dorsoventral axis reversal. In
403
the nematode Caenorhabditis elegans, two cells at the 3-cell
stage were manipulated so they exchanged positions, resulting
in the reversal of the dorsoventral axis. Since the embryos with
a reversed dorsoventral axis had normal asymmetry of the
left/right axis with respect to the new dorsoventral axis orientation, the left/right polarity had inverted too (Priess and
Thomson, 1987). Thus, the left/right axis is not committed
until the dorsoventral axis is established, which occurs between
the 3-cell and 6-cell stage (Wood, 1991). Thus, it was
concluded that in C. elegans the asymmetries along the
left/right axis are not specified at least until the dorsoventral
axis is specified and the differences between left and right are
due to cell interactions (Wood, 1991). Like C. elegans, sea
urchin embryos have a period during which reversal of the
dorsoventral axis results in reversal of the left/right axis. At
some point after this time, the asymmetric bilateral axis is
fixed. The experiments that we have described, as well as those
with C. elegans, again demonstrate that at the very least we
know the bilateral axis and the polarity of that axis is dependent
upon cell interactions, not segregation of cytoplasmic determinants.
A most perplexing aspect of axis specification in sea urchins
is that when the dorsoventral axis is reversed it is inverted by
180° and only in the dorsal half. Why it happens only in one
half and how the exact hemispherical inversion is controlled is
not understood. It was previously hypothesized that there is an
gradient from ventral to dorsal and the gradient is stabilized in
the ventral half, thus preventing axis inversion (Hörstadius,
1973). Presumably, in the dorsal half the molecular machinery
orients the position of this axis. The polarity of the axis, on the
other hand, is less stable than the orientation; thus, the act of
cutting the embryo in half disrupts the polarity but not the
position of the dorsoventral axis. We hypothesize that the
polarity is randomized when the dorsal half is removed from
the ventral half. This would explain the observation that some
dorsal half embryos (from the DiI injection/separation experiment) did not reverse this axis, while others did rotate the axis
180°. Our results agree with previous observations in H. erythrogramma embryos, where not all dorsal half embryos
reverse their dorsoventral axis (Raff, 1992; Henry and Raff,
1993). Whether first cleavage fixes the axis orientation but not
the polarity is not known.
How might left/right polarity become specified with respect
to the dorsoventral and anteroposterior axes? Recently, a
model for the establishment of handedness in animals was
proposed that consisted of three components: conversion,
random generation of asymmetry and interpretation. Conversion is the first step by which a molecular asymmetry is translated into handedness at a cellular level. Most importantly, it
requires that the handed molecules orient with respect to the
anteroposterior and dorsoventral axes (Brown and Wolpert,
1990). Therefore, if one of these primary axes reverses, two
changes are made: the handed molecules reverse polarity and,
as a result, the left/right axis also inverts. Our micromanipulation and lineage tracing experiments support this hypothesis.
At a molecular level, this interdependency of the dorsoventral
and left/right axes is not well understood. However, there are
two genes in mice which, when mutated, have an effect on
bilateral asymmetry. The iv gene effects the random generation of asymmetry, the second step of the Brown and Wolpert
(1990) model, (Layton, 1976) while the inv gene results in
404
E. R. McCain and D. R. McClay
complete inversion of left/right polarity (Yokoyama et al.,
1993). It is also known that the extracellular matrix in frog
embryos provides cues for bilateral axis asymmetry (Yost,
1990, 1992). In its simplest form, the establishment of the
anteroposterior and dorsoventral axes must create a differential, bilateral expression of factors, to the left side or to the
right. At a later stage, the embryo must be able to ‘read’ these
specified asymmetric differences and respond by generating an
asymmetric left/right pattern. This model must involve cell
interactions since, experimentally, axial specification is shown
to occur in the multicellular embryo.
We are particularly indebted to Norris Armstrong for the insight
and enthusiasm that he brought to the conversations between he and
E. R. M. concerning this research. Support provided by HD14483.
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(Accepted 1 November 1993)