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JEZ 845
Evagination of the Amniotic Cavity in Larvae
Derived from Lithium-Treated Embryos of a Direct
Developing Echinoid, Peronella japonica
Department of Biological Sciences, Graduate School of Science, University of
Tokyo, Tokyo 113, Japan
The effect of LiCl on the development of the sand dollar, Peronella japonica, a
direct developer, was examined. Embryos treated with LiCl were vegetalized and exogastrulated,
as reported for species showing indirect development. In addition, lithium treatment of Peronella
embryos resulted in evagination of the amniotic cavity. Larvae whose amniotic cavity was evaginated did not complete the adult rudiment, but formed the adult skeletal system. The ratio of
larvae with an evaginated amniotic cavity was not correlated with the concentration of LiCl enployed.
The present results suggest that treatment of echinoid embryos with LiCl causes evagination of
invaginating tissues as well as vegetalization. J. Exp. Zool. 279:309–312, 1997. © 1997 Wiley-Liss, Inc.
Typical echinoids develop through a feeding and
swimming larval stage called the pluteus, and
metamorphose into juveniles several weeks after
fertilization (Emlet et al., ’87). This mode of development is referred to as indirect development.
Another mode of development, direct development,
is a process in which part or most of the early
stages of development are omitted, and the larva
metamorphoses without feeding. This mode has
evolved independently in several phylogenetic lineages of echinoids (Strathmann, ’78; Raff, ’87).
Generally, the direct developers metamorphose
without completing the typical pluteus (Raff, ’87).
The development of the sand dollar, Peronella
japonica, is unique (Wray and Raff, ’91). A larva
of this species does not form the larval mouth,
and metamorphoses within 3 days without feeding after going through an abbreviated pluteuslike stage (Mortensen, ’21; Okazaki, ’75). As the
larvae metamorphoses within such a short time
after fertilization, this species is considered to be
well suited for studies of larval development. One
of the specific features in the development of this
species is that the amniotic cavity composing part
of the adult rudiment is formed at an early stage
(late gastrula) and invaginates to a markedly
deeper extent than that in the typical larva
(Mortensen, ’21; Okazaki and Dan, ’54). The amniotic cavity in sea urchins that are typical indirect developers is formed on the left lateral side
of the larval body. In P. japonica, however, it is
formed in the center of the ventral side, where
the larval stomodeum is formed in the embryos
of typical sea urchins (Okazaki and Dan, ’54; Okazaki, ’75). The amniotic cavity of the Peronella
embryo, after initial formation in the center of the
ventral side, shifts toward the left dorsal side (Kajihara et al., ’93). In spite of the many interesting
features in the development of this species, the
process and mechanism of its development and
metamorphosis have never been studied in detail,
except for the reports of Mortensen (’21), Okazaki
and Dan (’54) and Amemiya and Arakawa (’96).
Several substances which cause vegetalization
of sea urchin embryos have been reported so far.
Among them, LiCl is known to be the most effective vegetalizing substance (Herbst, 1893; von
Ubisch, ’29). von Ubisch (’29) examined the effect
of LiCl on the animal cap consisting of presumptive ectoderm (eight mesomeres) isolated from the
16-cell-stage embryo. The LiCl-treated animal cap
was vegetalized to form endo-mesodermal structures, which were expressed exclusively by the
vegetal-half cells in undisturbed embryos. Undissected whole embryos treated with LiCl were vegetalized and developed into exogastrulae in which
the area of the endodermal archenteron increased,
resulting in evagination toward the outside (Lallier,
’75; Mitsunaga et al., ’83). The vegetalized embryos
became less dissociable than normal embryos, because adhesiveness among cells in the endoderm
*Correspondence to: S. Amemiya, Dept. of Biol. Sciences, Graduate School of Science, University of Tokyo, 7-3-1, Hongo, Bunkyo-ku,
Tokyo 113, Japan. E-mail:
Received 13 May 1997; revision accepted 20 June 1997.
was stronger than that in the ectoderm (Fujisawa
and Amemiya, ’82). The effects of LiCl on echinoid
embryos have so far been studied using indirect developers; the effect of LiCl on the development of
direct developers has never been reported. Moreover, the effects of LiCl on echinoid embryos have
mainly been examined at early stages of development. Thus, the delayed effects of LiCl expressed
at later stages in larvae treated at an early stage
of development remain to be studied.
In the present investigation, embryos of a direct developer, P. japonica, were treated with LiCl
from cleavage to the early blastula stage, and examined at later stages. The results indicated that
lithium treatment caused evagination of the amniotic cavity as well as exogastrulation.
Adults of Peronella japonica were obtained in
the vicinity of the Noto Marine Laboratory on the
Japan Sea coast. Eggs and sperm were collected
by artificial spawning induced by intracoelomic
injection of a small amount of 100 mM acetylcholine chloride. The eggs were fertilized in artificial
seawater (Jamarin U, JSW, Jamarin Laboratory)
and the embryos were cultured at 25°C in JSW
in plastic petri dishes through to the 16-cell stage.
At this stage, aliquots of the embryos were transferred to 24-well plastic plates containing various
concentrations of LiCl dissolved in JSW, and cultured for 2 h when the embryos were early blastulae. The embryos were then rinsed with JSW and
removed into plastic petri dishes containing JSW
for continued culture. The embryos were examined using a stereoscopic microscope (Leica MZ8, Deerfield, IL) at various intervals to count the
numbers of embryos that had exogastrulated and/
or those with an evaginated amniotic cavity. The
embryos were photographed using a light microscope (Nikon Biophot).
The archenteron in normal embryos of Peronella
japonica invaginated from the vegetal pole, as in
embryos of other sea urchins. The amniotic cavity in Peronella embryos was formed on the area
corresponding to the presumptive stomodeum of
typical sea urchin embryos (Fig. 1A), confirming
the previous report (Mortensen, ’21; Okazaki and
Dan, ’54; Okazaki, ’75). Three types of morphological abnormalities were found in the Peronella
embryos treated with LiCl. Some embryos exogastrulated (Fig. 1B), others evaginated the amniotic cavity (Fig. 1C), and a few embryos showed
both phenomena (Fig. 1D). The endodermal area
in the exogastrulae was increased (Fig. 1B) in
comparison with that in normal embryos, indicating that lithium treatment caused vegetalization
of the Peronella embryos.
Embryos whose amniotic cavity became evaginated after treatment with LiCl developed into
pluteus-like larvae and formed adult skeletons (Fig.
1E). The evaginated amniotic cavity was easily detached from the embryo because it tended to stick
to the plastic dish during culture (Fig. 1E). Embryos with an evaginated amniotic cavity failed to
differentiate an adult rudiment, and to metamorphose into juveniles. This appears to be reasonable
because the adult rudiment is formed from a
hydrocoel derived from a coelomic pouch through
cooperation with the epidermis of the amniotic cavity. These results indicate that the adult skeleton
is formed independently of the adult rudiment.
It has been reported that the ratio of formation
of exogastrulae in indirect developers is correlated
with LiCl concentration (Lallier, ’75; Mitsunaga
et al., ’83). Therefore, the effects of various concentrations of LiCl on evagination of the amniotic cavity were examined for Peronella embryos.
The sensitivity to LiCl differed markedly among
embryos derived from different mothers. Therefore, a typical experimental example is shown in
Table 1. The experiments were carried out using
concentrations of LiCl ranging from 10 to 60 mM.
The highest ratio (53%) of the embryos developing an evaginated amniotic cavity was obtained
with 20 mM LiCl. A higher concentration did not
always produce a stronger effect, and in fact the
weakest effect (7%) was obtained with 30 mM
LiCl. The effect of 10 mM LiCl (30%) was weaker
than that of 20 mM, but higher than that of 30
mM. Thus, a quantitative relationship between
LiCl concentration and amniotic cavity evagination was not found in Peronella embryos. A total
of six experiments was carried out using embryos
obtained from six different batches, and the results were essentially similar to that shown in
Table 1. It is unclear why a quantitative relationship was not found. However, one possibility is
that the variation in lithium sensitivity among
the embryos derived from the same batch was too
great to allow detection of the effects resulting
form use of the different concentrations. The effect of LiCl on echinoid embryos showing typical
indirect development has been well studied, and
exogastrulation based on vegetalization of the embryos has been reported to occur (von Ubisch, ’29;
Mitsunaga et al., ’83; Lallier, ’75). In the present
Fig. 1. Normal and LiCl-treated embryos of Peronella
japonica (scale bars 50 µm). A: A normal embryo at 18 h after
fertilization. An arrow indicates the amniotic opening. B: An
embryo at 18 h after fertilization, treated with 60 mM LiCl.
The archenteron (arrow) evaginates to form an exogastrula.
Arrowheads indicate the boundary between the ectoderm and
endoderm. C: An embryo at 24 h after fertilization, treated
with 20 mM LiCl. The amniotic cavity (arrowhead) evaginates toward the outside of the embryo. D: An embryo at 20
h after fertilization, treated with 30 mM LiCl. The amniotic
cavity (arrowhead) as well as the archenteron (arrow)
evaginates from the embryo. E: A pluteus larva at 4 days
after fertilization derived from an embryo treated with 30
mM LiCl. The amniotic cavity has evaginated, but subsequently lost because it tended to stick to the plastic dish
during culture. An asterisk indicates the place where the
evaginated amniotic cavity was formed. Some spines (arrowheads) are formed in the larval body.
study, it was found that echinoid embryos showing direct development also underwent exogastrulation after treatment with LiCl. The increased
volume of tissue present in the exogastrulated
archenterons implies that the embryos were
vegetalized. In addition to exogastrulation, it was
demonstrated that lithium treatment of Peronella
embryos resulted in evagination of the amniotic
cavity. So far, it has been considered that an increase in the endodermal territory by treatment
of embryos with LiCl causes exogastrulation
(Giudice, ’73). However, the amniotic cavity is
formed in the animal half of the embryo, suggesting that evagination of the amniotic cavity does
not depend on the endodermalizing effect of LiCl.
We consider that LiCl has two different effects
TABLE 1. Relationship between concentration of LiCl and
formation of embryos with an evaginated amniotic cavity1
of LiCl
Number of individuals with
an evaginated amniotic cavity
10 mM
20 mM
30 mM
60 mM
9 (30%)
16 (53%)
2 (7%)
11 (37%)
All embryos were examined at 18 h after fertilization when control
larvae were at the pluteus stage.
on echinoid embryos: vegetalization and evagination of invaginating structures.
We thank the members of the Noto Marine Biological Station for supplying materials. We also
thank the stuff of the Misaki Marine Biological
Station where most of the present work has been
carried out. The present study has been covered
by a Grant-in-aid from the Ministry of Education,
Science and Culture of Japan (93640598) to S.A.
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