Develop. Growth Differ. (2008) 50, S239–S256 doi: 10.1111/j.1440-169X.2008.01035.

Review
Development of the appendicularian Oikopleura dioica:
Culture, genome, and cell lineages
Hiroki Nishida*
Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho,
Toyonaka, Osaka 560-0043, Japan

Appendicularians are planktonic tunicates (urochordates), and retain a swimming tadpole shape throughout
their life. Together with ascidians, they are the closest relatives of the vertebrates. Oikopleura dioica is
characterized by its simplified life habit and anatomical organization. It has a tiny genome, the smallest ever
found in a chordate. Its life cycle is extremely short – about 5 days – and it can be maintained in the laboratory
over many generations. Embryos and adults are transparent and consist of a small number of cells. The
anatomy of juveniles and adults has been described in detail. Cleavage pattern, cell lineages, and morpho-
genetic movements during embryogenesis have also been comprehensively documented. A draft genome
sequence is now available. These features make this organism a suitable experimental model animal in which
genetic manipulations would be feasible, as in Drosophila and Caenorhabditis elegans. In this review,
I summarize a hundred years’ knowledge on the development throughout the life cycle of this organism.
Oikopleura is an attractive organism for developmental and evolutionary studies of chordates. It offers
considerable promise for future genetic approaches.
Key words: appendicularian, cell lineage, fate map, life cycle, Oikopleura.

Reviews by Galt and Fenaux (1990) and Alldredge

Introduction
Recent advances in our understanding of the molecu-
lar and cellular mechanisms that govern development
owe much to model organisms with short life cycles
in which genetic approaches are feasible, such as
Drosophila and Caenorhabditis elegans. Oikopleura
dioica Fol 1872, an appendicularian (or larvacean), is
a candidate model organism for several reasons: (i) it
has a life cycle of only 5 days at 20°C (Fenaux 1998a);
(ii) inland and multigeneration culture methods have
been established; (iii) a draft genome sequence is
now available; (iv) the animal is transparent during
development; (v) morphogenesis and cell lineages
during embryogenesis are well described; and (vi) the
juvenile and adult anatomies have been comprehen-
sively described.
*Author to whom all correspondence should be addressed.
Email: hnishida@bio.sci.osaka-u.ac.jp
Received 31 January 2008; revised 29 February 2008;
accepted 29 February 2008.
© 2008 The Author
Journal compilation © 2008 Japanese Society of
Developmental Biologists
(1976) give a general introduction to appendicular-
ians. The Oikopleura tadpole builds and lives inside a
so-called house (oikos in Greek), which is made from
acelluar materials secreted from the oikoplastic
epithelium (trunk epidermis) (Fig. 1A,B; Fenaux 1986;
Flood & Deibel 1998; Thompson et al. 2001). The
house is a complex filtration apparatus by which the
animal can feed on a highly concentrated suspension
of particles filtered from seawater. The tail generates a
water current through the house when the tadpole is
within the house. The tadpole leaves and discards the
house five to 10 times per day, forming a new one
in just 10 min. Appendicularians are second or third
most abundant among the zooplankton community
and serve as food for other zooplankton and fishes
(Alldredge 1976). Their discarded houses are the main
component of marine snow. Thus, appendicularians
have ecological importance in marine food web.
Appendicularians are planktonic tunicates (urochor-
dates), and retain a swimming tadpole shape
throughout their life (Fig. 1A,B, see also Fig. 3),
whereas other tunicates resorb the tail during meta-
morphosis. Only three families and 69 species of
appendicularians have been described so far (Fenaux
S240
1993). Together with ascidians and thaliaceans, they
are the closest relatives of the vertebrates (Fig. 1C;
Garstang 1928; Bourlat et al. 2006; Delsuc et al.
2006). 18S rDNA sequences suggest that appendicu-
larians are the most basal among these tunicates
(Wada 1998; Swalla et al. 2000), although their phylo-
genetic position among the tunicates is still con-
troversial (Stach & Turbeville 2005). O. dioica is the
sole dioecious (or dioicous) species so far reported
among the Tunicata. They represent a simplified body
plan of chordates, characterized by a tail with a noto-
chord and dorsal neural tube, an endostyle, and a gill
aperture. The structure of the adult body is relatively
simple and consists of a small number of cells; for
example, there are only 20 notochord cells at hatching
and 20 non-fused muscle cells even in the tail of a
mature adult (Bassham & Postlethwait 2000; Nishino &
Satoh 2000, 2001; reviewed in Fenaux 1998b; Nishino
& Satoh 2001).
Laboratory culture
O. dioica is abundant worldwide in coastal regions. It
tolerates extremes of temperature and salinity, and
so far is the only species that has been cultured in
laboratories. Paffenhöfer (1973) first reported suc-
cessful culture in the laboratory. Now it is routinely
cultured in laboratories in Villefranche-sur-Mer
© 2008 The Author
Journal compilation © 2008 Japanese Society of Developmental Biologists
H. Nishida
Fig. 1. The appendicularian Oiko-
pleura dioica and its phylogenetic
position. Dorsal (A) and lateral (B)
views of the animal in its “house”.
Carbon particles from India ink were
added to seawater and were con-
centrated by the house. Seawater
comes into the house from the bilat-
eral inlet filters and goes out from
the ventral outlet pore. The particles
in seawater are highly concentrated
by the nets of the food-trap filter and
introduced into the trunk via the
mouth. Digestive organs within
the trunk are made visible by the
black particles. Bar, 2 mm. (C)
Phylogenetic tree within the phylum
Chordata.
(France), Bergen (Norway), and Eugene, Oregon
(USA), and in our lab.
The current culture methods were developed in
Bergen as described in Chioda et al. (2002). In our
lab, the animals are cultured in artificial seawater
(RohtoMarine, Rei-Sea Co., Tokyo, Japan) in 5 L
plastic beakers under constant stirring with an acrylic
paddle (15 r.p.m.) at 20°C (Fig. 2A; Fujii et al. 2008).
Ten grams of activated charcoal is added to each
beaker in order to keep the seawater in good condi-
tion. The life cycle is 5 days. Cultures are diluted five
fold on day 1, and then approximately 100 animals are
transferred to clean artificial seawater on day 3 by
pipetting, and allowed to naturally spawn on day 5
(Fig. 2C). The animals are fed with a cocktail of live
algae and cyanobacterium of various sizes (Fig. 2B),
namely Isochrysis galbana (approximately 4 mm, 3000
cells/mL), Chaetoceros calcitrans (3–6 mm, 3000 cells/
mL), Rhinomonas reticulata (12 mm, 1000 cells/mL),
and Synecococcus sp. (1.5 mm, 4.0 mL of culture/5 L)
on days 1 and 2, and then at double those concen-
trations from days 3 to 5. The animals grow very fast
each day, and grow well under these culture condi-
tions (see Fig. 11). Growing animals can trap only
foods that are intermediate in size between the mesh
size of the inlet filter and the food-trap filter of the
house (Fig. 1A,B). This is why they are fed foods of
various sizes. We have cultured the animals for 2
 Development of Oikopleura
Fig. 2. Laboratory culture. (A) The
animals are cultured in artificial sea-
water in 5 L plastic beakers with an
acrylic paddle at 20°C. Activated
charcoal (10 g) is added to each
beaker. (B) Culture of algal strains in
plastic bags. Oikopleura dioica is
fed with a mixture of Isochrysis
galbana, Chaetoceros calcitrans,
Synecococcus sp., and Rhinomo-
nas reticulata. (C) The animal’s life
cycle is 5 days (D1–D5). Cultures
are diluted on day 1, and then
approximately 100 animals are
transferred to clean artificial seawa-
ter on day 3. They naturally spawn
on day 5.
years over a hundred generations without introducing
new animals from the ocean.
The life cycle is shown in Figure 3. At 20°C, they
hatch 3 h after fertilization. Tail shift (see below) of
the larva takes place at 10 h, and then the juvenile
becomes fully functional, making its first house and
starting to take food. They mature to males or females
with full-grown gonads in 5 days, then spawn and die
soon after. Developmental time roughly doubles at
13°C (Fig. 3B). Each female spawns 40 to 400 eggs
depending on the amount of food available.
Genome
O. dioica has a tiny genome of only 72 Mb, the
smallest ever found in a chordate (ascidian Ciona,
160 Mb; amphioxus Branchiostoma, 550 Mb; fugu,
400 Mb; human 3000 Mb) (Seo et al. 2001). Shotgun
sequences obtained at a high coverage (14¥) are
estimated to represent 99.7% of all coding nucle-
otides. The first assembly of the draft genome is
available at Genoscope (http://www.genoscope.cns.
fr/externe/GenomeBrowser/Oikopleura/). In spite of
the small genome, the number of genes is estimated
as 15, 000 (comparable to Ciona, and approximately
half of human). This indicates a high gene density
within the genome (one gene every 5 kb). Indeed,
intergenic sequences are very short, and 62% of
introns are smaller than 50 bp. The positions of introns
in each gene diverge from those in other animals
Journal compilation © 2008 Japanese Society of Developmental Biologists
S241
(Edverdsen et al. 2004). Comparison of lengths of
some genes between O. dioica and human revealed
that genes measuring 0.8, 1.6, and 2.2 kb in O. dioica
correspond to genes measuring 15, 30, and 35 kb in
human, respectively. The small size of the genome
and high density of genes will facilitate chemical and
insertional mutagenesis in this organism. It is still pre-
mature to conclude a link between the small genome
and the short life cycle or rapid embryogenesis only
from the data so far available.
Several groups have used molecular and compara-
tive approaches for studying the development of this
animal by cloning several developmentally regulated
genes (Bassham & Postlethwait 2000, 2005;
Thompson et al. 2001; Cañestro et al. 2005; Cañestro
& Postlethwait 2007; see also Nishino et al. 2001 for
Oikopleura longicauda). A conspicuous character of
O. dioica is the partial loss of Hox genes (Seo et al.
2004; Edverdsen et al. 2005), specifically the central
genes Hox3, 5, 6, 7, and 8, and the Hox cluster is
totally fragmented and dispersed within the genome.
However, the anterior–posterior order of Hox gene
expression seems to keep spatial colinearity. A similar
situation is seen in ascidians (Ikuta et al. 2004; Ikuta &
Saiga 2005), although ascidians lost different Hox
cluster genes (Hox7, 8, 9, and 11). The body plan of
chordate ancestors (probably similar to that of cepha-
lochordates; Fig. 1C) may have been simplified in the
tunicate lineage, resulting in relaxed pressure to con-
serve a full set of the Hox gene cluster. Alternatively,
© 2008 The Author
S242
embryogenesis with determinate and invariant cell
lineages may reduce the requirement for several
Hox genes (Edverdsen et al. 2005; Seo et al. 2004).
Another unique character of O. dioica is the presence
of spliced-leader RNA trans-splicing (Ganot et al.
2004), which is known in a limited and sporadic dis-
tribution among eukaryotes, and only in C. elegans
and ascidians among metazoans so far (Vanden-
berghe et al. 2001; Satou et al. 2006). At least 25% of
O. dioica mRNA has spliced-leader RNA in the 5′
ends, although the biological significance of trans-
splicing is still unclear. These unique characters that
are commonly found in appendicularians and ascid-
ians support their close relationship.
Embryogenesis
Artificial fertilization can be done by mixing fully
matured eggs and sperm dissected from gonads, or
naturally spawned eggs and sperm. Sperm can be
kept frozen in liquid nitrogen (K. Ouchi and H. Nishida,
© 2008 The Author
Journal compilation © 2008 Japanese Society of Developmental Biologists
H. Nishida
Fig. 3. Life cycle of Oikopleura
dioica at (A) 20°C and (B) 13°C,
showing durations of embryogen-
esis and of larval and juvenile
development.
unpubl. data, 2008). The fine structure of sperm has
been described in Flood and Afzelius (1978). Each
sperm is 30 mm long and has a typical shape with
head and tail. The fine structure of eggs and events
at fertilization have been described in Holland et al.
(1988). Unfertilized eggs are approximately 80 mm in
diameter, and are arrested at meiotic metaphase I.
There are no accessory cells inside or outside the
vitelline membrane.
By 30 s after fertilization, the surface of the egg
becomes rough. This is the first sign of fertilization.
The narrow perivitelline space does not expand after
fertilization. In ascidians, movements of the egg cyto-
plasm, called ooplasmic segregation, take place
after fertilization (Conklin 1905; Nishida 1994). These
movements transform the symmetry of eggs from
radial to bilateral. During the movements, several
localized maternal mRNAs that play crucial roles in
cell fate specification and embryonic axis determina-
tion are brought to the future posterior pole and
establish the secondary embryonic axis perpendicu-
 Development of Oikopleura S243

Fig. 4. Embryogenesis of Oiko-

pleura dioica. Developmental times
and stages are indicated below
each photo. (A) Unfertilized egg,
80 mm in diameter. (B) Fertilized
egg after emission of the second
polar body. (C) 2-cell stage. (D)
4-cell stage. (E–G) 8-, 16-, and
32-cell embryos, respectively. The
name of each blastomere is indi-
cated in (E and F). The embryo in
(G) is gastrulating from the left side
of the photo. (H–L) Late gastrula to
tailbud stages. Photos were taken
every 30 min. (M, N) Right and
ventral views during tail elongation.
In the optical section of the tail,
muscle (Mu), notochord (Not),
nerve cord (NC), and endodermal
strand (ES) are visible. The tail has
rotated counterclockwise by 90°
when viewed from the posterior
direction with the nerve cord on the
left side. (O) A tadpole larva is
hatching from the vitelline mem-
brane (VM). Bar, 50 mm. ant,
anterior; pos, posterior; ani, animal
pole; veg, vegetal pole. (Modified
from Fujii et al. 2008 with
permission.)

lar to the primary animal–vegetal axis (Nishida &
Sawada 2001; Negishi et al. 2007; reviewed in Nishida
2002, 2005; Prodon et al. 2007). In O. dioica eggs,
however, ooplasmic movement is not obvious (Holland
et al. 1988), and there is no mitochondrion-rich myo-
plasm comparable to that in ascidian eggs (H.
Nishida, unpubl. data, 2007). So how unfertilized eggs
of O. dioica with radial symmetry turn into bilaterally
symmetrical embryos remains elusive. The embryo-
genesis of O. dioica is a kind of simplified form of that
of ascidians: the fate maps of both organisms are
similar, as mentioned below, but O. dioica does not
share the ooplasmic movements just after fertilization.
Identification of Oikopleura homologues of localized
mRNAs in ascidians and examination of their localiza-
tion will shed light on this issue.
Even though Oikopleura has a small number of
embryonic cells and can be cultured readily in the
laboratory, only few reports describing its embryo-
genesis have appeared so far (Delsman 1910;
Fenaux 1998a; Nishino & Satoh 2001). Among these,
that of Delsman (1910) is remarkably detailed and
comparable to that of Conklin’s (1905) monumental
paper on ascidian embryogenesis. Delsman investi-
gated the cleavage pattern in amazing detail using
embryos at various stages collected directly from the
ocean with a plankton net. He managed to identify
each blastomere during the cleavage and gastrula-
tion stages.
Eggs and embryos are highly transparent and thus
suitable for observation with differential interference
contrast (DIC) optics (Fig. 4; Fujii et al. 2008). The
emission of two polar bodies at 15 min is followed by
the first cleavage at 35 min at 13°C. Early cleavages
take place every 15 min. After the cleavage stage, the
first extensive morphogenetic movement, gastrulation,
starts at 2 h at the 32-cell stage (Fig. 4G). Every
vegetal blastomere is internalized during gastrulation.

© 2008 The Author

Journal compilation © 2008 Japanese Society of Developmental Biologists
S244
The tailbud stage is attained at 4 h (Fig. 4K), then the
tail elongates and the embryo becomes bent ventrally.
Notochord cells aligned in a single row become
evident at 4.5 h (Fig. 4L–N). Tailed larvae hatch from
the vitelline membrane at 6 h (Figure 4O; see also
supplemental movies of the embryogenesis in Fujii
et al. [2008]). There is no obvious structure in the head
and trunk regions, but the larvae occasionally beat
their tail. Fine structures of hatching larvae are
described in Stach (2007). The tail shows a remark-
able 90° counterclockwise rotation relative to the trunk
when viewed from the posterior, with the neural tube
on the left side (Fig. 4N). The rotation of the tail reflects
the direction of the tail beat. In ascidians, lancelets
(Amphioxus), and lower vertebrates, the tail beats in a
left–right direction, whereas in appendicularians it
beats in a dorsal–ventral direction.
Cleavages, gastrulation, and neurulation
Fujii et al. (2008) recently reassessed Delsman’s
(1910) descriptions of cleavage pattern and morpho-
genetic movements in detail using updated
techniques such as DIC observation, time-lapse video
recording, and 3-D reconstruction with confocal
microscopy. Movies of full embryogenesis, gastrula-
tion, and neurulation are available as supplementary
materials with the paper. We confirmed that the
descriptions by Delsman were both precise and accu-
rate, except for one new finding of unequal division
of B1 blastomeres, as described below. Cleavage
showed an invariant pattern among individuals. The
cleavage pattern is somewhat complicated, as the
pattern is roughly, but not precisely, bilateral. This
makes it difficult to recognize the orientation of
embryos, as they lack a straight midline, unlike
ascidian embryos. The detection of genes that
are expressed in specific subsets of blastomeres has
significantly advanced our understanding of early
cell-fate-specification processes in ascidian embryos
(Nishida 2005). Similarly, the precise identification of
blastomeres would be essential for clarifying patterns
of gene expression in O. dioica. For example, expres-
sion of brachyury and muscle actin is initiated in
notochord and muscle precursor blastomeres at
the 32- and 64-cell stages, respectively (Bassham &
Postlethwait 2000; Nishino & Satoh 2000, 2001).
The first two cleavage planes are meridional
(Fig. 4C,D). The first cleavage generates the left and
right blastomeres, and the second divides the anterior
and posterior blastomeres, as in ascidian embryos,
which are equivalent to the dorsal and ventral blas-
tomeres, respectively, in amphibian embryos. (Refer to
Nishida [2005] for differences in conventional defini-
© 2008 The Author
Journal compilation © 2008 Japanese Society of Developmental Biologists
H. Nishida
tions of the anterior–posterior and dorsal–ventral axes
in tunicate and amphibian embryos). The anterior-left
and posterior-right blastomeres of the 4-cell embryo
are invariably formed slightly closer to the vegetal pole,
where they are in contact with each other (Fig. 4D,
arrow). On the other hand, the anterior-right and
posterior-left blastomeres are in contact at the animal
pole. This is the first sign of invariant left–right asym-
metry in the O. dioica embryo. The third cleavage
occurs horizontally, generating four smaller vegetal
blastomeres and four larger animal blastomeres
(Fig. 4E). As described later, all the vegetal blastomere
descendants are internalized during gastrulation.
At the 8-cell stage, each blastomere is named as in
the ascidian system (Delsman 1910). Animal cells are
indicated by a lower-case letter, and vegetal cells by a
capital. Anterior cells are named “a” and “A” and
posterior cells are named “b” and “B”. The cells of the
right half when viewed from the vegetal pole (future
dorsal side) with the anterior pole upward are labeled
by underlining (Fig. 4E). From the 16-cell stage,
Delsman (1910) applied the nomenclature system
used in annelid and mollusc embryos. All the descen-
dant cells of a given blastomere in the 8-cell embryo
are indicated with the character of the blastomere.
After each division, a numeral (1 or 2) is added, 1 for
the daughter cell closer to the animal pole. For
instance, blastomere “A” divides into A1 and A2. Then
the A1 blastomere divides into A11 and A12, and A2
divides into A21 and A22. Details of the cleavage
pattern up to the 32-cell stage are reported in Fujii
et al. (2008) (see also Fig. 8).
Two rounds of unequal cell division occur in the
posterior-vegetal B-line blastomeres. The B blas-
tomere pair of the 8-cell embryo divides into the larger
B2 and the smaller B1 pair. In the second round, B1
blastomeres at the posterior pole of the 16-cell embryo
divide unequally into the larger B12 and the smaller
B11 again (see Fig. 8, posterior views) (Fujii et al.
2008; see also supplemental movies in Fujii et al.
2008). Thus, the cleavage pattern of the O. dioica
embryo shows striking similarity to that of ascidians, in
which three rounds of unequal division take place at
the posterior pole of the vegetal hemisphere from
the 8- to 64-cell stages (Conklin 1905). Eventually, the
smallest blastomeres at the posterior pole of the
64-cell ascidian embryo are fated to become primor-
dial germ cells (Tomioka et al. 2002; Shirae-
Kurabayashi et al. 2006). It is uncertain whether the
resulting tiny B11 cell (see Fig. 8, brown cell pair) of
O. dioica is a primordial germ cell, but comparison
with ascidian embryos suggests that this is plausible.
The posterior blastomeres in ascidians have a special
subcellular structure, the centrosome-attracting body
 Development of Oikopleura S245

Fig. 5. Gastrulation. Ingressing

cells during the first phase (A–D)
and the second phase (E–G) are
indicated by pseudo-yellow color.
(A–D) First phase. Sequential
images of time-lapse video taken
from 105 to 122 min after fertiliza-
tion at 13°C. The name of each
blastomere is indicated. Eight
yellow-colored blastomeres at the
vegetal pole are ingressing. The
embryonic surface becomes
covered by six green-colored blas-
tomeres by epiboly. These six green
cells are arranged like an asterisk at
the completion of ingression of the
yellow cells (D). B11 cells, sister
cells of B12, generated by unequal
division are shown in blue. Anterior
is up. (E–G) Second phase, from
122 to 145 min. The B12 cells have
divided into B122 and B121 and
are now colored yellow. Six yellow-
colored cells are internalized
sequentially in the order B121,
B122, and B11 and covered by the
b-line cells. (Reprinted from Fujii
et al. 2008 with permission.)

(CAB; Hibino et al. 1998;Iseto & Nishida 1999; Nish-
ikata et al. 1999; Negishi et al. 2007). During unequal
cleavages, the microtubule bundle extending from the
posterior-most centrosome of the pair is connected to
the CAB in the posterior cortex. Then, as the microtu-
bule bundle shortens, the interphase nucleus shifts
posteriorly and approaches the CAB. Consequently,
an asymmetrically located mitotic apparatus is
formed, and unequal division occurs. The CAB is
a multifunctional structure in ascidian embryos
(reviewed in Nishida et al. 1999; Nishida 2002; Prodon
et al. 2007), and it also serves as an mRNA localiza-
tion scaffold, where some maternal mRNA are
specifically localized. The CAB also contains an
electron-dense matrix similar to germplasm. It is likely
that appendicularians also posses the CAB structure,
as two rounds of unequal division take place at the
posterior pole.
In O. dioica, gastrulation is initiated as early as the
32-cell stage, 1 h 50 min after fertilization at 13°C,
much earlier than in ascidians (at the 110-cell stage in
Halocynthia roretzi, 9 h after fertilization at 13°C) and
vertebrates. Internalization of the vegetal cells
proceeds by ingression and/or epiboly, keeping the
embryonic outline almost spherical. There is no arch-
enteron. All vegetal cells, namely A- and B-line cells,
are internalized. The gastrulation process can be sub-
divided into three phases. First, eight cells at the
vegetal pole ingress (Fig. 5A–D), then descendants
of B1 blastomeres in the posterior region follow
(Fig. 5E–G; Fujii et al. 2008; see also supplemental
movies in Fujii et al. 2008). Finally, b22 descendants
in the animal hemisphere (muscle precursors) are
internalized later (Stach et al. 2008).
After the first phase of gastrulation, neurulation
starts at the 64-cell stage, again earlier than in other
chordates. It is difficult to precisely define the neurula
stage in O. dioica, as the second phase of gastrulation
takes place simultaneously with neurulation. In the
anterior region, two rows of four cells that originate
from the A1 blastomere pair are internalized (Fig. 6),
as previously observed in O. longicauda (Nishino &
Satoh 2001). There is no neural fold, and folding of the
neural plate is not observed (Fujii et al. 2008, see also
supplemental movies in Fujii et al. 2008). However, at
the tailbud stage, four cells constitute a neural tube in
optical transverse sections of the nerve cord in the tail
(Fig. 4N), as observed in ascidian tadpole larvae.
Therefore, tube formation would take place at a later
stage after neural precursor cells have completely
ingressed. In addition to the A1 descendants, a recent
cell lineage analysis showed that descendants of the

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Journal compilation © 2008 Japanese Society of Developmental Biologists
S246 H. Nishida
a222 cell (Fig. 6B,C) join the central nervous system
(CNS) at a later stage (Stach et al. 2008).
Thus, the initial morphogenetic movements –
gastrulation and neurulation – take place quite early
when the embryo still consists of only a small number
of cells. The cells are internalized by ingression, not by
folding of an epithelial sheet.
Cell lineages and fate map up to
hatching larvae
Detailed descriptions of cell lineages are especially
important for animal embryos that show invariant and
determinative cleavage patterns, as is the case for
C. elegans and ascidians (Sulston et al. 1983; Nishida
1987). Recently, Stach and his colleagues have traced
complete cell lineages of O. dioica up to the hatching
stage by using time-lapse recording with 4-D micros-
copy (Stach et al. 2008). The cell lineages and cell
fate were essentially invariant among three specimens
examined. A fertilized egg divides nine or 10 times on
average and generates approximately 550 cells at
hatching. The most striking feature of O. dioica
embryogenesis is that the fates of most blastomeres
are restricted to give rise to a single tissue type by the
32-cell stage (Fig. 7). The fate map shows bilateral
symmetry (Fig. 8) with the exception that the right A22
blastomere gives rise to notochord (pink) in addition
to endoderm (yellow). Every cell in the animal view
develops into larval epidermis (green). In the vegetal
view, cells at the vegetal pole form endoderm (yellow).
The posterior and lateral marginal cells give rise to tail
muscle (red). Notochord (pink) and CNS (light blue)
are derived from the anterior marginal blastomeres.
© 2008 The Author
Journal compilation © 2008 Japanese Society of Developmental Biologists
Fig. 6. Neurulation. Ingressing
neural precursor cells are indicated
by pseudo-yellow color. Sequential
images of time-lapse video taken
from 120 to 130 min after fertiliza-
tion at 13°C. Anterior is at the
bottom left, and the vegetal pole is
at the upper right. Four yellow cells
of A1 descendants in the anterior
region divide once along the
anterior–posterior axis and are then
internalized. The surface is covered
by green a-line cells. (Reprinted
from Fujii et al. 2008 with
permission.)
The topography in presumptive tissue territories
looks quite similar to that in the ascidian fate map
(Fig. 8, bottom left; Nishida 2005), which in turn shows
similarity to the amphibian fate map although each
territory differs in relative proportion (Fig. 8, bottom
right; Kourakis & Smith 2005). Therefore, the fate map
of O. dioica can be regarded as a typical one shared
by basal chordates. In ascidians, fate restriction in
most blastomeres is completed by the 110-cell stage,
in contrast to the 32-cell stage in O. dioica. However,
it is notable that in all these animals fate restriction is
almost completed just before gastrulation starts. The
Oikopleura fate map at the 32-cell stage represents
the simplest one in chordates and probably among all
animals so far observed.
Larval development
Larval development is subdivided into five stages,
finishing with the young juvenile at stage 6 (Figs 3B,9;
Fenaux 1976, 1977, 1998a; Galt & Fenaux 1990). Just
after hatching (stage 1), there is no obvious structure
in the trunk, but larvae develop into fully functional
juveniles (stage 6) in 13 h after hatching at 13°C, or in
about half the time at 20°C. During these stages the
tail continuously elongates and becomes flattened
laterally.
1. Stage 1: larvae occasionally move by tail beat,
although the nervous system is not evident yet.
They are surrounded by an acellular larval tunic,
which is discarded at stage 3.
2. Stage 2: the statocyte is forming in the brain. The
boundaries of organs begin to appear. Notochord
vacuolation starts.
 Development of Oikopleura S247

Fig. 7. Cell lineage tree of Oikopleura dioica

from fertilized egg to hatching larva. Each
lineage is color-coded after the developmental
fate of the cell is tissue-restricted. An arrow at
the bottom marks the left–right asymmetry in
the formation of notochord in the A22 lineage.
(Modified from Stach et al. 2008 with
permission.)

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Journal compilation © 2008 Japanese Society of Developmental Biologists
S248 H. Nishida

Fig. 8. Fate map and cell fate restriction during cleavage stages. Embryos are viewed from various directions indicated at the top.
Orientations of embryos are indicated at the bottom. Note that the vegetal pole (future dorsal side) is down in the anterior, posterior,
right, and left views. Arrowed bars connect sister blastomeres. At the 16-cell stages, blastomeres are colored when the fate of the
blastomere is restricted to give rise to a single tissue type. At the 32-cell stage, every tissue-forming territory is colored regardless of
fate restriction of blastomeres. Color codes are shown at the bottom. Note that most cells at the 32-cell stage are fated to give rise to
a single cell type. The fates of heart (purple) and germ cells (brown) are just speculation based on comparison with ascidian embryos,
and are not experimentally confirmed. At the bottom left, the fate map of the animal and vegetal halves of the 110-cell ascidian embryo
are shown with the same color codes for comparison (Nishida 2005). White blastomeres are precursors of mesenchyme and trunk
lateral cells, which are not present in Oikopleura dioica embryos. At the bottom right, the fate map of the left half of the Xenopus blastula
is shown with the same color codes (Kourakis & Smith 2005). Animal pole is up. HM, head mesoderm. Note the similarity of topography
in the presumptive tissue territories between appendicularian, ascidian, and amphibian fate maps, although each territory differs in
relative proportion. The fate map of O. dioica was drawn using the cleavage pattern data in Fujii et al. (2008) and lineage data in Stach
et al. (2008) with permission.

3. Stage 3: mouth and visceral formation is evident.
Larvae swim vigorously.
4. Stage 4: tube formation is initiated in the digestive
duct with cilia moving inside. Cilia of spiracles
(gill apertures) also start to move. The endostyle
evaginates from the floor of the pharynx. Heart
beat starts. Mouth opens, and notochord vacu-
oles are fusing with each other. Tail becomes
flattened to form lateral fins.
5. Stage 5: mouth and spiracle formation completes.
Water current starts inside the larva. Lumens of
the digestive duct are now continuous. The proxi-
mal region of the tail narrows, and the tail bends
slightly in the ventral direction. The trunk epider-
mis (oikoplastic epithelium) secretes house
materials (pre-house).
6. Stage 6: this stage is attained after the tail shift.
After a few seconds of intense movement, the tail
orientation suddenly changes 120°. Distal end of
tail is now in the same direction as the mouth. The
first house swells and forms in 10 min, and the
animal can start feeding. The tail shift is com-

© 2008 The Author

Journal compilation © 2008 Japanese Society of Developmental Biologists
 Development of Oikopleura
Fig. 9. Larval development is subdivided into five stages (1–5).
After the tail shift, stage 6 is the young juvenile stage. Develop-
mental times after fertilization at 13°C and the corresponding
stages are indicated above each photo. Larvae develop into fully
functional juveniles (stage 6) in 13 h after hatching at 13°C.
(B–E) Left-side views. (F) Right-side view after the tail shift. Bar,
50 mm. See details in text.
pleted in a few seconds; however, the event is
thought to be comparable to metamorphosis in
other invertebrates (Galt & Fenaux 1990).
Anatomy
The general organization of organs that are present
in the adult form is already achieved by stage 6
(Fig. 10). The only exception is the gonad, which is a
small rudiment in the posterior-ventral position
(Fig. 10I). Cell divisions in the epidermis and probably
in most of the other somatic tissues cease before tail
shift (Galt & Fenaux 1990). Therefore, growth of the
juvenile results solely from an increase of cell volume
(Fig. 11). The somatic cells increase their content of
DNA to become polyploid (Fenaux 1971; Ganot &
Journal compilation © 2008 Japanese Society of Developmental Biologists
S249
Thompson 2002), a result of successive rounds of
DNA replication in the absence of karyokinesis and
cytokinesis. Organ formation is completed after tail
shift (stage 6) while the animal is still small and com-
pletely transparent. Every structure in the adult form
can be easily observed in anesthetized whole animals
at this stage by changing the focal plane (Fig. 10). The
tail shift makes the definition of body axes difficult. In
this report, body axes that are defined for the trunk
region before the tail shift are used for the axes of the
whole body after the tail shift, so the mouth is still
anterior, although the tadpole swims backwards.
Here the characters of each tissue and organ are
briefly summarized. The anatomy of appendicularians
is reviewed in detail in Fenaux (1998b). Transverse
sections at various levels and an interactive computer
3D reconstruction are available at the EURAPP site
(http://www.obs-vlfr.fr/~eurapp/).
Epidermis
The entire epidermis consists of a single layer of epi-
thelium, with no mesodermal lining below it. The only
coelomic space is the pericardium. Most regions of
the trunk epidermis are highly patterned to secrete the
intricate house, and constitute the oikoplastic epithe-
lium; the posterior part of the trunk region, covering
the gonad and tail epidermis, does not secrete the
house. The oikoplastic epithelium consists of approxi-
mately 2000 cells, and is subdivided into bilateral
territories according to nuclear sizes and shapes, spe-
cific gene expression, and extent of polyploidization
(30–1300-fold) (Thompson et al. 2001; Ganot &
Thompson 2002). Each territory is related to certain
house structures; for example, the Eisen and Fol fields
are thought to secret the inlet and food-trap filter,
respectively. The tail epidermis is a single layer
of squamous epithelium. Left and right protrusions
make fins (Fig. 10W). This contrasts to the ascidian
larva, whose dorso-ventral fins are made of acellular
materials.
Nervous system
The nervous system of O. dioica shows significant
complexity, and has been intensively described
(Olsson et al. 1990). The brain (or sensory vesicle)
is present in the antero-dorsal region (Fig. 10I,J). A
cavity on the left side of the brain (Fig. 9F,10A) con-
tains a statocyte that senses gravity. The statocyte
contains a presumed calcareous statolith, and is con-
nected to the brain wall via a stalk and via some
sensory cilia emanating from the brain wall (Holmberg
1984). The whole brain consists of approximately 70
© 2008 The Author
S250 H. Nishida

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Journal compilation © 2008 Japanese Society of Developmental Biologists
 Development of Oikopleura S251

Fig. 10. Anatomy of stage 6 juveniles. (A–O) Serial optical sections. (A–E) Dorsal views of a juvenile from shallow to deep focal plane.
Bar, 50 mm. (F–I) Right views. (J, K) Left views. (L–O) Ventral views. Blue arrows indicate seawater current. Red arrows represent flow
of food. Seawater exits from the spiracles (blue circles). Fecal pellets are discarded from the anus (red circles). (P–V) Serial resin
(Technovit) sections from anterior to posterior levels viewed from the posterior side. Dorsal is up. Approximate level of each section is
indicated at the bottom of (E). (W) A section of the tail. Notochord is a tube with a lumen. Nerve cord is on the left of the notochord.
Each of the dorsal and ventral epidermises is lined by a single flattened muscle cell. See details in text.

cells (Olsson et al. 1990; Søviknes et al. 2005). Axons

from the brain are connected to sensory cells present
in mouth and pharynx regions, namely, upper lip cells,
lower lip cells, pharynx cells, and ventral sense organ
cells. The ventral sense organ contains 30 cells and
is suggested to be an olfactory organ (Bollner et al.
1986). In addition, a pair of bilateral epidermal
sensory bristles, the Langerhans receptors, project
out of the trunk (Fig. 10E) and probably sense
mechanical stimuli. These cells receive neurite projec-
tion from a single cell in the tail ganglion (Holmberg
1986). The descending axons from the brain are con-
nected to the spiracle to control its ciliar movement,
and to some epidermis cells in the Eisen and Fol
regions, plausibly to control house production.
The tail shows a remarkable 90° counterclockwise
rotation relative to the trunk when viewed from the
posterior, with neural tube on the left side as men-
tioned in the section on embryogenesis. A thick
caudal nerve runs posteriorly beyond the stomach
(Fig. 10A), turns in the ventral direction, and inner-
vates the tail ganglion (approximately 30 cells
including glial cells; Søviknes et al. 2005) on the left
side of the proximal region of the tail (Fig. 10J,L). A
nerve cord runs along the left side of the tail with
sporadic distributions of neuronal cell bodies
(Fig. 10E, approximately 30 cells; Søviknes et al.
2007). On the left side of the tail nerve cord, a small
hollow is surrounded by four (lateral and medial)
ependymal cells in transverse section (Holmberg &
Olsson 1984). The nerve cord innervates each dorsal
or ventral muscle cell by a single axon of a cholinergic
motor neuron (Flood 1973, 1975; Nishino et al. 2000;
Søviknes et al. 2007). Recently, the spatiotemporal
pattern of neurogenesis in O. dioica was described in
detail (Søviknes & Glover 2007).
Heart
Hemolymph circulates within the body through blood
vessels without endothelial cells, constituting an open
blood system. There are no blood cells in the
hemolymph. The heart is present ventrally between
the left and right stomachs (Fig. 10M). It beats very
fast, so it is easily recognizable in a living animal. The
anatomical description of the Oikopleura heart is
poor so far, and further investigations are definitely
required.
Notochord
The notochord is a synapomorphy characteristic of
chordates. It is present in the center of the tail through-
out its length. The notochord is a tube with a
continuous lumen surrounded by flattened notochord
cells, and serves as an axial endoskeleton
(Fig. 10E,W). It is surrounded by basement membrane
and outer fibrous extracellular matrix (Olsson 1965).
The notochord consists of 19 disc-shaped cells plus
one spherical terminal cell (t cell) at hatching, and
they further divide to form flattened epithelial sheet
surrounding the central lumen during larval develop-
ment (Fig. 10W; Nishino et al. 2001); in contrast, an
ordinary ascidian larva has 40 notochord cells that do
not divide further after hatching. The t cell is present
at the posterior tip of the notochord throughout life
(Fig. 10E) and is never integrated into a notochord
rod. Its function is as yet unknown.
Muscle
The dorsal and ventral epidermis of the tail is lined
with a single flattened layer of non-fused muscle cells
(Fig. 10E,W; Nishino et al. 2000). At hatching, a single
row of eight muscle cells is aligned along the anterior–
posterior axis on each side of the tail. During larval
development, two additional small muscle cells of
unknown origin appear at the tip of the tail (Nishino
et al. 2001). Thus, 10 muscle cells line each side. This
contrasts to ordinary ascidian larvae, which have 18 to
21 muscle cells arranged in three rows on each side.
Muscle fibers are present only on the inner side of
each muscle cell, and the epidermal side of the cells
is filled with mitochondria. Each muscle cell is inner-
vated by a single axon of a different cholinergic motor
neuron (Flood 1973, 1975; Nishino et al. 2000;
Søviknes et al. 2007). Cell bodies of ten pairs of the
bilateral motor neurons are present along most of
the length of the tail nerve cord.
Digestive tract
Concentrated food particles with some seawater is
introduced into the mouth from the food-trap filter of
the house (Figs 1A,B,10B,K,P). Within the pharynx,
the food is trapped by mucus secreted from the endo-

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Journal compilation © 2008 Japanese Society of Developmental Biologists
S252 H. Nishida

style (Fig. 10I,Q) and brought into the esophagus

(Fig. 10B,J,K, red arrows). The seawater goes
ventrally to a pair of spiracles (Fig. 10B,D,K,S,T), gill
apertures that open ventrally and directly to the
outside of the body. A ciliary band just inside the
opening drives the water current within the body to
draw food particles into the pharynx, and is readily
visible in living animals.
The digestive tract shows remarkable left–right
asymmetry. Food particles progress into the esopha-
gus, left stomach, and right stomach via a connection
by means of the inside cilia (Fig. 10K,H,U, red arrows).
Then processed food is delivered into intestine
(Fig. 10F,G), rectum, and anus (Fig. 10E,H,M,N,T).
Fecal pellets are discarded ventrally from the anus.
Each region of the digestive tract has a distinct thick-
ness of epithelium and shows specific fine-structure
characters reflecting different physiological roles
(Burighel et al. 2001).
In the tail of newly hatched larvae, an endodermal
strand is present on the right of the notochord
(Fig. 4N). According to Delsman (1912), most cells of
the endodermal strand are absorbed into the trunk
and give rise to a pair of oral (or buccal) glands
(Fig. 10C), although this fact needs verification. Only
two cells of the endodermal strand remain in the tail
and give rise to two subchordal cells in the adult
(Fig. 10E). The functions of oral gland and subchordal
cells remain elusive. The presence, number, and dis-
tribution of subchordal cells provide good criteria for
identifying species among the Oikopleurinae. Interest- Fig. 11. Juvenile development from the end of day 1 (after tail
ingly, only members of the genera Oikopleura, Folia, shift) to day 5 (sexual maturation) at 20°C. Bar, 1 mm. Photos are
and Stegosoma with oral gland and subchordal cells shown at the same scale. On day 5, the animals mature sexually,
show bioluminescence of the house (Galt & Fenaux and male and female become distinguishable by the gonad
filled with sperm or eggs. They spawn on day 5. See details
1990).
in text.

Juvenile development and gametogenesis

After the tail shift and initiation of feeding on the first
day, juveniles grow rapidly (Fig. 11). A gonadal rudi-
ment is present in the posterior-ventral position and
grows continuously to extend along the posterior epi-
dermis and eventually occupies the posterior half of
the body. Initially on day 5, males and females cannot
be distinguished. Sperm and oocyte formation is
remarkably rapid, taking a few hours (Nishino &
Morisawa 1998). Fine structure during spermatogen-
esis is described in detail in Martinucci et al. (2005).
Spermatogenesis proceeds in a highly synchronized
way in the whole testis (syncytium), and then a number
of sperm are produced.
Oogenesis proceeds in a rather unusual way. The
whole ovary is a single syncytium, called a coenocyst
(Ganot et al. 2007a,b) Oogenesis is divided into
five phases. After the proliferation of germ nuclei in
a common cytoplasm connected via cytoplasmic
bridges, the fate of each nucleus is determined to be
meiotic nucleus or nurse nucleus at phase 1 on day 2,
but all nuclei remain within the common cytoplasm.
Only meiotic nuclei are destined to be oocytes.
Meiotic nuclei enter into the zygotene stage, while
nurse nuclei start DNA synthesis to become polyploid,
gradually increasing nuclear size in phase 2. In phase
3 on day 4, meiotic nuclei are arrested at prophase I.
The coenocyst cytoplasm increases rapidly by active
transcription of nurse nuclei. In phase 4, some meiotic
nuclei are eventually selected to be oocytes, and grow
so that the cytoplasm of the coenocyst is transferred
through a ring canal. The ring canal marks the future
vegetal pole of the egg. Meiosis is resumed. In final
phase 5, full-grown oocytes are arrested at metaphase

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Journal compilation © 2008 Japanese Society of Developmental Biologists
 Development of Oikopleura
I (Fig. 11, day 5 female). Other nuclei undergo apop-
tosis. The size of full-grown oocytes is constant, and
the number of oocytes produced by a single female
depends on the available amount of food and thus on
the final volume of the coenocyst. Efficient numerical
adjustment of oocyte production to environmental
resources is important to the fitness of this species.
How animals select an adequate number of meiotic
nuclei to become oocyte nuclei during phase 4 is of
interest, but unknown.
Under natural conditions, oogenesis proceeds as
described above, and the animals spawn on day 5
without a specific spawning cue in lab culture.
However, when animals are stressed, for example by
high population density, they produce eggs earlier
than normal. In this case, the coenocyst is smaller and
the number of eggs becomes less. At spawning, the
gonad of the female bursts via rupture of the gonadal
wall at the specialized spawning anlagen on the
dorsal side of the gonad (Ganot et al. 2006). In con-
trast, sperm come out of a small hole in the sperm
duct on the dorsal side, and spawning lasts several
minutes. Animals of both sexes die a few hours after
spawning; thus the animal is semelparous.
Perspective
O. dioica is characterized by its simplified life habit
and organization, and shows the following features,
some of which are eminently advantageous for devel-
opmental and genetic studies: (i) it is a swimming
tadpole up to the adult stage; (ii) it is abundant in the
ocean; (iii) it is dioecious and semelparous; (iv) it has
quick development and a short life cycle; (v) closed
and inland culture over generations is feasible; (vi) it is
transparent; (vii) it has a small cell number in embryos
and adults; and (viii) it has a small genome and high
density of genes (one gene per 5 kb).
Taking advantage of the extremely short life cycle of
O. dioica, genetic approaches involving transgenesis
and mutagenesis would be fruitful. The syncytial
coenocyst of the female gonad is a good candidate for
a DNA injection site to yield a large number of off-
spring with introduced DNA. Maintenance of frozen
sperm will also facilitate studies using transgenesis.
The high density of genes and assembled genome
sequences will greatly help mutagenesis studies.
Enhancer trap and insertional mutagenesis with the
transposon Minos have recently succeeded in
ascidians (Sasakura et al. 2005; Awazu et al. 2007;
reviewed in Sasakura 2007). Minos should also work in
appendicularians. Mutagenesis using transposons
could facilitate the identification of mutated genes in
the assembled genome sequences. Therefore, it will
Journal compilation © 2008 Japanese Society of Developmental Biologists
S253
be a good start with this kind of approach in future
studies.
Several particularly interesting issues will make
good targets for studies on O. dioica:
Establishment of chordate body plan and cell fate
specification during embryogenesis
These have been extensively investigated in ascidians
(Satoh 1994; Nishida 2005). Comparison of these pro-
cesses in O. dioica would be interesting because its
eggs do not show directed movement of egg cyto-
plasm just after fertilization, which is required to
establish the secondary embryonic axis in ascidians.
In addition, cell fate restriction is completed much
earlier: at the 32-cell stage rather than at the 110-cell
stage in ascidians.
Left–right asymmetries
The cleavage pattern shows left–right asymmetry as
early as the 4-cell stage. Juveniles and adults also
show remarkable left–right asymmetries in the tail and
digestive tract. What factors are involved in the gen-
eration of these asymmetries and how are early and
late asymmetries causally related?
Pattern formation of oikoplastic epithelium
The epidermis is spatially patterned for the secretion
of the intricate house. Such an elaborate pattern
would involve various processes of pattern formation.
Analysis of these processes will contribute to our
understanding of how organisms create precise pat-
terns to generate highly complex but well organized
structures.
Polyploidy of somatic cells
How do cells transit from ordinary cell division to DNA
synthesis without cell division after the tail shift? What
is the cue, and how are the specific numbers of
endocycles of different cell types controlled?
Sex determination
O. dioica is the sole dioecious species so far reported
in the subphylum Tunicata. Therefore, this character is
apparently derived. Nothing is known about sex deter-
mination. Is sex genetically controlled? How did the
sexes diverge during evolution? Was the evolutional
transition from hermaphroditic to dioecious state pros-
ecuted by a few trivial steps? These questions are
© 2008 The Author
S254 H. Nishida
fundamental to an understanding of the evolution of
sex in animals.
Oogenesis
Oogenesis proceeds in a single giant syncytium, the
coenocyst. How oocyte production is adjusted to
the available resources is unknown. How are fates of
nurse cell and meiotic nuclei specified, and then how
do animals select an adequate number of meiotic
nuclei to become oocytes?
Control of swimming modes
The swimming pattern of larvae is developmentally
controlled. In addition, three kinds of swimming mode
are distinguishable, with distinct frequency and curva-
ture of tail beat, in juveniles and adults. Juveniles in
the house, outside the house, and expanding the
house show different swimming modes. How is swim-
ming mode switched by a neural circuit with a small
number of neuronal cells?
In addition, detailed further comparison of the
genomes of appendicularians, ascidians, and
Amphioxus will shed light on the evolutionary history
and mechanisms of chordate evolution. In conclusion,
O. dioica is an attractive organism for developmental,
evolutionary, and physiological studies of chordates,
offering considerable promise for future genetic
studies.
Acknowledgments
I thank Dr D. Chourrout for inviting me to his lab and
teaching me how to culture Oikopleura. Thanks are
also due to the members of the Misaki Marine Biologi-
cal Station and the Seto Marine Biological Laboratory
for help in collecting Oikopleura to start our culture.
I am grateful to Dr T. Stach and his colleagues for
sharing their results before publication. I also thank Dr
A. Nishino for critical reading of the manuscript. Some
of the work described here was supported by a
Grant-in-Aid from MEXT (17657073).
Conflict of Interest
No conflict of interest has been declared by H.
Nishida.
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