Reference: Biol. Bull. 221: 126 –136. (August 2011)
© 2011 Marine Biological Laboratory
Pattern of Cell Proliferation During Budding in the
Colonial Ascidian Diplosoma listerianum
HELEN NILSSON SKÖLD1,*, THOMAS STACH2, JOHN D. D. BISHOP3, EVA HERBST3,
AND MICHAEL C. THORNDYKE1,4,5
1
Marine Ecology-Kristineberg, University of Gothenburg, Kristineberg 566, 450 34 Fiskebäckskil,
Sweden; 2Freie Universität Berlin, Department of Biologie, Animal Systematics and Evolution, KöniginLuise-Str, 1-3, 14195 Berlin, Germany; 3Marine Biological Association, Citadel Hill, Plymouth, UK;
4
School of Biological Sciences, Royal Holloway University of London, Egham, Surrey, UK; and
5
Royal Swedish Academy of Sciences
Introduction
Abstract.
Many invertebrates reproduce asexually by
budding, but morphogenesis and the role of cell proliferation in this diverse and nonconserved regeneration-like process are generally poorly understood and particularly little
investigated in didemnid ascidians. We here analyzed cell
proliferation patterns and telomerase activity during budding in the colonial didemnid ascidian Diplosoma listerianum, with special focus on the thoracic bud where a new
brain develops de novo. To help define developmental
stages of the thoracic bud, the distribution of acetylated
tubulin was also examined. We found extensive cell proliferation in both the thoracic and abdominal buds of D.
listerianum as well as higher telomerase activity in bud
tissue compared to adult tissues. In the parent adult, proliferation was found in various tissues, but was especially
intense in the adult esophagus and epicardial structures that
protrude into the proliferating and developing buds, confirming these tissues as the primary source of the cells that
form the buds. The neural complex in the thoracic bud
forms from a hollow tube that appears to separate into the
neural gland and the cerebral ganglion. Whereas most of the
bud undergoes proliferation, including the hollow tube and
the neural gland, the cerebral ganglion shows little or no
proliferation. Pulse-chase labeling experiments indicate that
the ganglion, as well as the myocardium, in adult zooids are
instead composed of postmitotic cells.
Regeneration involves resumption of morphogenesis in
the adult body. Regeneration and repair of the nervous
system is limited in vertebrates. However, in tunicates,
which are close relatives of vertebrates within the phylum
Chordata, regeneration of adult tissues, including the brain,
is common. For example, the entire neural complex of the
solitary tunicate Ciona intestinalis is replaced completely 4
weeks after ablation (Bollner et al., 1995, 1997). Many
colonial tunicate taxa routinely generate whole new individuals from adult tissue in a budding process responsible
for colony survival and growth (Nakauchi, 1982; Satoh,
1994). Despite widespread and increasing interest in stem
cell biology, tissue renewal, and aging, there have been
surprisingly few detailed studies of the cellular and molecular mechanisms involved in vegetative propagation by
budding in tunicates, the closest relatives to vertebrates that
undergo natural adult cloning (Dunn et al., 2008; Nilsson
Sköld et al., 2009), including the de novo formation of a
new brain. Moreover, while there is great diversity between
ascidian families in the detailed modes of budding, the
overall bud morphogenesis has underlying similarities in
different ascidians (Nakauchi and Kawamura, 1986; Satoh,
1994). Buds normally inherit the parental epidermis, but the
remaining bulk of the new tissue derives from stem-cell-like
cells that can arise, depending upon the taxon, from several
different adult tissues (Nakauchi, 1982). There may also be
great plasticity in developmental routes for bud formation
even within the same species (Voskoboynik et al., 2007).
This diversity is markedly different from larval development, which is highly conserved among all deuterostomes.
Received 6 October 2010; accepted 17 December 2010.
* To whom correspondence should be addressed. E-mail: helen.skold@
marecol.gu.se
Abbreviations: BrdU, bromodeoxyuridine; TRAP, telomeric repeat amplification protocol.
126
BUDDING IN DIPLOSOMA LISTERIANUM
Classically, two basic modes of cellular mechanism are
distinguished in budding and regeneration events: epimorphosis, involving cell proliferation, and morphallaxis. In
morphallaxis, the new tissues are formed by cell migration
or direct transformation from existing cells without local
cell proliferation. In oligochaetes and sea stars, morphallaxis has a well-established role in regeneration and asexual
reproduction (Martinez et al., 2005; Hernroth et al., 2010).
In hydra, although regeneration after injury is morphallactic, budding instead involves local cell proliferation (Holstein et al., 1991). In the colonial ascidian Botryllus schlosseri, blood-borne stem cells are involved in budding (Laird
et al., 2005), and blood cells of the closely related Botrylloides leachi and Botrylloides violaceus proliferate (Ermak,
1981; Brown et al., 2009). However, cell proliferation levels are not particularly high in the developing buds of
botryllines (Rabinowitz and Rinkevich, 2004; Rinkevich et
al., 2007; Brown et al., 2009), and a recent conclusion is
that most cell proliferation occurs in the blood-borne stem
cells before they form the buds (Brown et al., 2009). In
Polyandrocarpa misakiensis, which belongs to the same
family within the Stolidobranchia, the Styelidae, as the
botryllines, budding involves mostly cell migration and
transformation of epithelial cells (thus morphallaxis), but
also some degree of cell division (Kawamura and Nakauchi,
1986; Kawamura and Fujiwara, 1994; Kawamura et al.,
1995). There have so far been no cell proliferation analyses
in the more distant and, in this respect, considerably less
well studied didemnid ascidians within Aplousobranchia.
Here, we investigate basic developmental processes and
cell proliferation patterns during adult budding in the colonial tunicate Diplosoma listerianum (Milne-Edwards, 1841).
This species belongs to the Didemnidae, a family in which
zooids form two morphologically distinctive buds—a thoracic
bud producing the new branchial basket and brain, and an
abdominal bud producing the new gastrointestinal tract and
heart. Here we especially focus on the thoracic bud for better
understanding of how the new brain is formed. Individual
asexually propagating clones of D. listerianum have been
maintained in our cultures for 19 years, so the cells forming the
buds in this species may also have characteristics for long-term
self-renewal such as up-regulation of telomerase activity, as
described for B. schlosseri (Laird and Weissman, 2004; Laird
et al., 2005). We therefore included comparative analysis of
telomerase activity in excised buds versus adult zooid tissue. To enhance a classification of the major developmental
stages of the thoracic bud, we also documented the expression pattern of acetylated tubulin as a marker for ciliation.
Materials and Method
Study animal and culture process
Experiments were carried out using laboratory cultures of
Diplosoma listerianum originally derived from wild United
127
Kingdom populations as described by Ryland and Bishop
(1990). Colonies kept in reproductive isolation were grown
on glass microscope slides in 800-ml tanks, fed with Isochrysis galbana and Rhinomonas reticulata, and maintained
at 16 °C with a regime of 15-h light and 9-h dark. Regular
reduction of colonies by cutting and transfer onto clean
glass in fresh tanks maintained healthy growth. For details
of culture protocols, see Bishop et al. (2001) and Ryland
and Bishop (1990).
During budding, a zooid of D. listerianum forms one
thoracic bud containing the branchial basket including the
ganglion, and an abdominal bud that includes the stomach
region (Fig. 1A–D). The two zooids derived from a budding
event separate so that the newly budded thorax unites with
the old abdomen, and the old thorax with the new. The old
abdomen is noticeably more pigmented than the newly
budded abdomen (Fig. 1B–D). Budding is not synchronized
between the zooids within a colony, so all stages of the
process may be present at the same time. When transplanted
into a suitable habitat in the field, small colonies from
culture are capable of 10-fold increase in the number of
zooids in a week (AD Sommerfeldt and JDDB, Marine
Biological Association, Plymouth, UK; unpubl.). In laboratory culture, growth is slower, with up to a 4-fold increase
per week at 16 °C. Here the budding process typically takes
3 d from the appearance of small buds visible in a live zooid
to the separation of the two zooids (3.14 d ⫾ 0.074 s.e., 83
budding events).
Telomerase activity (TRAP) assay
Buds and adult zooidal tissue from six different Diplosoma colonies were separated by manual dissection in
RNase-free PBS, quick-frozen in Eppendorf tubes on dry
ice and stored at – 80 °C. The budding materials were at
different budding stages. Telomerase activity using the telomeric repeat amplification protocol (TRAP) was done
using the TRAPeze Telomerase Detection kit S7700
(Chemicon International, Inc. Temecula, CA), according to
the manufacturer’s protocol, as described by Hernroth et al.
(2010). In brief, extracts of buds and adult tissue in each
clone were simultaneously homogenized on ice in CHAPS
lysis buffer including RNase inhibitor (RNasin: Promega,
Madison, WI), incubated on ice for 30 min, and centrifuged
for 20 min (12000 g, 4 °C); the supernatant was quickfrozen on dry ice and stored no longer than 20 d at – 80 °C
before analysis. Protein concentrations were determined
using the Micro BCA Protein Assay kit (Pierce Biotechnology, Inc., Rockford, IL). For the TRAP assay, reaction
mixtures containing 0.1 and 0.02 ␮g of protein from extracts of bud and adult tissue were analyzed for each colony.
As negative controls, extracts were heat-treated for 15 min
at 95 °C, and one reaction mixture was run without any
extract. The TSR8 reference template (0.1 amol l–1) was
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SKÖLD ET AL.
Figure 1. Diplosoma listerianum. (A–D) Zooids at successive budding stages isolated from fixed colonies.
During propagative budding, D. listerianum forms one anterior bud (arrow in A and B) containing the branchial
basket (including the cerebral ganglion: squares in B, C), and a somewhat later-formed posterior bud (arrow in
C) that contains the stomach region. Note the comparatively more pigmented old abdomen in the separating
zooids (B–D, arrowheads). Scale bar: 200 ␮m. (E, F) Telomerase activity analysis using the telomeric repeat
amplification protocol (TRAP assay). TRAP results from reactions containing 0.1 and 0.02 ␮g protein of adult
tissue or bud tissue from the same colony preparation were run in duplicate, with heat treatment (HT) of the two
samples to inactivate telomerase as negative controls. (E) Representative gel. TSR8 (0.1 amol l–1) is a positive
template control and P-D is a negative control containing no extract. (F) The telomerase activity in reactions
containing either 0.1 or 0.02 ␮g protein, expressed as total band intensity per lane, is significantly higher in buds
than in adult extracts (P ⫽ 0.031 as indicated by *, n ⫽ 6).
used as positive control. The reaction tubes were incubated
for 30 min at 30 °C and then amplified by polymerase chain
reaction (15 min hot start at 94 °C, cycled 33 times at 94 °C
for 30 s, at 59 °C for 30 s, and at 72 °C for 1 min) using
HotStarTaq DNA polymerase (Qiagen, Hilden, Germany).
The products were separated by 10% polyacrylamide gel
electrophoresis, stained with SYBR green (Invitrogen), and
visualized under ultraviolet light using a Gel Doc 2000
(Bio-Rad, Hercules, CA). The Wilcoxon signed-ranks test
was used as a nonparametric pairwise test to compare values
of total band intensity (summary of peak density values
from all bands) after background subtraction, in bud versus
adult tissue extracts from each colony preparation, as a
measure of relative telomerase activity (Sigma Stat, ver. 3.5;
Jandel Scientific, San Rafael, CA). The bud and adult material to be compared pairwise were loaded on the same gel.
BrdU labeling, immunohistochemistry, and microscopy
Budding colonies were transferred into 5-cm petri dishes
for bromodeoxyuridine (BrdU) incorporation using the RPN
202 Cell Proliferation kit (Amersham Biosciences, Uppsala,
Sweden). Colonies were initially treated with 25, 100, and
250 mol l–1 BrdU in filtered seawater for 5 h, 1 d, or 3 d at
12 °C. The major difference in labeling was found to
depend on the time of BrdU incorporation rather than the
administered dose, and 100 mol l–1 BrdU was chosen for
standardization. Nonlabeled colonies showed no staining.
Pulse-chase experiments were done by post-incubation of
BrdU-treated colonies in pure seawater. The colonies were
then anesthetized using cold, freshly prepared 0.3% propylene phenoxetol (Clariat GmbH, Sulzbach, Germany) in
seawater as described by Bishop and Sommerfeldt (1996)
BUDDING IN DIPLOSOMA LISTERIANUM
and fixed overnight in 4% paraformaldehyde in filtered
seawater or phosphate-buffered saline (PBS, pH 7.4.) at 4
°C. BrdU incorporation was analyzed by preparing wholemounts of zooids dissected from treated colonies stored in
70% ethanol, or by processing the colonies for histology.
For histology, labeled colonies were rinsed in PBS and
dehydrated in an ethanol series (50%, 70%, 90%, 95%, and
100%) for embedding in paraffin wax. After 100% ethanol,
the colonies were transferred to Histoclear (National Diagnostics, Hessle, UK), 1:1 Histoclear/melted wax (melting
temperature 55–58 °C; from Merck, Darmstadt, Germany),
and finally to 100% melted wax for solidification at room
temperature. Series of sections of 7– 8 m were transferred
onto SuperFrost Plus glass slides. Dried slides were dewaxed in Histoclear and rehydrated in an ethanol series
(100%, 95%, 90%, 80%, 70%, and 50%). Endogenous
peroxidase activity was quenched by a 10-min treatment in
0.3% H2O2, and the slide washed in PBS and processed for
detection BrdU by following the RPN 202 protocol using
nickel in the diaminobenzidine solution to produce black/
brownish staining. For immunohistochemical analysis of
ciliation, a monoclonal acetylated tubulin antibody (clone
6-11B-1 from Sigma-Aldrich, Stockholm, Sweden) and a
secondary peroxidise conjugated anti-mouse antibody were
instead used. Eosin or Mayer’s hematoxylin, both from
Sigma Diagnostics, were used as counterstains. Stained
slides were subsequently dehydrated in an ethanol series
(50%, 70%, 90%, 95%, and 100%) and finally Histoclear
before mounting (Polymount, Polysciences, Inc, Warrington, PA). Slides were analyzed using a Leica microscope adapted for digital imaging with a Canon S40 color
camera and Canon imaging software (Leica Microsystems).
Results
Relative telomerase activity
Analysis of telomerase activity using the TRAP assay
showed slightly but significantly higher labeling intensity in
bands from tissue extracts of buds compared to the corresponding adult zooid tissue extracts from the same colonial
preparation (P ⫽ 0.031 for both 0.1 and 0.02 ␮g protein
reactions, n ⫽ 6), indicating higher telomerase activity in
buds than in adult tissue (Figs. 1E, F). The only modest
increase in telomerase activity in the more concentrated
extracts (0.1 ␮g) compared to in the less concentrated (0.02
␮g) suggests polymerase inhibition, saturation, or both, in
the former.
Cell proliferation pattern in adult zooids
The branchial basket in adult Diplosoma listerianum zooids possesses several zones of active cell proliferation (Fig.
2). The endostyle consists of a series of distinct groups of
cells in eight major longitudinal zones (Burighel and Clo-
129
ney, 1997). BrdU labeling was detected in the heavily
ciliated zones 3 and 5, when animals were incubated in
BrdU for 5 h or 1 d (Fig. 2A), whereas 3-d incubations
revealed incorporation in all endostyle zones. A regular
pattern of positive labeling can be detected in the epithelium
surrounding the stigmata (Fig. 2B). After 5 h of labeling,
individual cells in the most anterior and posterior parts of
the stigmata are stained. After 1 d, labeling has spread
outward from the extreme anterior and posterior points,
resulting in a chevron pattern of labeling of the stigmata in
both adult zooids and late buds. After 3 d, the lateral walls
of the stigmata are also labeled. After 1 d, individual cells in
the epithelium of the oral siphon are positively labeled, as
are cells at the base of the oral tentacles (not shown). In
addition, the epithelium of the branchial basket that surrounds the entrance to the esophagus is positively labeled
after 5 h in BrdU.
In the abdomen, proliferating cells are situated in the
space surrounding the heart (Fig. 2C, arrowhead) and stomach (Fig. 3A, arrowhead). These cells are presumably blood
cells, given that didemnids have an open circulatory system.
The myocardium was unstained after 1 d in BrdU but
stained after pulse-chase labeling (Fig. 2C, D), indicative of
postmitotic cells in the myocardium.
Analysis of cell proliferation during budding indicates
that both the thoracic and abdominal buds that grow out
from the abdominal area undergo massive cell proliferation
during their development (Fig. 2E, arrows). Epithelial cells
around the entrance and exit of the adult stomach also
incorporate BrdU (Fig. 2E, arrowheads). Proliferation is,
however, comparatively higher at the entrance to the stomach after labeling with BrdU for 5 h. The testis is stained
after 5 h and more, in agreement with previous research
using tritiated thymidine to label D. listerianum sperm
(Bishop, 1996). A schematic summary of the proliferation
pattern in adult zooids after 1 d of BrdU labeling is shown
in Figure 2F.
Cell proliferation pattern in buds and adjacent adult
tissues
In addition to the developing thoracic and abdominal
buds, extensive cell proliferation is also seen in the epidermis, esophagus, and epicardium that protrude into the buds
(Fig. 3). When incubated for 24 h in BrdU, the epithelium
of the esophagus is strongly labeled in the area where the
thoracic and abdominal buds are connected to the parental
esophagus (Fig. 3A). Most of the epicardium is strongly
labeled, with the exception of the more basal cells (Fig. 3B,
arrowhead), suggesting presence of more slowly dividing
cells in this region of the epicardium. A schematic summary
of the tissues involved in thoracic bud development is
depicted in Figure 3C.
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SKÖLD ET AL.
Figure 2. Diplosoma listerianum. (A–E) BrdU incorporation in sections of selected adult tissues. (A)
Endostyle (end), cells of zones 3, 5, and 8 show labeling (arrows), while the heavily ciliated zone 1 or the more
polarized cells of the other endostyle zones are unlabeled. (B) Branchial basket: a regular pattern of labeling can
be detected in the anterior and posterior parts of the epithelium surrounding the stigmata. (C) Abdomen of the
adult zooid: BrdU incorporation is evident in blood cells around the heart (arrowhead), but not in the
myocardium (arrow). (D) After pulse-chase labeling with 5 h of BrdU labeling followed by 3 days of pure
seawater, the myocardium also showed BrdU incorporation (arrow). (E) Section of a whole zooid: intense
labeling is seen in both thoracic and abdominal early buds (arrows), epithelial cells around the entrance and exit
of the stomach (arrowheads), and stigmata. In all pictures except D, colonies were fixed directly after 1 d of BrdU
labeling. (F) Semi-schematic summary of the cell proliferation pattern during budding, where incorporation of
BrdU during 1 d of labeling is shown in red. nc ⫽ neural complex; es ⫽ esophagus. Scale bars in A–E, 100 ␮m.
Cell proliferation pattern in the developing neural
complex
The central neural complex in D. listerianum is positioned dorsally, close to the oral siphon and opposite to the
endostyle (which is ventral) (Fig. 4A–C), as in ascidians in
general (Burighel and Cloney, 1997). The ganglion in adult
zooids of D. listerianum is distinctive and spherical, with an
outer cortex one or two cells thick. Just ventral to the
ganglion, the discrete and densely ciliated funnel, or dorsal
tubercle, connects to the branchial basket at one end and
continues at the other into a group of larger globular cells,
the neural gland body. A disk-shaped area of pigmented
BUDDING IN DIPLOSOMA LISTERIANUM
131
Figure 3. Diplosoma listerianum. (A, B) Longitudinal sections of developing thoracic bud (insert in A,
arrow indicates thoracic bud in section of whole zooid), showing intense BrdU incorporation in areas of the
esophagus and in epicardial structures that protrude into the bud, as well as in the outer epithelia that cover the
bud (A, arrows). A population of proliferating cells is also situated more distant from the bud in the space
surrounding the stomach (A, arrowhead). The epicardial structures show noticeably more BrdU incorporation in
the areas adjacent to the bud than in cells more distant from the bud (B, arrowhead shows unlabeled nuclei). (C)
Schematic modified from Groepler (1992, Zoologische Beiträge 34: 3–33), presenting the parental tissues
involved in development of a thoracic bud—i.e., epithelium, epicardium (ep), and esophagus (es)— based on the
results of the BrdU labeling. Cell proliferation is indicated as thicker lines. Scale bar in A, 100 ␮m.
cells is located dorsal to the brain. Cell proliferation was
seen in the ciliated funnel/neural gland after 5 h to 3 d of
incubation with BrdU. The labeling was especially evident
in the area of the ciliated funnel/neural gland close to the
ganglion (Fig. 4C). The adult ganglion itself showed no
proliferating cells. Neither did the neural gland body (sections with 1 d of BrdU incorporation analyzed only).
Analyses of buds at different developmental stages and
after different incubation times in BrdU made it possible to
examine the development of the thoracic bud and neural
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SKÖLD ET AL.
Figure 4. The neuronal complex in adult Diplosoma listerianum. (A) Square indicates position of neuronal
complex in section of whole zooid. (B) Enlargement of the neuronal complex in A shows the ganglion (g)
composed of an outer cortical layer 1–2 nuclei deep and an inner nucleus-free core. Facing inward from the
ganglion is the ciliated funnel (cf), which connects with the cavity of the branchial basket at one side and with
the neural gland body (ngb) on the other side. Facing outward from the ganglion is a group of drop-shaped
pigmented cells (p). (C) After 1 day of treatment with the mitotic marker BrdU, cell proliferation is observed in
the adult neural gland (arrow), whereas the ganglion is unstained. Sections A and B are counterstained with
hematoxylin and section; C with eosin. Scale bars: 100 (A) and 10 ␮m (B, C).
complex in detail. In early thoracic buds (Phase 2–3), there
is a hollow dorsal tube in the area where the nervous system
will be located. After 5 h of BrdU incubation, this tube
shows evenly distributed proliferation (Fig. 5A, arrow). In
Phase 4, when early signs of stigmata can be seen but the
endostyle has not yet reached its typical adult form, proliferation is concentrated in the floor of the dorsal tube,
leaving an unlabeled dorsal area (Fig. 5B, arrow). Whereas
the inner structures of the bud show extensive proliferation,
the epidermis is at this stage less labeled (Fig. 5C, arrows).
In sections of slightly later buds with developed stigmata
(Phase 5), the neural complex now comprises two structures, the neural gland and the ganglion (Fig. 5D, E). In later
buds that morphologically have an adult-like endostyle
(Phases 6 and 7), the ganglion is spherical and the gland/
funnel beneath has the shape seen in the adult. After 3 d of
continuous BrdU incubation, the ganglion in later buds
shows few or no proliferating cells, while the funnel of the
neural gland is highly stained (not shown). However, pulsechase experiments with 5 h of labeling and 3 d in pure
seawater show labeling of the ganglion (Fig. 5F, G). This
indicates that the mature ganglion is composed of cells that
are no longer dividing—that is, postmitotic. Longitudinal
sections reveal an initial BrdU-labeled thickening (Fig. 5H,
arrow) in Phase 3 that will form the more distinct hollow
tube. At this stage and the next stage (Phase 4) the hollow
tube is asymmetrical and leaf-shaped, with the stalk directed
posteriorly (Fig. 5I, arrow). BrdU labeling is evident. In the
next stage (Phase 5), the neural complex separates into the
neural gland and the ganglion, both having elliptical and not
yet spherical shapes (Fig. 5J, in square). The formation of
the neural complex from the original hollow tube during
budding is schematically summarized in Figure 5K, with
BrdU labeling in black.
Ciliation pattern in thoracic bud by presence of
acetylated tubulin
For further examination and staging of development of
the thoracic bud, including the neural complex, we also
analyzed timing of the appearance of ciliation in the endostyle and ciliated funnel by the presence of acetylated tubulin (Fig. 6). Adult ciliation is shown in A–C of Figure 6,
and the progressive ciliation of the endostyle, stigmata, and
ciliated funnel during development of the thoracic bud is
shown in D–G.
Discussion
Coloniality is an asexual reproductive strategy that has
independently evolved in different families within the ascidians. Of those, budding in the didemnid ascidians is
particularly little characterized. Here, we have investigated
basic developmental processes during budding in the colonial didemnid ascidian Diplosoma listerianum. Specifically,
we analyzed cell proliferation patterns and telomerase
BUDDING IN DIPLOSOMA LISTERIANUM
Figure 5. Diplosoma listerianum. Investigation of development of the neural complex by BrdU incorporation during thoracic bud development. (A–E) Frontal/transverse sections of thoracic buds of successive
developmental stages in colonies labeled with BrdU for 5 h (A) or 1 d (B–E). (A) Bud phase 2–3: arrow indicates
the dorsal hollow tube that will become the neural complex. (B, C) Bud phase 4: arrows indicate unstained areas
in the dorsal part of the tube, the developing ganglion, and outer epithelia cells. (D, E) Bud phase 5: arrows in
E indicate labeled nuclei of the ganglion (g) and in the dorsal cells of the ciliated funnel (cf). Free unlabeled cells
are also seen in proximity to the ganglion (arrowhead in E). (F, G) Fully developed thorax: result of pulse-chase
experiment with 5 h of BrdU treatment followed by 3 d of pure seawater, showing labeling of the ganglion
(arrows), but not of the ciliated funnel. (H–J) Longitudinal sections of progressive stages of bud development
after 1 d of BrdU labeling, showing the neural complex located opposite the developing endostyle (end). The
neural complex (nc) originates as a thickening (arrow in H, phase 3), followed by a not yet spherical neural
complex (I, phase 4) that subsequently becomes divided into two compartments composed of the elliptical
ganglion and the ciliated funnel (J, phase 5, in square). Sections A–G are counterstained with eosin and H–J with
hematoxylin. (K) Schematic of the development of the neural complex based on the sections, suggesting
formation of the ganglion and the ciliated funnel by separation of the cells of the initial dorsal hollow tube. Scale
bars: 50 (A–D, F, H–J) and 10 ␮m (E, G).
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Figure 6. Diplosoma listerianum. Analysis of ciliation during thoracic bud development by presence of
acetylated tubulin. (A) Longitudinal section of an adult zooid undergoing budding, showing intense ciliation in
the stigmata of the branchial basket (arrowhead) and in the esophagus, but not in a developing thoracic bud
(arrow). (B) Enlargement of the adult neuronal complex showing the ganglion (g) and the ciliated funnel (c f)
above the cavity of the ciliated branchial basket. (C) The endostyle of a fully developed zooid, also heavily
ciliated. (D–G) Sections of thoracic buds at progressive developmental stages showing absence of ciliation at bud
phase 3 (D). In phase 6 (E, F), stigmata rudiments have just started to become ciliated (arrow in F). In a later
bud, phase 7 (G), ciliation is established in the ciliated funnel beneath the ganglion, plus the branchial basket,
endostyle (end), and stigmata. Scale bars: 10 (A, D–F) and 50 ␮m (B, C).
activity to gain insight into mitotic capacity and the role of
cell proliferation, particularly in thoracic budding including
formation of the new brain. We also investigated the distribution of acetylated tubulin as a marker for cilia. Together,
our data allow us to present the first picture of the cell
proliferation pattern in didemnids, exemplified by D. listerianum, and a classification of the developmental stages of
the thoracic bud (Table 1). In this classification, we have
divided the developmental events into “stages” that are recognizable under stereomicroscopy and more detailed bud or adult
“phases” that are recognizable in histological material.
We show highly organized patterns of continuing cell
proliferation in mature zooids of D. listerianum. From studies based primarily on the unitary stolidobranch Styela
clava, Ermak (1982) reported renewing cell populations in
various epithelia including those of the ciliated funnel,
endostyle, stigmata, esophagus, and stomach; these are areas in which proliferation was also noted in D. listerianum.
This suggests substantial similarity in adult zooid cell and
tissue homeostasis between these two distantly related species.
We also show particularly strong and widespread BrdU
staining in the buds, indicating that cell proliferation and
thus epimorphosis is a major mechanism for budding in D.
listerianum. Our proliferation data clearly confirm the role
of the epithelium, esophagus, and epicardium as sources of
cells during didemnid budding, as suggested earlier by
histological examination lacking explicit proliferation analysis (Berrill, 1935; Brien and Van den Breede, 1948; Kitazawa, 1967; Groepler, 1992). Thus budding in D. listerianum involves multiple tissues. The cells involved could be
either differentiated cells, stem cells, or de-differentiated
cells, and further work is needed to better understand the
relative prevalence of the respective differentiation levels in
the cells involved in D. listerianum budding.
We found telomerase activity in both adult and bud
extracts, but the level of telomerase activity was significantly higher in buds. Our result from D. listerianum is
therefore similar to data obtained from Botryllus schlosseri
(Laird and Weissman, 2004). Since budding in both botryllines, and perhaps particularly in D. listerianum, involves
cell proliferation, elevation of telomerase activity in their
developing buds might enable colonial ascidians to maintain
the proliferative capacity necessary long-term for asexual
135
BUDDING IN DIPLOSOMA LISTERIANUM
Table 1
Stages and major events of thoracic bud development in Diplosoma listerianum
Stages*
1
Phases**
Bud
1
2 Fig. 5A (late)
3 Fig. 5A (early)
2
4 Fig. 5B, C, I
5 Fig. 5D, J
6
3
7
4
5
Adult
1
2
Major developmental events
Evagination and local proliferation of anterior gut epithelium. Protrusions of proliferating epicardial appendages
into the evaginating bud.
Formation and folding of the proliferating inner vesicle. Protrusion into the bud from esophagus.
Lateral pockets formed. Thickening of anterior part of inner vesicle to neural complex rudiment and formation
of the dorsal hollow tube. Endostyle rudiment as a large evagination of inner vesicle opposite to dorsal
hollow tube.
Lateral pockets fuse with inner vesicle to form brachial basket rudiments. Stratification of cells within the
dorsal hollow tube to the formation of dorsal postmitotic cells and ventral mitotic cells.
Ventral mitotic cells of the dorsal hollow tube separate and form the ciliated funnel/neural gland rudiment. The
dorsal postmitotic cells separate and form the ganglion.
Endostyle rudiment takes adult form. Stigmata form in the branchial basket rudiment. Nerve fibers extend from
ganglion. Evidence of cilia*** in branchial basket.
Number of stigmata formations increase. Ciliation of branchial basket, endostyle, and ciliated funnel.
Pigmented cells anterior to ganglion formed. Oral siphon opens.
Filtering. Thorax contracts upon contact. Pigmented siphon. Spherical ganglion. Sperm production.
Thoracic regression phase. No cell proliferation. Increased pigmentation, also in abdomen. Retraction and
disintegration of thorax.
* Stages: morphology recognizable under stereomicroscopy.
** Phases: morphology recognizable in sectioned colonies.
*** Ciliation identified by presence of acetylated protein in sectioned colonies.
propagation. In many mammals, including humans, the
drastic decline in overall telomerase activity is associated
with cessation of somatic growth, onset of somatic senescence, and loss of cellular renewal (Chang and Harley,
1995; Iwama et al., 1998; Kinugawa et al., 2000; Cawthon
et al., 2003; Herbig et al., 2006). However, animals that
constantly grow, like some fish, or have extensive regenerative capacity, like sea stars (Elmore et al., 2008; Hernroth
et al, 2010), show high telomerase activity in adult tissues,
thereby providing proliferative potential.
During asexual budding by colonial tunicates, a new
neural complex is formed from pre-existing adult tissue.
Work on budding in B. schlosseri indicates that a dorsal
tube formed by invagination eventually differentiates into
the ciliated funnel, neural gland, and dorsal organ; “pioneer
cells” delaminate from the dorsal tube, and subsequently
from the neural gland, and migrate to form the cerebral
ganglion, differentiating into neurons (Burighel et al., 1998;
Mackie and Burighel, 2005). The solitary tunicate C. intestinalis is similarly able to rebuild a complete neural complex after excision. The cellular source (or sources) for this
neural regeneration is not clear, but stem-cell-like hemocytes and dorsal strand tissues have been suggested (Bollner
et al., 1997). In a late (Phase 5) bud of D. listerianum, some
nuclei incorporating BrdU were observed in the ganglion
close to the ciliated funnel (Fig. 5E). While it is possible
that there is a proliferation zone there, offering some support to the possibility that the ciliated funnel/neural gland
gives rise to cells forming the ganglion, the timing of the
overall cell proliferation pattern differs widely between the
ganglion and the ciliated funnel. From our work, it appears
that the ganglion is mainly composed of postmitotic cells, in
contrast to the ciliated funnel. The differences in BrdU
labeling in the cerebral ganglion compared to the ciliated
funnel suggest that in D. listerianum both the ganglion and
the ciliated funnel derive from cells of the dorsal hollow
tube. Cell-tracking experiments would, however, be necessary for a definite understanding of the de novo formation of
ascidian brains.
Our results demonstrate regionalized cell proliferation in
the adult and extensive cell proliferation in the buds. The
ganglion, which forms early, and the heart are composed of
cells that do not divide in the adult zooid—that is, postmitotic cells. Extensive proliferation was found in the epithelia
and in the apparently pluripotent adult esophagus and epicardial structures that protrude into the buds and into the
inner structures such as the brachial basket of the new zooid,
strongly supporting their contributions in bud formation.
We further show that budding tissue has relatively higher
telomerase activity than adult tissue, providing elevated
mitotic potential that should be important for the capacity
and longevity of the epimorphic budding process in D.
listerianum.
Acknowledgments
We thank Christine Wood, Carolina Oweson, and Maria
Asplund for technical assistance. The study was supported
136
SKÖLD ET AL.
by BBSRC grant 111/COD15494 to MT and JDDB, and by
The Swedish Research Council, C.F. Lundströms Stiftelse,
Helge Ax:son Johnsons Stiftelse, Craafordska Stiftelsen,
and Journal of Cell Science to HNS. Additional support is
acknowledged from the Royal Swedish Academy of Sciences and HMK Wallenbergs Fonden to MT.
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