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Organisms, Diversity & Evolution 5 (2005) 15-24
ORGANISMS
DIVERSITY &
EVOLUTION
www.elsevier.dejode
Comparison of the serotonergic nervous system among Tunicata:
implications for its evolution within Chordata
Thomas Stach
Royal Swedish Academy of Sciences, Kristincberq's Marine Researchstation, 45034 Kristineberq. Sweden
Received 3 March 2004; accepted 4 May 2004
Abstract
Using immunohistochemistry in combination with confocal laser scanning microscopy, the serotonergic nervous
systems of major tunicate taxa were studied in three-dimensional detail. Organisms analyzed included
aplousobranchiate, phlebobranchiate, and stolidobranchiate ascidian larvae, appendicularian juveniles and adults,
and doliolid oozooids. Outgroup comparisons to notochordates showed that the serotonergic nervous system of the
last common ancestor of Chordata consisted of two elements: (i) an anterior concentration of serotonergic cell bodies,
and (ii) a fiber network that followed posteriorly and gave rise to fiber tracts that descended towards the effective
somatic lateral musculature. Within Tunicata, the nervous systems of Appendicularia and Aplousobranchiata appear
serotonin-reduced or negative. This could be interpreted as a heterochronic reduction and a synapomorphy between
Appendicularia and Aplousobranchiata. In this hypothesis, free-living Appendicularia are derived within Tunicata,
and a biphasic life cycle with a free-swimming larva and a sessile, ascidian-like adult is most parsimoniously
reconstructed for the last common ancestor of Tunicata. The close spatial relation of the serotonergic cell cluster with
the statocyte complex suggests a function as an integrative control center for the coordination of locomotion. A similar
anterior concentration of serotonergic nerve cells is known from tornaria larvae.
CO 2005 Elsevier GmbH. All rights reserved.
Keywords: Chordate: Urochordate; Tunicate; Phylogeny; Seroton in; 5-HT
See also Electronic Supplement at: http://www.senckenberg.de/odesj05-02.htm
Introduction
Structures of the nervous system can preserve
phylogenetically informative characters even in other?
wise disparate taxa (e.g. Hanstrorn 1928; Hessling and
Westheide 2002; for Chordata see reviews by Nielsen
1999; Meinertzhagen and Okamura 200 I; Nieuwenhuys
2002). Serotonin is an ancient neurotransmitter present
in most bilaterian taxa (see; e.g., Hay-Schmidt 2000). To
date, the organization of the serotonergic nervous
E-mail address:thomas.stach(iI;kmf.gu.se (T. Stach).
1439-6092/$ - see front matter ~-:: 2005 Elsevier GmbH. All rights reserved.
doi: 10.1016/j.ode.2004.05.004
system in Chordata is known for Petromyzontida (e.g.
van Dongen et al. 1985; Hay-Schmidt 2000), Gnathos?
tomata (e.g. Ekstrom et al. 1985; van Mier et al. 1986),
and, to some extent, Cephalochordata (Holland and
Holland 1993; Candiani et al. 200 I; Morikawa et al.
2001). For the craniate taxa functional data on the
serotonergic part of the nervous system are also available
(van Mier et a!. 1986; Grillner and Matsushima J991).
However, for the third major taxon of the Chordata, the
Tunicata, the serotonergic nervous system remains hardly
known. Pennati et al. (200 I) demonstra ted the presence of
serotonin in the larval brain and also the tail of Phallusia
16 T . Stach / Organisms. Diversity & Evolution 5 (2005) 15-24
mammillata, a phlebobranch ascidian species. Previous
attempts to study the distribution of serotonin using
immunofluorescence in adult nervous systems of several
ascidian species have returned largely negative results
(Fritsch et al. 1982; Georges 1985).
Tunicates display an enormous diversity of life cycles,
and recent molecular studies led to two conflicting
hypotheses concerning the phylogenetic position of
Appendicularia. Wada (1998), Swalla et al. (2000) , and
Winchell et al. (2002) supported the hypothesis that
Appendicularia were the sister group to the remaining
tunicatcs, and thus suggested that the last common
ancestor of Tunicata was free-living . Stach and Turbe?
ville (2002, in press) included Aplousobranchiata in their
analyses and found that Appendicularia were the sister
group to aplousobranchiate ascidians. Besides the
analysis of 18S rDNA sequences these authors pointed
out that aplousobranchiate ascidians and appendicular?
ians shared a horizontally oriented tail in their early
developmental stages. However, this character is also
found in Perophoridae, a taxon that is nested within the
phlebobranchiate ascidians. Thus there are currently no
undisputed morphological characters known that sup?
port either of the two hypotheses.
Tunicata is one of three major taxa of Chordata.
Chordata, Echinodermata, Hemichordata, and possibly
Xenoturbella sp. (Bourlat et al. 2003), constitute the
Deuterostomia . Knowledge of the last common ancestor
of Tunicata influences our perception of the last
common ancestor of Chordata, which in turn is
important to understand the evolution of Deuterosto?
mia . A major controversy in the evolution of Deuter?
ostomia concerns the phylogenetic position of
Hemichordata. Molecular studies strongly support a
sister group relationship between Hemichordata and
Echinodermata (recently reviewed in Jenner and Schram
1999; Winchell et al. 2002). Morphological studies often
find arguments to support a sister group relationship
between Enteropneusta and Chordata (e.g. Ax 200 I;
Nielsen 200 I; Lowe et al. 2003; Nezlin and Yushin
2004). though not all morphological studies agree.
The potentially conservative aspect of the nervous
system, the ubiquitous distribution of the serotonin
neurotransmitter, and the scarcity of data on the
serotonergic nervous system in tunicates, make this
system a promising candidate to contribute to phyloge?
netic discussions . Here. I present a comparative study of
the serotonergic part of the central nervous system for
phlebobranch, stolidobra nch, and aplousobranch asci?
dians, as well as for appendicularians and doliolids . The
results allow the reconstruction of details of the
serotonergic nervous system for the last common
ancestor of Tunicata and Chordata. show some
similarities to the serotonergic nervous system of
tornaria larvae, and in addition suggest an integral
function in chordate locomotion .
Colour versions of Figs . I??A and 6, as well as
additional illustrations are available from an Organisms
Diversity and Evolution Electronic Supplement
(www.senckenberg.de/odes/05-02.htm).
Material and methods
Specimens
Table I lists the species examined in the present study
along with information on the respective number and
stages of animals analyzed.
Origin of animals, fixation, storage
Solitary ascidians: Gametes of two individuals were
dissected and mixed on a 220 urn mesh gauze on top of a
beaker containing filtered seawater (pore size 0.4 urn).
Thus, fertilized eggs sank to the bottom and superfluous
sperm in the water column was decanted three times.
Development of animals was followed under a dissecting
microscope. Timed stages were fixed in 1.5% parafor?
maldehyde in seawater for at least 2 h to a maximum
fixation time of 12h. If animals were stored, they were
washed in distilled water for 10 min, transferred to
100%, methanol, and kept in a refrigerator at 4 ?C. All
animals were used within 6 weeks after fixation . Larvae
of colonial ascidian species were dissected from the atria
or brooding pouches, respectively, and processed in the
same way. Planktonic species were caught with a 15-min
drift tow at about 5 m depth . Mesh size of the plankton
net was 220 um. All animals were immunolabeled
against serotonin for microscopic examination.
Immunohistochemistry
If necessary, animals were rehydrated in distilled
water for 10min. The larval tunic of ascidian species
proved to be a major obstacle against antibody
penetration. Various methods - e.g., acetone treatment,
microwaving, sonication , freeze-and-thaw cycles, pro?
tease digestion - were evaluated as to their effectiveness
in enhancing antibody penetration. The most successful
procedure was as follows. Larvae were incubated for
5 min in 5% cellulase (Sigma, St. Louis, Missouri, USA)
in phosphate-buffered saline (PBS, 0.15 M , pH 5.5) at
37 "C. After subsequent heating to 72 QC, larvae were
cooled down on ice and washed with PBS (0.15 M, pH
7.4) 3 times for 10min each. Prior to incubation with the
primary antibody, all specimens were treated for I h at
room temperature in PBS (0.15 M, pH 7.4) containing
0.1'Yo Triton-X-IOO, 2% Bovine Serum Albumine
(I3SA), and 0.05?;(1 NaN,. Incubation in primary anti?
body anti-serotonin (Sigma, St. Louis, Missouri, USA)
T. Stach I Organisms, Diversity & Evolution 5 (2005) 15-24 17
Fig. I. (A- D) . Herdmania momus (Pyuridae, Stolid obran chiata). Larval sta ge 21.5 h pos t-fert iliza tion, approx . 20 "c. (A) G ener al
ligh t-mi cr oscopic aspect. (B) Sup erimposed ima ges o f tran smitting ligh t-m icroscopic image with pr oject ion of sero to nergic ner vou s
sys tem; scro to nergic ner vou s system: back ground-rem oved part of a projection of 104 confocal optica l sec tions tak en a t 0.5 11m
int ervals. (C) Detail s of the sero to ner gic nervou s sys tem show ing the entire project ion of optica l sec tions used in (B). (D )
Serot on ergic ner vou s system, o blique ventra l aspect ; projection as in (B,C) but rot ated by 45". (E- H) Ascidia interrupta (Ascidiid ae,
Phlcbobran ch iata ). La rval stage 2 1.5 h post -fertilizat ion , approx. 20 "c. (E) General light-microscopi c as pect. (F) Superimposed
images o f tr an smitting light-microscopic image with projection of seroto nergic nervou s system; sero to nergic nervous system:
background-removed part of a projecti on of 93 confocal o ptica l sectio ns tak en at 0.5 11m in tervals. (G ) Det ails of the serot onergic
nerv ou s system showing the entire pr oject ion of optical sections used in (F). (H) Dorsal aspect ; pr ojecti on as in F, G , but rotated by
90". Abbreviations: nn = fiber net , oc = ocellus, pa = papilla, pp = posterior projection , serNS = sero to ncrgic nervous syst em,
sc = sc ro to nergic cell, st = statocyte complex, tu = tunic.
--------------------- - - --- - - - - - - - - - -- - - - -- -
18 T. Stach I Organ isms. Diversit y & Evolutio n 5 (2005) 15-14
Fig. 2. Oozooid of Doliolum nationalis labeled with antibodies against serotonin; projections of confocal optical sections. (A)
Overview, anterior to the right, dorsal to the top; 97 optica l sections taken at 5 urn intervals; [ ] = area enlarged in (B). (B) Dorsal
ganglion; 67 optical sections taken at 1.511m intervals. Abbreviations: cf = ciliated funnel , dg = dorsal ganglion, in = intestine,
nn = fiber net , pp = posterior projection, sc = serotonergic cell, I-VIII = muscle bands .
Fig. 3. Clave/ina oblonqa larva released from atrium. (A) General light-m icroscopic aspect. (B) Confocal optical sect ion of same
larva shown in (A) labeled with antibodies against serotonin; no projection shown, because outer tunic was labeled at various
po sition s, thereby masking internal signal ; representative section from a series of 57 confocal optical sections taken at 111m intervals.
Insets: area indicated by [] enlarged, two confocal optical sections taken 7 urn apart ; arrows point to immunopositive cells lining the
sensory vesicle; double arrowheads point to immunopositive cells in the pharyngeal epithelium. Abbreviations: en = endostyle,
oc = ocellus, os = oral siphon, pa = papilla , so = stomach, st = stutocyte complex, ta = tail , tu = tunic .
was carried out in preincubation fluid at a dilution of
approximately I: 100 for 12-24 h in a humid chamber at
room temperature. Animals were washed six times for
10 min in PBS (0.15 M , pH 7.4). Incubation in secondary
antibody Alexa Fluorqc 546 goat anti-rabbit (Molecular
Probes, Eugene, Oregon, USA) was carried out in
preincubation fluid without BSA at a dilution of 1:200
for 6-24 h in a humid chamber at room temperature.
Specimens were washed 12 times for 10min , transferred
to distilled water, and dehydrated through an ethanol
series . Animals were mounted on polylysine (Sigma, St.
Louis, Missouri, USA) covered microscope slides and
cleared with Murray's fluid (2 parts benzyl benzoate: I
part benzyl alcohol). Two different controls were
processed the same way, one with primary antibodies
omitted, the second with secondary antibodies omitted.
Microscopy
All specimens were studied with a Nikon Eclipse E800
microscope equipped with a Radiance2000 confocal
laser scanning system (BioRad). Stacks of confocal
optical sections were recorded , applying appropriate
T. Stach / Organisms, Diversity & Evolution 5 (2005) 15-24 19
Fig, 4. Oikopleura fusiformis (Appcndicularia) labeled with antibodies against serotonin. (A) Light-microscopic aspect. (B)
Projection of 71 confocal optical sections taken at 211m intervals. (C-E) Three confocal optical sections taken at 20 urn intervals;
double arrowhead and arrow mark cells labeled in the dorsal ganglion. Abbreviations: cf = ciliated funnel , dg = dorsal ganglion,
en = endostyle, ep = epidermis, fo =Fol's oikoplast, in = intestine, mo = mouth, ms = mucus strand, ta = tail.
Table I. Information on specimens examined
Species Family Order # specimens Ontogenetic Serotonin in CNS"
examined stage
Oikopleura rufescens Fol, 1872 Oikopleuridae Appendicularia 4 Adult Not detected
Oikopleura [usiformis Fol, 1872 Oikopleuridae Appendicularia 3 Juvenile/adul t Few cellsh
Fritillaria sp. (Quoy & Gaimard 1833) Fritillariidae Appendicularia 1 Adult Not detected
Claoelina oblonqa Herdmann, 1880 Clavelinidae Aplousobranchiata 23 Larva Few cells"
Eudistoma olioaceum (Van Name, 1902) Clavelinidac Aplousobranchiata 7 Larva Not detected
Aplidium constellatum (Verrill, 1871) Polyclinidae Aplousobranchiata \3 Larva Not detected
Didemnum cf candidum Savigny, 1816 Didemnidae Aplousobranchiata 4 Larva Not detected
Ascidia interrupta Heller, 1878 Ascidiidae Phlebobranchiata II Larva Distinctly present"
Ciona intestinalis Linne, 1767 Cionidae Phlebobranchiata 9 Larva Present"
Hcrdmania momus (Savigny, 1816) Pyuridae Stolidobranchiata \8 Larva Distinctly present
Microcosmus exasperatus Heller, 1878 Pyuridae Stolidobranchiata 5 Larva Present
Styela plicata Leseur, 1823 Styelidae Stolidobranchiata 7 Larva Distinctly present
Molqula occidentalis Traustedt, 1883 Molgulidae Stolidobranchiata 4 Larva Present
Doliolum nationalis Borgert, 1894 Doliolidae Thaliacea 4 Oozooid Distinctly present
Thalia democratica (Forskal, 1775) Salpidae Thaliacea 3 Oozooid Present
"eNS = central nervous system.
"Few cells tOikopleura fusiformisi = two cells in dorsal ganglion .
"Few cells (Clooelina oblonqa; = two cells next to sensory vesicles plus five cells in pharyngeal epithelium.
"Distinctly present = anterior cell aggregation, posterior fiber net. posterior projection.
"Present = signal of several cells detected, morphology unclear; see text for details.
20 T. Stach / Organisms, Diversit y & Evolution 5 (2005) 15-24
- --- - - - - - -
filter settings. A transmitting light microscopic picture
of the same frame and magnification was recorded
immediately after collection of the stacks of confocal
optical sections. BioRad's LaserSharp2000 and Con?
focal Assistant software was used to analyze the images.
Results
Stolidobranchiata
Fig. I A-D shows light-microscopic images of a larva
of Here/mania monius after hatching (21.5 h post?
fertilization, at 20 QC) . The part of the nervous system
in the larval trunk that is immunopositive to labeling
with antibodies against serotonin in H. momus is seen in
Fig.IB-D.
The serotonergic nervous system consists of two
parts. Anteriorly, about 20 cells form a cluster of
approximately 20 urn length, 15 urn height, and 15 urn
depth. Perikarya stain intensely and are of a spherical
shape with a diameter of approx . 2 um (Fig. IC, D). Cell
counts in this anterior region of different specimens
va ried between J 7 and 20 (11 = 4). The discrepancy
might be related to differences in staining intensity. The
anterior cell bodies are closely packed and seem to
possess short fibers establishing contact with one
another. Note the close spatial relationship of this
cluster of serotonergic perikarya to the larval statocyte
complex that becomes obvious when the fluorescent
image is projected onto the same light-microscopic
frame (Fig. I B).
Several of the serotonergic neurons have fibers
projecting posteriorly. These fibers form a conspicuous
network immediately posterior to the cluster of perikar?
ya , and neurons seem to form lateral connections in it
(Fig. IC, D). The entire network is of conical shape; it is
wide anteriorly and narrows at its posterior end, where
one single longer nerve fiber emerges. This fiber projects
in a slightly ventrally bent curve for another 30 urn
further posteriorly.
Labeling with antibodies against serotonin in the
larvae of Microcosmus exa..speratus (Pyuridae), Styela
plicata (Styelidae), and Molqula occidentalis (Molguli ?
dae), was detected close to the cerebral vesicle. However,
only S. plicata showed sufficient preservation to
ascertain the presence of an anterior concentration of
cells followed by a posterior fiber net and a posteriorly
projecting fiber.
Phlebobranchiata
Fig. I E-H shows light-microscopic images of a larva
of Ascidia interrupta after hatching (2 1.5 h post-fertiliza?
tion, 20 ?C). The part of the nervous system in the larval
trunk that is imrnunopositivc to labeling with antibodies
against serotonin in A. interrupta is depicted in Fig .
1F-H . Again, the serotonergic nervous system consists
of two parts . The anterior perikarya form a cluster of
approximately 40 urn length, 22 um height, and 20 urn
depth . Individual cells are harder to discern in this
species, because spheres of the appropriate size range
(around 2 urn) display various intensities of antibody
labeling. Estimates of the number of cells in the anterior
region in different specimens varied between 36 and 41
(11 = 3). The anterior cell bodies are closely packed .
Note the close spatial relationship of this cluster of
serotonergic cells to the larval statocyte complex
(Fig. I F).
Fibers projecting posteriorly from the serotonergic
cells form a Jess extensive network compared to H.
tnomus larvae. In this network several fibers run almost
parallel to one another for about 15 urn (Fig. 1G, H),
although some lateral connections seem to be present.
One single longer fiber projects in a slightly ventrally
bent curve for another 12 urn further posteriorly
(Fig. I F).
Nervous structures in the larvae of Ciona intestinalis
stained positively with antibodies against serotonin;
fixation quality was too poor to record the detailed
morphology.
Doliolidae
Labeling with antibodies against serotonin in the
oozooid of Dolioluni nationalis is seen in Fig. 2.
Immunoreactivity is confined to three areas: the dorsal
ganglion, the ciliated funnel , and the intestinal tract.
In the dorsal ganglion approximately 20 serotonergic
cells are distributed in a u-shaped pattern (Fig. 2B).
There arc 4-5 positively labeled cells laterally on each
side. About 5 cells with fainter staining are situated at
the anterior border of the ganglion. Immediately behind
this group of anterior cells lie another 5 cells that stain
intensely with antibodies against serotonin . From these
latter cells short fibers emerge that form a network from
which a slightly longer fiber projects posteriorly.
Preliminary investigation demonstrates that several
cells but no fibers were labeled with antibodies against
serotonin in the oozooid of Thalia democratica.
Aplousobranchiata
Fig. 3 shows light microscopic images of a larva of
Clave/ina oblonqa. Immunoreactivity for antibodies
against serotonin is seen in Fig. 3B. There are numerous
serotonergic cells labeled in the stomach wall. Inset in
Fig. 3B shows immunoreactive cells close to the larval
statocyte complex. Anterior to the statocyte complex on
its ventral side are cell bodies of two cells that show
T. Stach / Organisms, Diversity & Evolution 5 (2005) 15-24 21
I
, ~
intense labeling (arrows in Fig, 3B, left inset), One of
these cells bears an apical process that projects dorsally
and in the direction of the statocyte complex. Next to
these two serotonergic cells is a row of 5 cells that react
with the antibodies against serotonin (double arrow?
heads in Fig. 3B, right inset). These cells are situated in
the dorsal epithelium of the branchial basket. This is the
site where in the adults the ciliated funnel that connects
the neural gland to the pharynx opens. No labeling with
antibodies against serotonin was detected in earlier
ontogenetic stages of C. oblonqa (Clavelinidae).
Staining experiments with antibodies against seroto?
nin in the larvae of Eudistoma olioaceum (Clavelinidac),
the larvae of Aplidium stellatum (Polyclinidae), and the
larvae of Didemnum cf. candidum (Didemnidae), re?
turned negative results.
Appendicularia
The reactivity with antibodies against serotonin in an
oikopleurid appendicularian is seen in Fig. 4. Two cells
in the posterior part of the neural ganglion are positively
stained in Oikopleura fusiformis (double arrowhead and
arrow in Fig. 4D, E). No other cellular structures stain
in the nervous system of this species.
No labeling was detected in the central nervous
system of Oikopleura rufescens (Oikopleuridae) and a
single specimen of Fritillaria sp. (Fritillariidae).
Discussion
A morphologically distinct serotonergic nervous
system with an anterior aggregation of cell bodies, a
posterior network of fibers, and a posterior fiber
projection is present in the phlebobranch and stolido?
branch ascidian larvae and in the doliolid oozooid. A
comparable serotonergie complex was not detected in
appendieularians and aplousobraneh ascidian larvae.
Homology of the serotonergic nervous systems
Several lines of evidence suggest homology of the
serotonergic nervous systems present in Tunicata. (i)
They are characterized by epitopes that are labeled by
monoclonal antibodies against serotonin. (ii) All ser?
otonergic components of nervous systems are situated in
the anterior region of the dorsal central nervous system.
(iii) Moreover, in phlebobranch and stolidobranch
larvae, doliolid oozooids, and Notochordata, they are
similar in organization and possibly in function. These
similarities support the hypothesis that the observed
serotonergic nervous systems are homologous.
Garstang (1928) suggested that Doliolida might be
derived by neoteny from sessile ascidians. The similarity
of the serotonergic cel1s in the dorsal ganglion of
Doliolida to the serotonergic nervous system in larval
stages could corroborate this hypothesis. Investigations
of the nervous system of doliolid larvae will be of special
interest.
Reduced serotonergic nervous system in
aplousobranchiate larvae and Appendicularia
The lack of labeling with antibodies against serotonin
in several species of aplousobranchiate ascidians and
appendicularians could be the result of technical
problems. For example, it is well known that penetra?
tion of antibodies into animals invested with cuticles can
be problematic (e.g. Harlow and Lane 1998). However,
in larvae of Clave/ina lepadiformis and in 0. fusiformis,
cells in the dorsal nervous system were positively
labeled. In C. lepadiformis, cells in the pharyngeal
epithelium and the stomach were labeled as well. This
indicates that the observed reduction of signal in the
central nervous system was real. Nevertheless, it seems
unlikely that the almost ubiquitous neurotransmitter
serotonin would be reduced completely in the two
clades. Serotonin could also be modified in these two
groups. It is noteworthy in this context that serotonin
was not detected by immunofluorescence in adult
ascidians (Fritsch et al. 1982; Georges 1985), and that
Appendicularia and Aplousobranchiata have been
considered to show heterochrony. Thus, it is possible
that a heterochronic modification of the serotonergic
nervous system in the two groups is an evolutionarily
derived shared character.
Reconstruction of the serotonergic nervous system of
the last common ancestor of Tunicata and Chordata
The presence of a distinct serotonergic nervous system
in phlebobranch and stolidobranch ascidian larvae and
doliolid oozooids, but absence in Appendicularia and
Aplousobranchiata, poses the question what character
state was present in the last common ancestor of the
Tunicata. Outgroup comparison can answer this ques?
tion.
Fig. 5 depicts phylogenetic relationships between
higher chordate taxa (Stach 2000; Nielsen 200 I; Stach
and Turbeville 2002). The relevant outgroup, the sister
group of Tunicata, is Notochordata consisting of
Cephalochordata + Craniata.
Both, the serotonergic nervous system in larval
Cephalochordata (Hol1and and Holland 1993: p. 200,
Fig. 4F, arrowhead) and Petromyzontida (Hay-Schmidt
2000: p. 1072, Fig. Ik), show an anterior aggregation of
neurons with a posterior fiber network, and fibers
projecting posteriorly. The same was reported from
ontogenetic stages of Xenopus /aevis (van Mier et a!.
22 T. Slach I Organism s. Diversity & Evolut ion 5 (2005) 15-24
Fig. 5. Phylogenetic hypothesis for higher chorda te taxa ;
relationships of higher tunicate taxa as suggested by a
parsimony anal ysis of 18S rONA sequences (Stach and
Turbeville 2002); two indicated potential synapomorphies
are : I = horizontal orientation of larval tail , 2 = reduction of
scrotonergic nervous system.
Rf,
Fig. 6. Schematic representation of the tunicate larval central
nervous system; based on Nicol and Meinertzhagen (1991);
with selected morphological features added. Abbreviations:
mn = motoneuron (after Okada et a!. 2002), nt = neural tube,
oc = ocellus (after Burighel and Cloney 1997), ps = pressure
sensitive cells (after Nicol and Meinertzhagen 1991),
Rf =Reissner's fiber (after Olsson 1993), serNS =
serotonergic nervous system (present study), st = statocyte
complex (after Dilly 1962), sv = sensory vesicle, vg = vegetal
ganglion cells.
1986). Such a distinctly organized serotonergic nervous
system therefore can be considered to be homologous
and present in the last common ancestor of Notochor?
data, Tunicata, and Chordata.
The only study on the serotonergic nervous system of
an ascidian larva found immunoreactivity against
serotonin in the sensory vesicle, diffused in the ventral
trunk region , in the papillae, and some celIs in the tail of
P. mammillata (Phlebobranchiata; Pennati et al. 200 I).
From the documentation published by these authors it is
difficult to assess the morphology of the labeled cells
and structures.
Comparison of the serotonergic nervous system to
other deuterostomes
Cladistic analyses of morphological characters sug?
gest that Enteropneusta might constitute the sister
group to Chordata (Hay-Schmidt 2000; Nielsen 2001),
whereas many molecular studies propose that a taxon
comprising Hcmichordata plus Echinodermata is the
sister taxon to Chordata (recently reviewed; e.g., by
Jenner and Schram 1999; Giribet 2002). Hay-Schmidt
(2000) suggested that two major types of larval
serotonergic nervous system could be distinguished in
bilaterians. The first type consists of a fixed low number
(around 5) of serotonergic cells and is found in
protostomian taxa. The second type features numerous
(> 15) serotonergic neurons in the apical ganglion and
posterior projecting axons, and is found in deuteros?
tomes. The scrotoncrgic nervous system in the ground?
plan of the Tunicata and Chordata reconstructed in the
previous paragraphs clearly belongs to the deuteros?
tome-like serotonergic nervous systems. Nezlin and
Yushin (2004) demonstrated that serotonergic cells in
tornaria larvae are concentrated in the apical ganglion,
whereas in echinoderm larvae they arc scattered along
the ciliary band. Thus, the serotonergic nervous system
in Enteropneusta seems more similar to that in the
groundplan of Chordata .
Functional considerations
Fig. 6 shows a schematic indicating the position of the
serotonergic nervous system as deduced from the
present study. Position and cell numbers of the anterior
aggregate of serotonergic cell bodies are similar to the
cells called NO (dorsal neurons) and NV (ventral
neurons) in the posterior sensory vesicle of C. intestinalis
(Nicol and Meinertzhagen 1991). The structure named
PSV by Takamura (1998) is also similar to the
serotonergic group of cells.
The larval stage of sessile ascidians reacts to various
environmental stimuli and plays a crucial role in
dispersal and substrate selection (Svane and Young
1989; Vazquez and Young 1996; Meinertzhagen and
Okamura 2001). In addition, McHenry and Strother
have recently demonstrated that the locomotion of
aplousobrunchiate larvae is rather complicated and
flexible (Mel-leary 200 1; McHenry and Strother 2002).
The position of the serotonergic cell bodies close to
the statocyte complex, and the posterior network, point
to an integrative function for sensory input. Signal
output could follow the posteriormost projection, which
is directed toward the visceral ganglion, where indivi?
dual motor neurons were characterized by Okada et al.
(2002). The serotonergic neurons might therefore func?
tion in the coordination of locomotion, as is known for
\
T. Stach I Organisms, Diversity & Evolution 5 (2005) 15-24 23
a variety of taxa, such as molluscs (Norekian and
Satterlie 200 I; Yu et al. 200 I) or hirudinean annelids
(Nusbaum and Kristan 1986). Serotonin also modulates
the locomotor behavior in lampreys (Grillner and
Matsushima 1991), and serotonergie neurons project
towards motoneurons during X. laevis ontogeny (van
Mier et al. 1986). However, serotonin serves a wide
range of functions as a signal molecule during ontogeny.
It modulates ciliary activity, regulates metamorphosis,
and in craniates functions in modulation of pain
reception, regulation of sleep-waking cycles, autonomic
functions and reproductive behavior.
Based on the association of serotonergic cells with the
sensory vesicle, Pennati et al. (200 1) suggested that
serotonergic cells were involved in light detection in the
larva of P. mammillata (Phlebobranchiata). The higher
resolution as well as better fixation and labeling
achieved in the present study demonstrate that the
serotonergic perikarya are actually closely associated
with the statocyte complex. However, the possibility
that the serotonergic nervous system receives inputs
from the light-sensitive ocellus cannot be excluded. In
fact, Takarnura (1998) revealed fibers emerging from the
ocellus directed towards the region of the central
nervous system where the serotonergic neurons were
discovered in the present study. Electrophysiolcgical
experiments will be necessary to decide between the two
hypotheses.
Acknowledgements
This research was conducted at the Smithsonian
Marine Station at Fort Pierce, Florida. It is contribution
No. 585 from that institute. A postdoctoral fellowship
from the Smithsonian Institution is greatly appreciated.
Special thanks are due to Dr. Mary E. Rice for
generously supplying excellent research facilities. Eric
Edsinger-Gonzales' help, advice, and encouragement is
appreciated.
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