Download Neurogenic and non-neurogenic placodes in ascidians

JOURNAL OF EXPERIMENTAL ZOOLOGY (MOL DEV EVOL) 302B:483–504 (2004)
Neurogenic and Non-neurogenic Placodes in
Ascidians
LUCIA MANNI1n, NANCY J. LANE2, JEAN-STÉPHANE JOLY3, FABIO
GASPARINI1, STEFANO TIOZZO1, FEDERICO CAICCI1, GIOVANNA ZANIOLO1,
and PAOLO BURIGHEL1
1
Dipartimento di Biologia, Università di Padova, I-35121 Padova, Italy
2
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ,
United Kingdom
3
UPR 2197 DEPSN, Institut Fessard, CNRS, Gif-sur-Ivette 91198, France
ABSTRACT
The late differentiation of the ectodermal layer is analysed in the ascidians Ciona
intestinalis and Botryllus schlosseri, by means of light and electron microscopy, in order to verify the
possible presence of placodal structures. Cranial placodes, ectodermal regions giving rise to
nonepidermal cell types, are classically found exclusively in vertebrates; however, data are
accumulating to demonstrate that the nonvertebrate chordates possess both the genetic machinery
involved in placode differentiation, and ectodermal structures that are possible homologues of
vertebrate placodes. Here, the term ‘‘placode’’ is used in a broad sense and defines thickenings of the
ectodermal layer that can exhibit an interruption of the basal lamina where cells delaminate, and so
are able to acquire a nonepidermal fate. A number of neurogenic placodes, ones capable of producing
neurons, have been recognised; their derivatives have been analysed and their possible homologies
with vertebrate placodes are discussed. In particular, the stomodeal placode may be considered a
multiple placode, being composed of different sorts of placodes: part of it, which differentiates hair
cells, is discussed as homologous to the octavo-lateralis placodes, while the remaining portion, giving
rise to the ciliated duct of the neural gland, is considered homologous to the adenohypophyseal
placode. The neurohypophyseal placode may include the homologues of the hypothalamus and
vertebrate olfactory placode; the rostral placode, producing the sensorial papillae, may possibly be
homologous to the placodes of the adhesive gland of vertebrates. J. Exp. Zool. (Mol. Dev. Evol.)
302B:483–504, 2004. r 2004 Wiley-Liss, Inc.
INTRODUCTION
Vertebrate placodes are transient specialized
regions of the embryonic ectoderm that give rise to
a variety of nonepidermal cell types and are
subject to morphogenetic movements, such as
invagination of cell sheets and epithelial-mesenchymal transactions (Schlosser and Northcutt, 2000;
Schlosser, 2002). They are recognisable as ectodermal thickenings characterised by columnar
cells or areas possessing an interruption of the
underlying basal lamina in which cells delaminate
and subsequently adopt a nonepidermal (e.g.,
neuronal) fate (Schlosser and Northcutt, 2000).
Although the focal ectodermal thickenings that
give rise to hairs, feathers, and teeth are also
called placodes, usually researchers restrict the
term ‘‘placodes’’ to the cranial placodes associated
with the nervous system. These arise from the
lateral borders of the neural folds and areas
r 2004 WILEY-LISS, INC.
immediately adjacent to them. With the exceptions
of lens placodes and adenohypophyseal placodes
which do not give rise to migratory cells, all the
above placodes possess individual cells able to
delaminate and migrate, and are neurogenic, i.e.,
include neurons among their derivative cell-type.
These neurons are represented by a wide variety
of mechano- and chemosensory structures, including the olfactory epithelium of the nose, parts of
the cranial ganglia, sensory cells of the inner ear
and lateral line (hair cells), and some gonadotropin-releasing-hormone (GnRH)-containing neurons (reviewed in Baker and Bronner-Fraser,
Grant information: grant from Ministero dell’Istruzione, dell’Università e della Ricerca and University of Padova (P.B.) and from the
E.M.P. Musgrave Fund (N.J.L.).
n
Correspondence to: Lucia Manni, Dipartimento di Biologia,
Università di Padova, via U. Bassi 58/B, I-35121 Padova, Italy.
E-mail: [email protected]
Received 6 April 2004; Accepted 23 June 2004
Published online 3 September 2004 in Wiley Interscience (www.
interscience.wiley.com). DOI: 10.1002/jez.b.21013
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2001). Thus, from neurogenic placodes both
primary sensory cells sending axons into the
central nervous system and secondary sensory
cells, i.e., dedicated receptors lacking axons,
derive; the latter are connected to the central
nervous system by sensory neurons of placodal
origin.
Cranial placodes, together with the neural crest,
are considered evolutionary innovations of craniates (Northcutt and Gans, ’83; Shimeld and
Holland, 2000). Nevertheless, in recent years,
comparative data from electron microscopy and
the results of developmental gene expression have
suggested that invertebrate chordates (amphioxus
and urochordates), and by extension the ancestral
chordates, have populations of cells with some of
the properties of placodes.
Amphioxus, the closest living relative to vertebrates, has a larva with numerous ectodermal
sensory cells, both primary and secondary (Bone
and Best, ’78; Lacalli and Hou, ’99; Holland and
Holland, 2001; Holland and Yu, 2002; Lacalli,
2002); for some of them, a possible homology with
the vertebrate taste buds and olfactory organs has
been proposed (Lacalli et al., ’99). Comparisons of
developmental gene expression (Pax-6, Neurogenin,
Msx and Sox1/2/3) between vertebrates and amphioxus also suggest possible homologies (Manzanares et al., 2000; Holland and Holland, 2001). The
anterior ectoderm in amphioxus expresses genes
that are considered specific markers of the vertebrate olfactory epithelium, althought, the presence
of olfactory or other telencephalic regions in the
brain of protochordates is debated (Holland and
Holland, ’99; Nieuwenhuys, 2002). Moreover, the
hypothesis that Hatschek’s pit is homologous to the
adenohypophysis has also been proposed (Nozaki
and Gorbman, ’92; Holland and Holland, 2001;
Boorman and Shimeld, 2002; Kawamura et al.,
2002; Satoh et al., 2002).
In many ascidians, such as Ciona intestinalis,
the atrium originates from a pair of dorsal
ectodermal invaginations comparable in position
to the otic placodes of vertebrates. In the atria of a
number of ascidians a variety of mechanoreceptors
have been found (Fedele, ’23; Millar, ’53; Goodbody, ’74; Bone and Ryan, ’78; Mackie and Singla,
2003). Among them, the cupular organs of C.
intestinalis have been considered possible candidates for placodal derivatives owing to their
similarities with the vertebrate otic and lateral
line receptors. Hence, a possible homology between the atrial primordia and the otic placodes
has been proposed by several authors (Katz, ’83;
Baker and Bronner-Fraser, ’97; Wada et al., ’98;
Shimeld and Holland, 2000; Jefferies, 2001).
However, unlike vertebrate hair cells, the cupular
organs contain primary sensory neurons and this
marked difference induced Mackie and Singla
(2003) to doubt that they are actually homologous
to the vertebrate hair cells. Recently, a new
mechanoreceptor organ, the coronal organ, was
found in the oral siphon of botryllid ascidians
(Burighel et al., 2003). It is composed of hair cells,
which are similar to those of the vertebrate octavolateralis system, because they are secondary
sensory cells, each bearing a single cilium eccentric to a bundle of stereovilli. Thus, the coronal
organ was considered a possible candidate as a
homologue of the vertebrate lateral line. Moreover, there are some indications of another
possible homology between three vertebrate structures, the hypothalamus, the adenohypophysealand the olfactory placode, and the neurohypophyseal duct, an embryonic structure in the ascidians,
which represents the rudiment of the cerebral
ganglion and the neural gland of the adult (Manni
et al., ’99, 2001; Christiaen et al., 2002). Nevertheless, the genome sequence recently performed
for C. intestinalis contains a number of genes
orthologous to those involved in vertebrate placode development (Cañestro et al., 2003).
In the present study we re-examined, by light
and transmission electron microscopy, the ectoderm-derived tissues, with attention to the possible presence of placodal structures, in two species,
C. intestinalis and Botryllus schlosseri, considered
representative models of solitary and colonial
ascidians, respectively (Dehal et al., 2002; Rinkevich, 2002). The two species differ in anatomical
aspects of their larvae. In C. intestinalis the atrial
chamber originates from a pair of dorsal ectodermal invaginations (Willey, 1893a; Katz, ’83),
whereas in B. schlosseri it arises from a single
ectodermal mid-dorsal anlage (Scott, ’34). Moreover, C. intestinalis possesses a dorsal ectodermal
invagination, the stomodeum, which represents
the rudiment of the oral siphon, that fuses and
perforates with the pharyngeal endoderm, in a
way reminiscent of the perforation of the vertebrate oral membrane. In contrast, in botryllids a
similar stomodeum is not recognizable and the
oral siphon rudiment forms in an antero-dorsal
region of the pharyngeal wall, which has ectodermal characteristics (Manni et al., ’99).
In our study, we identified a number of thickenings of the ectodermal layer, which have neurogenic derivatives and/or exhibited interruptions
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of the underlying basal lamina for the delamination of cells which were adopting a non epidermal
fate. Their possible homology with vertebrate
placodes is analysed.
MATERIALS AND METHODS
Specimens of Ciona intestinalis (family Cionidae, order Phlebobranchia) and Botryllus schlosseri (family Styelidae, order Stolidobranchia) used
in this study were originally collected from the
lagoon of Venice.
Adults of C. intestinalis were maintained in
tanks in the laboratory (at 181C). Eggs and sperm
were obtained surgically from the gonoducts. After
external cross fertilisation, embryos were maintained in tanks where they developed into swimming larvae (early larvae) within 18–20 hours that
further (24 h after fertilisation) differentiated into
mature larvae, adhered to substrate and metamorphose. They were selected at appropriate
stages under a stereomicroscope and pipetted
directly into the fixative liquid.
Colonies of B. schlosseri are composed of a large
number of small blastozooids embedded in a
common tunic and arranged in star-shaped systems. They were cultured on glass in the laboratory (at 181C) following Sabbadin’s (’55)
technique. They have internal fertilisation and
are ovoviviparous. The transparency of the colonies allowed us to follow the daily development in
vivo of embryos under the stereomicroscope, and
select them at appropriate stages. Larvae develop
in a week and, after a short period of swimming
life, undergo metamorphosis, giving rise to the
sessile oozooids, which reproduce asexually by
budding. After some blastogenetic generations,
blastozooids reach maturity and can reproduce
sexually, producing new larvae. Embryos were
dissected from the parents using a thin tungsten
needle, whereas free swimming and metamorphosing larvae were directly pipetted into the
fixative liquid. The developmental stages of embryos were based on the developmental stage of
the colony and the gross anatomy of embryos and
larva, as described in Manni et al. (’99).
Light and transmission electron
microscopy
Embryos and larvae were fixed in 1.5% glutaraldehyde buffered with 0.2 M sodium cacodylate,
pH 7.4, plus 1.6 % NaCl. After washing in buffer
and postfixation in 1% OsO4 in 0.2 M cacodylate
buffer, the specimens were dehydrated and em-
bedded in Araldite. Thick sections (1 mm) were
counterstained with toluidine blue; thin sections
(60 nm) were given contrast by staining with
uranyl acetate and lead citrate. Transverse serial
thin sections of embryos and larvae were cut and
analysed in order to verify the differentiation of
ectodermal tissues. Micrographs were taken with
a Hitachi H-600 electron microscope operated at
80 kV.
All photos were acquired with a Duoscan (Agfa),
and were collected and typeset in Corel Draw 9.
RESULTS
Larvae of Ciona intestinalis and Botryllus
schlosseri have a similar basic organisation, but
also exhibit differences (Fig. 1A, C), with regard to
both the size and the anatomical complexity. The
larva of C. intestinalis has a trunk about 340 mm
and a tail about 850 mm long, while the larva of B.
schlosseri has a trunk 400 mm long and a tail 1 mm
long. The larvae possess transitory structures for
larval life, larval-juvenile organs and prospective
juvenile organs (Burighel and Cloney, ’97), and
the last two kinds of organ are particularly well
developed in the mature larvae of B. schlosseri. In
both the species, several ectodermal derivatives
can be recognised (Willey, 1893a, b; Grave and
Woodbridge, ’24; Scott, ’34): some of them are
transitory larval structures, such as the outer
cuticular layer and outer compartment of the
tunic (fins), the adhesive organ with three papillae
(or palps), the sensory vesicle containing the
sensory organs, the visceral ganglion and the
nerves. The epidermis, inner cuticular layer and
inner compartment of the tunic are larval-juvenile
structures; others are prospective juvenile organs,
such as the oral and atrial siphons, the atrium
(paired in C. intestinalis, single in B. schlosseri),
the neurohypophyseal duct (or its neural complex
derivatives), the preoral lobe and epidermal
ampullae, and the primordium of the first bud
(in B. schlosseri).
In the following paragraphs we will describe the
ectodermal regions differentiating into placodes,
starting from the early larval stage (18 hours post
fertilisation) for C. intestinalis, and early tail-bud
embryos (three days before hatching) for
B. schlosseri (Table 1, Fig. 1B, D). At these stages
most of the rudiments of the main internal
structures are developed (the pharynx, the gut
rudiment, the mesenchyme cells), the ectodermal
organs are differentiating and the monolayered
epidermis is secreting the tunic. In agreement
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Fig. 1. Schematic drawing of longitudinally sectioned mature (A) and early (B) larva of Ciona intestinalis and mature larva
(C) and early tail-bud embryo (D) of Botryllus schlosseri. In A, the left atrial rudiment is visible from the external side; siphons
are not perforated. Blue, ectodermal derivatives; red, mesodermal derivatives; green, notochord; yellow, endodermal derivatives.
Larvae and embryos are not drawn to the same scale. In B and D, the lined areas mark some of the recognised placodes (rostral-,
stomodeal-, atrial-, neurohypophyseal placode). The peribranchial chambers are out of the plane of the section, while the tunic
and mesenchymal cells were omitted.
PLACODES IN ASCIDIANS
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TABLE 1. Ectodermal derivatives in C. intestinalis and B. schlosseri
n
In other compound ascidians, the bud can derive from other embryonic layers.
with Crowther and Whittaker (’84) and Nishida
(’92) we consider the ability to secrete the tunic a
valid marker specifying cells of ectodermal origin
even when they are not at the surface of body.
Thus, in B. schlosseri, we consider the dorsal
anterior region of the pharynx, representing the
rudiment of the oral siphon and ciliated duct, to be
of ectodermal origin, because in the embryo it is
initially covered by a thin layer of tunic (although
in the larva only the rudiment of the oral siphon
produces it) (Manni et al., ’99). Embryonic cells
are generally large and expanded, but in some
cases they are flat, such as are those limiting part
of the sensory vesicle; moreover, they possess
small yolk globules, particularly abundant in some
endodermal derived tissues. Generally they exhibit large nuclei with prominent nucleolus, with a
cytoplasm rich in ribosomes.
We identified several thickenings of the ectodermal leaflet capable of differentiating into nonepidermal cell types and also sites characterised by
an interruption of the basal lamina in which cells
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delaminate acquiring a nonepidermal fate. In the
broad sense of the definition proposed in our
Introduction, we discuss these ectodermal domains as possible placodes.
Rostral placode
The rostral ectoderm thickens in a placodal
region destined to form a complex sensory structure, the adhesive organ, and epidermal sensory
neurons, which are scattered in C. intestinalis
(Takamura, ’98) and grouped in B. schlosseri
(Grave and Riley, ’35; Scott, ’34). This region also
differentiates epidermal derivatives, such as the
ampullae or preoral lobe.
At three sites of the anterior tip, two dorsal and
one ventral, the columnar ectoderm evaginates in
conical projections, which elongate to give rise to
three sensory adhesive papillae, while the delimited central area remains depressed (Fig. 2A-D).
At the onset of metamorphosis the papillae play a
role in substrate selection and serve as attachment
devices by secretion of sticky substances (Burighel
and Cloney, ’97). In C. intestinalis (Fig. 2A, B), the
papillae are simple, coniform, noneversible and
are constituted of secreting cells, axial columnar
cells, primary sensory neurons and undifferentiated ectodermal cells (Groppelli et al., 2001). In
B. schlosseri (Fig. 2C, D), the papillae are more
complex because although they are coniform and
noneversible, they are ganglionated. The sensory
cells develop apical specialisations for reception,
while they sink with their basal portions below the
epidermis to form a ‘‘papillary ganglion’’ (Scott,
’34; Grave and Riley, ’35; Torrence, ’83; Burighel
and Cloney, ’97). Sensory cells send their axons to
the visceral ganglion; these fasciculate to form two
papillary branches, one with fibres of ventral and
right dorsal papillae, the other with fibres of the
dorsal left papilla (Fig. 2E). The two branches,
ultimately, join together in a papillary nerve
entering the visceral ganglion.
In B. schlosseri a wide area of the anterior
epidermis all around the triangle labelled by the
three papillae pushes out forming a circle of eight
pocket-like evaginations (Fig. 2C, F), the ampullae, which only in their anteriormost region have
columnar cells. At metamorphosis, the ampullae
elongate into the tunic but remain interconnected
by means of the marginal vessel running at the
periphery of the tunic (Burighel and Brunetti,
’71). In C. intestinalis, typical ectodermal ampullae are not recognisable. However, in the fully
developed larva, a wide anterior body-cavity, the
pre-oral lobe, is formed behind the papillae. At
metamorphosis, the anterior extremity of the preoral lobe thickens into a disc of ectoderm for
adhesion of the juvenile to the substratum (Fig.
2G, H). It is of note, that possibly nerve circuitries
connecting papillary neurons, epidermal neurons
and central nervous system exist in the larval
anterior region. With the use of monoclonal
antibodies, Takamura (’98) described in C. intestinalis a group of neurons, called rostral trunk
epidermal neurons, and nerve fibres connecting
them with the papillary neurons and the posterior
part of the sensory vesicle. In B. schlosseri Grave
and Riley (’35) described a ‘‘hold-fast mechanism,’’ constituted of two epidermal thickenings,
connected by means of nerve fibres to the papillary
nerve branches, hence to the central nervous
system. We were unable to follow these circuitries
to their ends; however, based on our observations
and anatomical relationships, the unique domain
which gives rise to them is the area of the rostral
placode.
Stomodeal placode
In the early larva of C. intestinalis, the anterior
dorsal ectoderm is thickened and invaginated to
Fig. 2. Rostral placode. A, B. Frontal section of early larva (A) and sagittal section of a mature (B) larva of C. intestinalis at
level of the papillae (p). ep, epidermis; mp, mesodermal pocket; nc, notocord; ns, nervous system; sv, sensory vesicle with otolith;
ph, pharynx; t, tunic. Toluidine blue. Scale bar¼50 mm in Figure A; scale bar¼20 mm in Figure B. C, D. Sagittal sections of
mature larvae of B. schlosseri. In (C), the ventral papilla (p) and three blood ampullae (a) are shown. In (D), a papillary nerve
(arrowheads) directed toward the visceral ganglion (vg) is recognisable. asr, atrial siphon rudiment; dg, dorsal groove; ep,
epidermis; osr, oral siphon rudiment; pb, primordium of bud; ps, protostigma; sv, sensory vesicle; t, tunic. Toluidine blue. Scale
bar¼60 mm in Figure C; scale bar¼35 mm in Figure D. Toluidine blue. E. Papillary nerves in the anterior blood lacuna of a larva
of B. schlosseri. Three branches joint to form a papillary nerve (pn) directed to the central nervous system (not visible on the
upper right side). Transmission electron microscopy. ep, epidermis; t, tunic. Scale bar¼5 mm. F. Electron micrograph of an
ampulla containing blood cells (bc). Note that the apical ampullar epidermis (ep) is thickened. t, tunic. Scale bar¼10 mm. G, H.
Stalk in the juvenile of C. intestinalis under light (G) and electron (H) microscopy, showing the ectodermal disc (ed) for the
adhesion at the substrate. bc, blood cells; t, tunic; tr, tail remnant. Scale bar¼50 mm in Figure G, toluidine blue; scale bar¼5 mm
in Figure H.
PLACODES IN ASCIDIANS
form a pocket of columnar cells, the stomodeum,
which includes the territories forming the oral
siphon with the velum and tentacles and the
ciliated duct with its aperture area. Its floor
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contacts the antero-dorsal end of the pharyngeal
endoderm. The lumen of the stomodeum is filled
with tunic (Fig. 3A-C). During metamorphosis, the
stomodeal and pharyngeal epithelia fuse and
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Fig. 3. Stomodeal placode. A, B. Transverse (A) and frontal (B) sections of C. intestinalis early larva showing the stomodeum
(st). Its wall is thick, the lumen is filled with tunic (t) and its bottom contacts the endodermal pharynx (ph), while posteriorly it
is connected to the sensory vesicle (sv) by means of the neurohypophyseal duct (nd). Anterior at top. ap, atrium primordium; ep,
epidermis; mp, mesodermal pocket; oc, ocellus; vg, visceral ganglion. Toluidine blue. Scale bar¼30 mm in Figure A; scale bar¼25
mm in Figure B. C. Stomodeum (st) of an early larva of C. intestinalis. Note the columnar cells (whose border is marked by the
white dotted line) contacting the thin epithelium (small arrows) lining the sensory vesicle (sv). Part of the lumen of the
neurohypophyseal duct (large arrows) is visible in tangential section; its anterior portion is ciliated (arrowhead). t, tunic.
Transmission electron microscopy. Scale bar¼5 mm. D, E. Transverse (D) and sagittal (E) sections of a juvenile of C. intestinalis
at the level of the oral siphon (os). In D, the rudiment (arrows) of the velum and tentacles is recognisable as a thickened area
bordering the base of the siphon. In E, the neurohypophyseal duct (nd) with its ciliated duct (cd) is opened in the branchial
chamber (bch). Toluidine blue. ach, atrial chamber; ep, epidermis; ps, protostigma; t, tunic. Toluidine blue. Scale bar¼25 mm in
Figure D and E. F, G. Sagittal section of a tail-bud embryo (F) and mature larva (G) of B. schlosseri. An invagination of
thickened epidermis forms the dorsal groove (dg), whose border is marked by the white dotted line. In the larva (G), the oral
siphon (os) is already opened and the tunic (t) occupies its lumen; note the thickened epithelium of the atrial siphon rudiment
(arrowheads), whilst the remaining atrial epithelium is very flat. Anterior at left. nd, neurohypophyseal duct. Toluidine blue.
Scale bar¼30 mm in Figure F; scale bar¼40 mm in Figure G.
break in preparation for communication of the
pharynx with the outside world, via the oral
siphon (Figs. 3D, E, 4). In the early juvenile, at
the base of the oral siphon the ectodermal layer
thickens and rises all around to form the rudiment
of the velar ring and tentacles. This rudiment is
located anterior to the ciliated duct aperture. In
contrast, in the B. schlosseri embryo, the dorsal
ectoderm, lying above the brain, thickens and
forms a deep longitudinal depression, the dorsal
groove, filled with tunic (Scott, ’34; Manni et al.,
’99) (Fig. 3F, G). Anteriorly, immediately in front
of the sensory vesicle, the groove makes contact
with the epithelium which participates in the
formation of the rudiment of the oral siphon. This
region has ectodermal characteristics because it
PLACODES IN ASCIDIANS
secretes a thin layer of tunic; its columnar
epithelium evaginates and contacts the dorsal
groove (Figs. 3F, G, 4, 5A). Thus, this area and
the anterior region of the dorsal groove represent
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a stomodeum. In the larva, their epithelia fuse
with each other and perforate opening into the
oral siphon, which remains closed for a while by
the tunic (Figs. 3G, 4). During metamorphosis, the
Fig. 4. Diagram comparing the main events of morphogenesis of the stomodeal and neurohypophyseal placodes (marked by
lines in A and D) in C. intestinalis and B. schlosseri. The sensory vesicle of C. intestinalis and the ganglionic vesicle of
B. schlosseri are resorbed during metamorphosis and are not present in the juveniles. Light grey, tunic; medium grey,
ectodermal tissues; dark grey, endodermal tissues.
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Fig. 5. Stomodeal placode in B. schlosseri. Transmission electron microscopy. A. Tangential section of the stomodeal region
of a larva. Note that the oral siphon (os) epithelium is thick, produces tunic (t) towards the pharynx lumen (ph) and fuses with
the anterior dorsal groove (dg) epithelium. Arrow, ciliated duct aperture. Scale bar¼5 mm. B. Oral siphon (os) in oozooid (late
metamorphosis); toluidine blue. Arrowheads, tentacle rudiments. Scale bar¼100 mm . C. Detail of the velum rudiment (v) in the
metamorphosing larva. ep, epidermis; ph, pharynx; t, tunic. Scale bar¼5 mm. D. Section of tentacle in an oozooid showing a hair
cell (hc) of the coronal organ. Some nerve fibres (nf) are recognisable at the base of the sensory cell. Arrowheads, streocilia of the
hair bundle; arrow, sensory cilium; sc, supporting cells. Scale bar¼2 mm.
PLACODES IN ASCIDIANS
inner wall of the siphon thickens further and folds
to form the rudiments of the velum and tentacles
(Fig. 5B). In the latter, secondary sensory cells
differentiate into hair cells: they bear an offcenter, single cilium and a bundle of stereovilli;
moreover, at their base they form synapses with
nerve fibres coming from the cerebral ganglion
(Figs. 5D, 6). These hair cells are arranged along a
line running on the upper side of the tentacles and
along the velum in the oozooid to form a coronal
organ, like that described in the blastozooid by
Burighel et al. (2003).
In C. intestinalis the stomodeum communicates
with the sensory vesicle by means of a very short
ectodermal duct, the neurohypophyseal duct,
which, in its anterior portion, possesses cilia and
represents the rudiment of the ciliated duct of the
neural gland (Fig. 3B, C, E). In B. schlosseri
(Manni et al., ’99), the relationship between the
oral siphon rudiment, the pharynx and the neurohypophyseal duct is different. In the early tail-bud
Fig. 6. Schematic drawing of a sensory coronal cell and
supporting cells. (Modified from Burighel et al., 2003).
493
embryo (Fig. 5A), the rudiment of the ciliated duct
is recognisable as a dorsal blind, evagination, below
the siphon rudiment, made up of columnar
epithelium, covered by tunic. During the successive
stages, the rudiment of the duct fuses with the
neurohypophyseal duct and produces cilia (Fig.
3G). The diagrams in Figure 4 compare the
morphogenetic events of the stomodeal area in
C. intestinalis with that of B. schlosseri.
Neurohypophyseal placode
During embryogenesis, cells from the anterior
area of the neural plate develop a short tube which
grows toward the stomodeum (Torrence, ’83;
Lemaire et al., 2002). This tube is the neurohypophyseal duct and represents the rudiment of the
adult neural complex components, e.g the cerebral
ganglion and the neural gland body with its
posterior elongation, the dorsal strand. The anterior part of the neural gland, the ciliated duct,
derives from the stomodeum (Fig. 7). In the early
larva of C. intestinalis, the neurohypophyseal duct
is composed of a very short and narrow canal, with
columnar cells lying on a basal lamina continuous
with that of the sensory vesicle and stomodeum
(Fig. 3B, C). During metamorphosis, the neurohypophyseal duct elongates into the dorsal wall of
the sensory vesicle, which meanwhile begins to
degenerate; hence, the duct grows backward into
the blood lacuna located on the roof of the
branchial chamber to approach the gut rudiment
(Fig. 3E). The neurohypophyseal duct wall proliferates intensively: some pioneer cells delaminate from it becoming neurons to form the
rudiment of the cerebral ganglion; at the same
time the neurohypophyseal duct differentiates
into the neural gland body and dorsal strand,
while the ciliated duct develops further. In the
juvenile, the cerebral ganglion is dorsal to the
neural gland and distinct from it, although areas
Fig. 7. Scheme of placodes (in italics) and structures derived from the stomodeal and neurohypophyseal placodes. The
contribution of stomodeal placode to the ciliated duct is based on B. schlosseri data.
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L. MANNI ET AL.
of contact between the two structures remain for a
time (Fig. 8C): here the basal lamina of the neural
gland cells is interrupted owing to the delamination of neuroblasts which are abandoning their
site of origin in the gland. In the adult of C.
intestinalis, or other ascidians, the dorsal strand
contains cells immunoreactive to vertebrate pituitary hormones (Terakado et al., ’97; Kawahara
et al., 2002). Moreover, it is thought to be the
source of the neurons either forming the GnRH
immunoreactive dorsal strand plexus (Fedele, ’23;
Mackie, ’95) or regenerating the ablated cerebral
ganglion (Bollner et al., ’55; Koyama, 2002). In the
mature larva of B. schlosseri (Fig. 8D, E), the
neurohypophyseal duct is more conspicuous than
it is in C. intestinalis. It is made up of a single
layer of cuboidal cells, separated from the overlying dorsal groove by a thin basal lamina, while
ventrally its wall is close to the visceral ganglion
(Manni et al., ’99). The lumen of the duct is
posteriorly continuous with that of the left ganglionic vesicle (Fig. 8D), a small cavity in the
visceral ganglion being considered the antimere of
the sensory vesicle (Sorrentino et al., 2000).
Fig. 8. Neurohypophyseal placode. A-C. Neural complex in juvenile of C. intestinalis. A. The neurohypophyseal duct is
differentiating into the neural gland, whose ciliated duct (cd) is visible. Its wall is continuous with the cerebral ganglion, formed
by a cortex (cx) of neuronal somata and a medulla (md) of neurites. In B, the arrows mark the area of contact between neural
gland (ng) and cerebral ganglion (cg). C represents an enlargement of the squared area in A, showing a proliferating region. The
basal lamina (arrowheads) of the neural gland is recognizable, except in the area of delaminating neurons. A, C: transmission
electron microscopy. B: toluidine blue. Scale bar¼10 mm in Figure A; scale bar¼25 mm in Figure B; scale bar¼1.5mm in Figure C.
D-F. B. schlosseri. Sagittal (D) and transversal (E) sections of the neurohypophyseal duct (nd) in the mature larva. The duct is
ventral to the dorsal groove (dg), opened into the pharynx (ph) by means of the ciliated duct rudiment (cd). F. Neural complex in
metamorphosing larva. The neurohypophyseal duct is differentiating into the neural gland (ng) and is proliferating neuroblasts,
aggregated to form the cerebral ganglion (cg). ep, epidermis; gv, ganglionic vesicle; vg, visceral ganglion; ps, protostigma; t,
tunic. Figure D, E: toluidine blue; Figure F: transmission electron microscopy. Scale bar¼40 mm in Figure D; scale bar¼50 mm in
Figure E; scale bar¼5 mm in Figure F.
PLACODES IN ASCIDIANS
Anteriorly the neurohypophyseal duct is blind and
its wall contacts the ciliated duct rudiment. In the
stages that follow, the neurohypophyseal duct
fuses anteriorly with the rudiment of the ciliated
duct while posteriorly it looses the original
connection with the ganglionic vesicle (Fig. 4). It
proliferates pioneer cells which coalesce ventral to
it to form the rudiment of the cerebral ganglion
(Fig. 8F). During metamorphosis, its wall acquires
the characteristics of the neural gland epithelium;
posteriorly it forms a hollow dorsal organ, homologous to the dorsal strand, which detaches from
the gland. A neural strand plexus has not been
observed in B. schlosseri (Manni et al., ’99).
Atrial placode
The rudiments of the atria are recognisable in
the early larva of C. intestinalis in the form of
two dorsal, symmetrical invaginations of the
ectoderm, one on the left and one on the right,
located posterior to the stomodeum (Figs. 3B,
9A). They are composed of columnar cells
contacting the posterior-most wall of the pharyngeal wings, which embrace the central nervous
system laterally. In the mature larva, the two
atrial invaginations are still opened outward, but
soon they loose their aperture, becoming two
small, closed chambers, at the bottom of which
the protostigmata perforate (Fig. 3D). On the
dorsal side, each atrial chamber forms the
rudiment of the atrial siphon, which are recognisable in section because their ectodermal cells
thicken and secrete tunic toward the lumen of
the atrial chambers (Fig. 9A). At the end of
metamorphosis, the atrial siphons open dorsally
by fusion and perforation of their wall with the
overlying epidermis. In the juvenile, the two
atrial chambers elongate antero-posteriorly following the enlargement of the branchial sac while
stigmata proliferate. They converge dorso-medially and fuse with each other, creating the
unpaired atrial cavity. Later, the two atrial
siphons also fuse to form the unique atrial siphon
of the adult. Some cells of the atrial chamber wall
differentiate into the cupular organs, mechanoreceptors containing primary sensory neurons
with cilia embedded in a gelatinous cupula
(Fedele, ’23; Millar, ’53; Goodbody, ’74; Bone
and Ryan, ’78).
In the embryo of B. schlosseri, the formation
of the atrial chamber differs completely from
C. intestinalis: it originates as a unique, dorsal
invagination of ectodermal columnar cells in the
495
posterior region of the trunk, during the early
differentiation of the larval nervous system (Scott,
’34; Manni et al., 2002). The primordial invagination sinks into the body and bifurcates over the
nervous system to form the rudiment of the left
and right peribranchial chambers. These later
descend ventrally and, on both sides, abut their
anterior walls against the pharyngeal walls. In the
early tail-bud embryo, the primordial invagination
closes its opening and separates from the epidermis. Then, its wall protrudes toward the dorsal
groove to form a dorsal cupuliform evagination
representing the rudiment of the atrial siphon,
whose cells are columnar and which secrete a thin
layer of tunic toward the lumen of the atrial
chamber (Figs. 3G, 9B). At metamorphosis, the
atrial siphon opens and becomes functional. No
cupular organs or other mechanoreceptor systems
have been found in the atrial chamber of B.
schlosseri (Burighel et al., 2003).
The cellular aspects of the mechanism of
perforation in branchial fissures was recently
elucidated in B. schlosseri (Manni et al., 2002):
in the early tail-bud embryo, stigmata primordia
appear as contiguous thickened discs of palisade
cells of ectodermal peribranchial epithelia. The
cells have a thin basal lamina and are lateroapically joined by typical tight junctions. During
larval development, in each primordium, the
peribranchial disc invaginates to contact the
branchial wall. Here, the basal laminae intermingle,
compact and are degraded, while the space
separating the two epithelia is reduced; cells
rapidly change their polarity and a remodelling
of tight junctions occurs permitting the structural
continuum of the two epithelia. Later, during
metamorphosis, the stigmata enlarge and develop
both apical cilia and microvilli and become functional in the oozooid.
In C. intestinalis, the four primary branchial
fissures originate during metamorphosis, hence
later than in B. schlosseri: at specific sites, the
ectodermal cells of the two atrial chambers contact
the endodermal cells of the pharynx becoming
columnar. Then, with a mechanism recalling that
in B. schlosseri, the branchial fissures are formed
and cilia and microvilli differentiate (Fig. 3D). The
juvenile increases its number of stigmata by
subdivision of the primary stigmata (Willey,
1893a). In the larva of B. schlosseri the primordium of the first bud is recognisable by a
thickening of the ectodermal cells on the right
ventro-lateral wall of the peribranchial epithelium
covered by epidermis (Fig. 2C). This thickening
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L. MANNI ET AL.
Fig. 9. Atrial placode. A. Transmission electron microscopy of the atrial placode (bordered by the white dotted line) in the
early larva of C. intestinalis. The placode (ap), obliquely sectioned, is in the form of an invagination of columnar cells close to
mesenchymal cells (mc) and the endodermal wall of the pharynx (ph). Inset: transverse section of early larva at level of the two
rudiments of the atria. Inset, toluidine blue. Scale bar¼2.5 mm; scale bar¼55 mm in inset. B. Sagittal section of atrial siphon
rudiment in the larva of B. schlosseri. The atrial dorsal epithelium is thickened and produces tunic (t) toward the atrial chamber
(ach). ep, epidermis of the posterior dorsal groove. Scale bar¼5.5 mm.
497
PLACODES IN ASCIDIANS
folds to form an inner vesicle surrounded by an
outer vesicle of epidermis. The bud grows during
metamorphosis and oozooid life to become the first
blastozooid of the new colony (Fig. 10).
Table 2 presents the ectodermal thickenings
interpreted as placodes, listing the organs and cell
types derived from them, as deduced from our
results.
Fig. 10.
placode.
Scheme of structures derived from the atrial
DISCUSSION
Neurogenic placodes, as discrete regions of
columnar ectoderm that form neurons, are common in all the main bilaterian groups (Baker and
Bronner-Fraser, ’97), but cranial placodes seem to
be exclusive of vertebrates. However, from an
evolutionary point of view, it is interesting to
analyze whether any/all of the series of cranial
placodes (adenohypophyseal, olfactory, lens, profundal, trigeminal, epibranchial, hypobranchial,
lateral line and ear placodes) are vertebrate
innovation, or whether any/all have homologous
in nonvertebrate chordates. The possibility that
cranial placodes can be present in the common
chordate ancestor was recently taken into consideration on the basis of data coming from extant
lower chordates (Baker and Bronner-Fraser, ’97;
Burighel et al., ’98, 2003; Graham and Begbie,
2000; Shimeld and Holland, 2000; Holland and
Holland, 2001; Manni et al., 2001). Amphioxus
possesses ectodermal sensory cells, including
primary and secondary sensory cells, the latter
connected to sensory neurons located in the brain.
Moreover, developmental gene expression in this
species suggests the presence of ectodermal territories homologous to the vertebrate olfactory and
TABLE 2. Placodes, and the organs and cell kinds derived from them in C. intestinalis and B. schlosseri
Derived organs
Placode
Rostral
Stomodeal
Neurohypophyseal
Atrial
Ciona
Cellular derivatives
Botryllus
Adhesive organ
with papillae
Adhesive organ with
papillae
Pre-oral lobe
Epidermis
Blood ampullae
Epidermis
Oral siphon, velum,
tentacles
Oral siphon, velum,
tentacles, coronal
organ
Ciliated duct
Cerebral ganglion,
neural gland and
dorsal organ
Ciliated duct
Cerebral ganglion,
neural gland and
dorsal strand, dorsal
strand plexus
Atrial chamber, atrial
siphon, atrial velum,
peribranchial
chambers, cupular
organs,
protostigmata
Atrial chamber, atrial
siphon, atrial velum,
peribranchial
chambers,
protostigmata
Ciona
Epidermal cells, axial
columnar cells, secreting
cells, primary sensory
neurons
Epidermal cells
Epidermal cells, epidermal
neurons
Oral secreting tunic cells,
sensory ciliated cells of the
tentacles
Ciliated cells
Motor neurons, GnRH+
neurons, neural gland cells,
epithelial cells of the dorsal
strand, GnRH+ neurons
of the dorsal strand plexus
Peribranchial/atrial cells,
atrial secreting tunic cells,
primary sensory neurons,
supporting cells secreting
the cupula, stigmatal
ciliated and parietal cells
Botryllus
Epidermal cells, primary
sensory neurons (other,
not identi¢ed, papillary
cell types)
vessel cells
Epidermal cells,
epidermal neurons
Oral secreting tunic
cells, supporting cells,
hair cells
Ciliated cells
Motor neurons, sensory
neurons, GnRH+
neurons, neural gland
cells, epithelial cells of
the dorsal organ
Peribranchial/atrial
cells, atrial secreting
tunic cells, stigmatal
ciliated and parietal
cells
In italics: neuronal derivatives. See Figures 7 and 10 for details regarding the pathway of di¡erentiation of stomodeal and atrial placodes.
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L. MANNI ET AL.
adenohypophyseal placodes (Holland and Holland,
2001). However, at the moment, clear morphological evidences of placodes are lacking in amphioxus. We cannot exclude that they, although
present, were not found, or that their absence
reprents not a primitive, but a secondarily derived
condition. Indeed, amphioxus, despite its close
relation with vertebrates, shows important characteristics which seem to be derived, such as the
absence of a true heart in vertebrate-like circulatory system, almost absence of hemocytes, and
absence of tight and gap juntions, present in the
other deuterostomes (Lane et al., ’87).
This report represents the first attempt to
clarify the anatomical signs of the appearance of
placodal structures during the development of C.
intestinalis and B. schlosseri, model species of
solitary and colonial ascidians, respectively. We
considered both the thickenings of the ectodermal
epithelium capable of differentiating nonepidermal cell types, and the ectodermal territories
whose basal lamina showed interruption permitting the delamination of cells acquiring neurogenic characteristics, as ‘‘neurogenic placodes.’’
Similar criteria were suggested by Schlosser and
Northcutt (2000) to identify the neurogenic
placodes in Xenopus laevis. We have been able to
recognize a number of neurogenic placodes in
ascidian larvae: the rostral-, the stomodeal-, the
neurohypophyseal-, and the atrial placode. These
placodes, for their position as respect to the
regionalisation of the ascidian larval nervous system
and a series of properties (positional, morphological,
embryonic, molecular, developmental), can be directly compared to vertebrate cranial placodes. In
particular, the early position of these ascidian
placodes with respect to the neural plate is of
extreme importance in the comparison with the
cranial placodes in early vertebrate embryos, because a direct comparison between the central and
peripheral nervous system of adults of ascidian and
vertebrate has scarce significance. It could be a
matter for discussion if other thickenings of the
ectodermal epithelium, e.g., branchial ones, not
producing neuronal derivatives nor delaminating
cells, might be considered placodes in a broad sense.
Actually, the branchial thickenings, together with
the thickenings we found at the level of oral siphon
rudiment in B. schlosseri or the atrial siphon
rudiments, can represent cell shape changes in
relation to fusion and perforation of two leaflets,
then they could not be considered placodes in the
strict sense such as for the hair and feather
rudiments of vertebrates. Moreover, the bud thick-
ening of B. schlosseri despite its morphological
characteristics, cannot be included among the
placodes for its ability to produce an entire organism.
Our study caused us to reconsider some pioneer
histological reports (Willey, 1893a,b; Grave and
Woodbridge, ’24; Garstang and Garstang, ’28;
Grave, ’34; Scott, ’34; Grave and Riley, ’35; Katz,
’83), which today serve as the basis for comparative anatomical studies. Actually, the larvae of
ascidians exhibit a wide variability among species
(Millar, ’71; Burighel and Cloney, ’97), and the
detailed organisation of their anatomy is little
known, although they are a popular model among
embryologists and impressive progress has been
made in the application of molecular tools to their
study. As a consequence, current researchers often
refer to the old, schematic drawings during
discussion and interpretation of their results. This
is also true for the larva of C. intestinalis, which,
despite recent improved knowledge of its genome
(Dehal et al., 2002; Cañestro et al., 2003) and
detailed observations on its nervous system
(Nicol and Meinerthagen, ’88; Meinertzhagen et al.,
2000; Okada et al., 2001), has morphological
aspects, which need to be better investigated.
Rostral placode
The rostral placode may be considered to be a
neurogenic placode, since it gives rise to the
ampullae/preoral lobe, epidermal neurons and
sensory adhesive organs (papillae). Some Authors
discussed the possibility that the latter are
homologous to the cement and hatching glands
of vertebrates because they share the ventral
position as respect to mouth and some developmental genes (e.g., BMPs-5-8) (Katz, ’83; Miya
et al., ’96; De Bernardi, 2002; Groppelli et al.,
2001, 2003). Thus homology between the rostral
placode and the ectodermal thickenings of the
anterior region of vertebrates, originating the
cement and hatching glands, may be proposed.
However, Baker and Bronner-Fraser (2001) and
Schlosser (2002) do not consider the territories of
cement glands and hatching glands of amphibians
to be placodes in the strict sense, because unlike
typical placodes, which arise from the deep
ectodermal layer, they develop from a superficial,
ectodermal layer. An homology of the ascidian
rostral placode with the vertebrate olfactory
placode is less teneable for differences in position
(the rostral placode is ventral to the mouth, the
olfactory is dorsal) and in cell constituents (i.e.,
PLACODES IN ASCIDIANS
the rostral placode lacks GnRH producing elements).
The papillae of B. schlosseri are noteworthy
because they possess a sort of ganglion at their
base (Grave, ’34; Torrence, ’83). In vertebrates,
ganglionic neurons derive by aggregation of cells,
delaminated and migrated from the cranial placodes or neural crest. In contrast, in B. schlosseri,
the ‘‘papillary ganglia’’ are formed from the basal
portion of primary sensory cells originating from
the rostral placode and maintaining the original
relationships with the contiguous cells, comprising
tight junctions and the basal lamina. Thus, these
ganglia are not true ganglia, because no process of
delamination and migration, nor basal lamina
interruption, has occurred during their formation.
In B. schlosseri the ampullae attach the zooid to
the substrate, whereas in the C. intestinalis
juvenile they are absent and a unique evagination,
the stalk, substitutes for them. Both the adhesive
edges of the ampullae and the stalk are apically
endowed with a thickened epithelium. However,
they cannot be considered placodes because they
are not transitory structures and do not show
epithelial-mesenchymal transaction. The adhesive
edges maintain their columnar aspect in the adult
for an efficient secretory activity (Zaniolo and
Trentin, ’87). It is noteworthy that the developing
ampullae in Molgula occidentalis, expressing the
gene Dll in their distal region during metamorphosis, have been compared to outgrowth in other
animal appendages (Panganiban et al., ’97).
Stomodeal placode
The definition of the stomodeal area in ascidians
raises some questions because different situations
occur and are exemplified in C. intestinalis and
B. schlosseri. In C. intestinalis the stomodeum
appears as a typical ectodermal invagination
contributing to the formation of the oral siphon,
while in B. schlosseri it corresponds to the area of
the anterior dorsal groove plus the end of the
epithelium of the pharyngeal cavity which forms
the ciliated duct of the neural gland. This anterodorsal ‘‘pharyngeal region’’ has the ability to
secrete the tunic, i.e., a property which marks
epidermal cells. Thus, this evidence demonstrates that the ciliated duct is not endodermal
in origin, as originally believed to be the case
for B. schlosseri and other species (Seeliger, ’93;
Garstang and Garstang, ’28; Elwin, ’37), but is
ectodermal in origin (Manni et al., ’99), such as in
C. intestinalis. The homology between the ascidian
499
ciliated duct rudiment and the vertebrate Rathke’s
pocket has long been debated and recently was reproposed (Manni et al., ’99). Molecular data
(Christiaen et al., 2002; Boorman and Shimeld,
2002)
reinforce
this
hypothesis:
both in C. intestinalis and in B. schlosseri, a gene,
homologous to the vertebrate Pituitary homeobox
gene (Pitx), is expressed during development in the
anterior neural ridge. In vertebrates, this area
represents the presumptive territory of the adenohypophysis (Couly and Le Douarin, ’88; Kawamura
et al., 2002). Moreover, in C. intestinalis the gene is
expressed in the ciliated duct of the neural gland of
adult zooids (Boorman and Shimeld, 2002).
We have shown that the velum and the tentacles
of the oral siphon derive from a thickened area,
which may be considered a placodal structure,
secondarily derived from the stomodeal placode.
This has the ability to differentiate sensory cells,
whose presence was observed by Seeliger (1893) in
adult C. intestinalis, and recently also in botryllid
ascidians where they are secondary sensory cells
and form the coronal organ (Burighel et al., 2003).
Our data demonstrate that in B. schlosseri, the
oozooid, like the blastozooid, possesses a coronal
organ, considered as an homologous to the vertebrate octavo-lateralis system on the basis of
morphological and molecular data, in particular,
for the structural characteristics of their cells
equipped with an eccentric cilium, their afferent
and efferent synapses with neurites connected to
the brain, and their alignement (see Burighel et al.,
2003). This proposed homology could rise a possible
criticism because 1) the coronal organ is into the
mouth, whereas the lateral line elements of
vertebrates lie on the head and trunk skin, and 2)
the ascidian hair cells do not possess polarized
stair-step steeovilli, a common derived characteristic of vertebrate hair cells. However, it is to
consider: 1) in several aquatic vertebrates the
lateral line elements reach and extend around the
mouth (Coombs et al., ’88); the coronal organ is
located external to the oro-pharyngeal limit (floor
of the stomodeum/pharynx) and anterior to the
aperture of the ciliated duct (derived from the
possible homologous of the adehypohyseal placode);
the ascidian body is completely covered by the tunic
and the coronal organ lies just in the most exterior
tunic-free available surface; the coronal organ is of
epidermal origin in that it derives from a region
able to produce tunic in embryo (Manni et al., ’99);
from a molecular point of view, the ascidian
stomodeum expresses early the developmental
Pax 2/5/8 gene, which marks the otic placode in
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L. MANNI ET AL.
vertebrates (Wada et al., ’98); in vertebrates much
of the embryonic ectoderm (included the most
anterior) is competent to generate an otic placode if
taken at an early age (Gallagher et al., ’96; Groves
and Bronner-Fraser, 2000a). 2) As regard the
absence of polarized microvilli in coronal hair cells,
it must be considered that there is variability
among vertebrate hair cells and some taxa, as the
hagfishes (Braun and Northcutt, ’97), lack descending step-like series of microvilli.
The stomodeal placode can be considered to be a
multiple placode, with a nonneurogenic component giving rise to the ciliated duct (Manni et al.,
’99), and a neurogenic component differentiating
into the sensory cells. Multiple placodes are
present in vertebrates, as in the case of the
olfactory and adenohypophyseal placodes, which
are grouped together in the anterior neural plate,
close to the presumptive hypothalamic region (Couly
and Le Douarin, ’88; Whitlock and Westerfield,
2000; Kawamura et al., 2002). It would be of interest
to investigate if the stomodeal placode is also able to
delaminate sensory neurons which migrate to the
developing cerebral ganglion, similar to that which
occurs in the formation of the vertebrate acousticolateralis ganglia. This hypothesis seems tenable and
should be considered, especially since the stomodeal
placode is very close to the neurohypophyseal
placode, which undergoes a massive proliferation
and delamination of pioneer cells responsible for
building up the cerebral ganglion.
Neurohypophyseal placode
The neurohypophyseal duct is a structure
derived from the anterior part of the embryonic
neural plate and represents the rudiment of the
neural complex of the adult ascidian. Its wall,
differentiating into the neural gland, proliferates
and delaminates pioneer cells, which coalesce to
form the cerebral ganglion (Torrence, ’83; Manni
et al., ’99). Despite the apparent diversity in
timing of differentiation, shape and dimension of
the neurohypophyseal duct in the two species we
have studied, its morphogenesis and the mechanism of neurogenesis occur mainly with the same
modality. Some differences regard the formation
of the dorsal strand, which is missing in B.
schlosseri and is substituted by the dorsal organ,
homologous to the dorsal strand. A number of cell
types are recognizable in the structures derived
from the neurohypophyseal duct in ascidians
(reviewed in: Manni et al., ’99; Kawamura et al.,
2002). These include all the motor neurons,
concentrated in the ganglion; sensory neurons
associated to the coronal organ; ganglionic neurons immunoreactive to GnRH, adrenocorticotropin, and prolactin; mesenchymatous cells of the
neural gland, some of them immunoreactive to
adenohypophyseal hormones and epithelial cells of
the dorsal strand with neurogenic potential.
Moreover, epithelial cells of the dorsal strand are
probably involved in the formation of the somatic
gonad rudiment (Takamura et al., 2002); and
GnRH-positive neurons are in the dorsal strand
plexus (Mackie, ’95). Because of its embryonic
origin from the anterior neural plate and its
ability to differentiate the above mentioned cell
kinds, the neurohypophyseal duct has been considered to includes components homologous to the
hypothalamus and the olfactory placode (Burighel
et al., ’98; Manni et al., ’99). The strict correspondence between the various regions of the neurohypophyseal duct and the ciliated duct rudiment,
with the primordia of adenohypophysis, hypothalamus and olfactory epithelium should be further
investigated.
Atrial placode
In 1983, Katz proposed that the atrial primordia
of C. intestinalis were comparable to the otic
placodes of vertebrates: this idea was based both
on the topological relationship of the two atrial
invaginations and the brain, and the presence of
the cupular organs in the atrial chamber (Fedele,
’23; Bone and Ryan, ’78), reminiscent of the
vertebrate neuromasts. The possible homology
was successively analysed by other authors on
the basis of paleontological interpretations (Jefferies, 2001) and data coming from molecular
biology (Baker and Bronner-Fraser, ’97; Wada
et al., ’98; Shimeld and Holland, 2000). At first,
the cupular organs were of special importance in
supporting this hypothesis; but recently, doubts
have been raised about the homology between
cupular sensory cells, which are primary sensory
neurons, and vertebrate hair cells (Mackie and
Singla, 2003). Moreover, the recent evidence that
other mechanoreceptors, composed of primary
sensory cells, the capsular organs and the cupular
strand, are present in the atrium of some species,
suggests that ascidians possessed different mechanoreceptors, probably evolving from clusters of
ciliated mechanoreceptors cells (Mackie and Singla, 2003, 2004). We propose that the paired atrial
rudiments represent a neurogenic placode owing
to their ability to differentiate sensory cells. Their
PLACODES IN ASCIDIANS
homology with the vertebrate otic placodes remains to be further investigated, although at the
moment it is supported by developmental and
molecular data. Considering that vertebrate embryos exhibit the potential to form otic derivatives
from both the ectoderm close to the hindbrain and
the anterior epiblast (Gallagher et al., ’96; Groves
and Bronner-Fraser, 2000), according to the
hypothesis that the chordate ancestor possessed
a wide placodal field, we propose that in ascidians
the capacity to produce specialised mechanoreceptors as the hair cells was restricted to the anterior
region, while the posterior region maintained the
neurogenic ability to differentiate other mechanoreceptors.
In B. schlosseri the atrial rudiment arises as a
single ectodermal mid-dorsal anlage. Nevertheless, in both the species examined, each atrial
rudiment closes to form a small atrial cavity,
which only secondarily re-opens, owing to the
formation and aperture of the atrial siphon. The
latter, in B. schlosseri, forms at the level of the
epidermis of the posterior region of the dorsal
groove. As in the case of the stomodeal placode,
the atrial placode is involved in the formation of
sub-areas (Fig. 10): the thickening of the atrial
siphon rudiment, derived from the dorsal atrial
cells secreting tunic, and the thickenings of the
protostigmata rudiments, derived from the peribranchial epithelia.
The perforation of pharyngeal fissures during
embryonic development represents a distinctive
characteristic of all chordates. The process of
adhesion of the ectodermal atrial epithelium to
the endodermal pharyngeal epithelium is considered homologous to pharyngeal plate formation
during vertebrate embryogenesis (Manni et al.,
2002). The development of the protostigmata (or
primary gill slits) has been described in both the
species analysed (Willey, 1893a; Berrill, ’47;
Manni et al., 2002) and no sensory cells have been
recognised. We cannot consider the protostigmata
thickenings as placodes in strict sense, since their
presence could be only related to the process of
fusion. This differs from the situation in vertebrates, in which two series of neurogenic placodes,
the epibranchial and the hypobranchial, are
associated with pharyngeal pouches (Schlosser
and Northcutt, 2000; Schlosser, 2003).
In colonial ascidians the young bud forms an
outer epithelial vesicle containing another inner
one, whose embryonic origin varies among species
(Satoh, ’94). In botryllids, the inner vesicle is
ectodermal, deriving from a specialised region of
501
the atrial epithelium, and appears as a thickened
button-like area, so that it could be considered,
from a morphological point of view, to be a
placode, able to develop into many organs and
different cell types, including nerve elements.
However, this kind of ability is typical of ascidian
buds, irrespective of the tissue (which can be
either mesodermal or endodermal) that gave rise
to them; thus, we do not consider the thickening of
the bud primordium as a placode.
A common primordium for all placodes?
Recent data from vertebrates suggest that all
placodes arise, from an ontogenetic point of view,
from a common placodal primordium located at
the border of the neural plate/neural crest and
future epidermis (reviewed in Schlosser and
Northcutt, 2000; Baker and Bronner-Fraser,
2001; Noramly and Grainger, 2002; Schlosser,
2002). This suggests that placodes may also have
been derived over evolutionary time from a
common precursor. In this respect molecular data
on developmental gene expression and cell lineage
of blastomeres in ascidians should be taken into
consideration. Genes involved in vertebrate placodes (Pax 2/5/8, XAG, Pax6, Pitx) and neural crest
differentiation (Pax3/7, Distal-less, Msx, Bmp2/4,
Snail) were found to be expressed in corresponding
territories in ascidians (reviewed in Holland and
Holland, 2001; Meinerthagen and Okamura, 2001).
Moreover, on the basis of the lineage of the
ascidian nervous system (Lemaire et al., 2002 for
review) some of structures we interpret as placodes
derive from the anteriormost regions of the neural
plate: this region do not roll up to contribute to the
central nervous system but differentiates into the
dorsoanterior epidermis, which includes the derivatives of the rostral placode and the stomodeal
placode. The neural plate region immediately
posterior participates to the formation of the
neurohypophyseal placode. The cell lineage of
blastomeres involved in the atrial placode formation was not determined; however, their topographic localisation, their relation with contiguous
tissues and the expression of Pax 2/5/8 gene in
their area (Wada et al., ’98), indicate that they
derive from a territory at the lateral border of the
neural plate. Taken together, the available data on
organisation and lineage of the ascidian nervous
system suggest that the presence of a common
embryological territory, from which all placodes
differentiate, may be a feature shared by all the
chordate early embryos.
502
L. MANNI ET AL.
LITERATURE CITED
Fig. 11. Cladogram illustrating the proposed origin of the
placodes in chordates. Black bras indicate appearance of
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The hypothetical homology between ascidian
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ascidian stomodeal placode contains homologous
elements to the vertebrate adenohypophyseal and
lateral line placodes; the ascidian neurohypophyseal placode contains elements homologous to the
olfactory placode and hypothalamus; ascidian
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addition to the rostral placode for adhesive organs,
whereas, the lens, profundal, trigeminal, epibranchial and ipobranchial placodes are synapomorphies of vertebrates (Fig. 11).
ACKNOWLEDGMENTS
We thank C.V.H. Baker and G. Schlosser for
discussions and comments on the manuscript. We
thank Mr. C. Friso for the drawings. This
investigation was supported by grants from
Ministero della Università e Ricerca Scientifica e
Tecnologica and by University of Padova to P.B.
and L.M., and by a grant from the E.M.P.
Musgrave Fund to N.J.L.
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