Download Novel, Secondary Sensory Cell Organ in Ascidians: In Search of the

THE JOURNAL OF COMPARATIVE NEUROLOGY 461:236 –249 (2003)
Novel, Secondary Sensory Cell Organ
in Ascidians: In Search of the Ancestor
of the Vertebrate Lateral Line
PAOLO BURIGHEL,1* NANCY J. LANE,2 GASPARINI FABIO,1 TIOZZO STEFANO,1
GIOVANNA ZANIOLO,1 MARIA DANIELA CANDIA CARNEVALI,3
1
AND LUCIA MANNI
1
Dipartimento di Biologia, Università di Padova, I-35121 Padova, Italy
2
Department of Zoology, University of Cambridge, Cambridge CB2 3EJ, United Kingdom
3
Dipartimento di Biologia, Università di Milano, I-20133 Milano, Italy
ABSTRACT
A new mechanoreceptor organ, the “coronal organ,” located in the oral siphon, is described by light and electron microscopy in the colonial ascidians Botryllus schlosseri and
Botrylloides violaceus. It is composed of a line of sensory cells (hair cells), accompanied by
supporting cells, that runs continuously along the margin of the velum and tentacles of the
siphon. These hair cells resemble those of the vertebrate lateral line or, in general, the
acoustico-lateralis system, because they bear a single cilium, located centrally or eccentrically
to a hair bundle of numerous stereovilli. In contrast to other sensory cells of ascidians, the
coronal hair cells are secondary sensory cells, since they lack axonal processes directed
towards the cerebral ganglion. Moreover, at their base they form synapses with nerve fibers,
most of which exhibit acetylcholinesterase activity. The absence of axonal extensions was
confirmed by experiments with lipophilic dyes. Different kinds of synapses were recognized:
usually, each hair cell forms a few afferent synapses with dendrites of neurons located in the
ganglion; efferent synapses, both axo-somatic (between an axon coming from the ganglion
and the hair cell) and axo-dendritic (between an axon coming from the ganglion and an
afferent fiber) were occasionally found. The presence of secondary sensory cells in ascidians
is discussed in relation to the evolution of sensory cells and placodes in vertebrates. It is
proposed that the coronal organ in urochordates is homologous to the vertebrate acousticolateralis system. J. Comp. Neurol. 461:236 –249, 2003. © 2003 Wiley-Liss, Inc.
Indexing terms: Urochordata; hair cells; tentacles; oral siphon; sensory system; placodes
Ascidians (Urochordata) are filter-feeding invertebrates
possessing a swimming tadpole larva which exhibits the
structural body plan of chordates (Burighel and Cloney,
1997). The larval central nervous system (CNS) derives
from a typical neural tube and shows a close relationship
with the vertebrate nervous system, sharing with the
latter similarities in organization and developmental patterning mechanisms (Wada et al. 1996, 1998; Shimeld and
Holland, 2000; Manni et al., 2001). At metamorphosis,
this nervous system degenerates and a remnant of the
neural tube, the neurohypophyseal duct, produces the
neural complex of the sessile adult (Burighel et al., 1998;
Manni et al., 1999, 2001). This consists of an invertebratelike cerebral ganglion associated with a neural gland
which, posteriorly, continues with a dorsal strand flanked
by a plexus of neurons reacting positively to antibody
for the hypothalamic gonadotropin-releasing hormone
© 2003 WILEY-LISS, INC.
(Mackie, 1995). The peripheral nervous system derives
from nerves extending from the ganglion and from sensory
cell bodies located at the periphery of the body; nerves are
mixed, containing axons of both sensory and motor neu-
Grant sponsor: Ministero della Università e Ricerca Scientifica e Tecnologica and University of Padova (P.B.); Grant sponsor: Emily Mary Pratt
Musgrave Fund (N.J.L.).
*Correspondence to: Paolo Burighel, Dipartimento di Biologia, Università di Padova, via U. Bassi 58/B, I-35121 Padova, Italy.
E-mail: [email protected]
Received 17 October 2002; Revised 21 January 2003; Accepted 1 February 2003
DOI 10.1002/cne.10666
Published online the week of May 5, 2003 in Wiley InterScience (www.
interscience.wiley.com).
SECONDARY SENSORY CELLS IN ASCIDIANS
rons (Mackie and Wyeth, 2000; Burighel et al., 2001; Zaniolo et al., 2002).
Ascidians possess a variety of receptors, whose sensory
cells usually bear modified cilia. These are generally classified as mechano-, vibration-, gravity-, chemo-, photo-,
and proprio-receptors (Bone and Mackie, 1982). As commonly occurs in invertebrates (Budelman, 1989), all these
receptor cells are primary sensory cells, since they have
their cell body located at the periphery and send their
axon into the CNS. An exception to this pattern is seen in
the appendicularians, planktonic animals considered to be
the earliest lineage to diverge within the urochordates
(Wada, 1998). Appendicularians possess unusual secondary sensory cells which form electrical synapses at their
base with peripheral nerve fibers coming from the CNS
(Bone and Ryan, 1979; Bone et al., 1979; Bollner et al.,
1986; Bone and Mackie, 1982).
Among the mechanoreceptors of tunicates, the cupular
sense organs are of special interest. These are found in the
atrium of the adult of the ascidian Ciona intestinalis
(Fedele, 1923; Millar, 1953; Goodbody, 1974; Bone and
Ryan, 1978) and salps (thaliacean urochordates) (Fedele,
1920). Their name derives from the presence of groups of
ciliated cells embedded in a gelatinous cupula. Their importance derives from their similarities with the vertebrate otic and lateral line receptors. Unlike vertebrate
hair cells, however, which are dedicated receptors that
require secondary innervation from neurons located in the
sensory ganglia of the VIIIth cranial nerve, the cupular
sense organs are constituted of primary sensory neurons
whose axons project directly to the CNS. Another sense
organ, the capsular organ, analogous to the cupular organ,
was recently described by Mackie and Singla (2003) in the
atrium of Chelyosoma productum. Its sensory cells are
primary receptors bearing a collar of microvilli surrounding a long cilium which projects into the capsule cavity.
Cupular and capsular mechanoreceptors differentiate in
the atrial chamber which receives the water coming from
the pharynx through the gill slits. In botryllids, the atrial
chamber arises as an ectodermal mid-dorsal anlage which
extends ventrally to surround the pharynx at right and
left (Manni et al. 2002). In more primitive ascidians, such
as Ciona intestinalis, it originates from a pair of dorsal
ectodermal invaginations which may be compared to the
otic placodes of vertebrates, being topographically located
in a corresponding position (Katz, 1983). The possible
homology between the atrial primordia and the otic placodes was proposed by different authors on the basis of
paleontological evidence (Jefferies, 2001), the anatomical
relationships of the atrial primordia, the morphology of
the cupular organs, and data coming from molecular biology, in that both the developing atria and otic placodes
express members of the Pax2/5/8 gene family (Baker and
Bronner-Fraser, 1997; Wada et al., 1998; Shimeld and
Holland, 2000).
In vertebrates, sensory cells are derived, together with
the sensory neurons, from neural crest or placodes, traditionally thought to be unique to vertebrates (Northcutt
and Gans, 1983). In particular, neural crest-derived receptors belong to primary sensory neurons, while placodederived receptors may be primary neurons (the olfactory
sensory cells) or secondary sensory cells (the hair cells)
associated with neurons. The hair cells are ciliary, highly
differentiated mechanoreceptors and their name derives
from the peculiar microvilli, called stereovilli (synony-
237
mous with stereocilia), that protrude into the fluid-filled
cavities of the inner ear or lateral line organs. Secondary
sensory cells are considered to be phylogenetically derived
from primary sensory cells (Baker and Bonner-Fraser,
1997; Fritzsch and Beisel, 2001).
The possibility that precursors of placodal cells may
occur in modern invertebrate chordates, cephalochordates
and urochordates, is of interest in relation to ideas concerning the evolutionary origin of vertebrate sensory cell
types. In the cephalochordate Amphioxus, considered to be
the closest living relative of vertebrates, different sensory
cells, type I cells and type II cells, are found scattered in
the rostral epithelium. Type I cells are primary sensory
cells, while type II cells are secondary sensory cells, with
synaptic terminals borne out on short extensions of the
cell body (Bone and Best, 1978; Lacalli and Hou, 1999;
Holland and Holland 2001; Holland and Yu, 2002). For
both types of cells, neither their embryological origin nor
their functional properties are known, although they are
generally assumed to be either mechano- or chemoreceptors on the basis of their morphology. On the oral spines
which project across the mouth there are secondary mechanoreceptor cells, the oral spine cells, involved in the
rejection response (Lacalli et al., 1999). Despite differences in function, these sensory cells were considered homologous to the vertebrate taste buds which, on the other
hand, are neither placodally nor neural crest-derived
(Barlow and Northcutt, 1995). Thus, the available data do
not resolve the evolutionary issue of how amphioxus sensory cells relate to the receptor cells of vertebrates.
The possible homology between placodes (the hypothalamic-hypophyseal-olfactory placode) and an embryonic structure of ascidians (the neurohyphyseal duct) has
recently been proposed (Manni et al., 1999, 2001). Moreover, in ascidians Burighel and Cloney (1997) have reported the presence of secondary sensory cells in the oral
siphon of botryllids; such cells bear a short cilium with
microvilli and their basal plasmalemma is in contact with
a number of fine nerve fibers. The oral siphon of most
ascidians bears, at its base, a crown of tentacles capable of
responding to different kinds of stimuli (mechanical, thermal, chemical) (Hecht, 1918a, b; Day, 1919), but the presence of sensory cells on them has only been previously
mentioned by Seeliger (1893).
In the present study we examined, by scanning and
transmission electron microscopy, the oral and atrial siphons in two ascidians, Botryllus schlosseri and Botrylloides violaceus, in order to verify unequivocally the presence of sensory cells. Here we describe a novel sensory
organ, the coronal organ, located on the inner wall of the
oral siphon. Its ciliated receptor cells are innervated by
secondary sensory neurons, located in the cerebral ganglion, and aligned in such a way so as to form a continuous
sensorial line, strongly reminiscent of the lateral line system of vertebrates.
MATERIALS AND METHODS
Specimens of Botryllus schlosseri and Botrylloides violaceus Oka, 1927 (family Styelidae, order Stolidobranchia)
used in this study were from the laboratory, where colonies adhering to glass are routinely cultured according to
Sabbadin’s (1955) technique. These colonies were originally collected from the lagoon of Venice. Each colony
contains numerous blastozooids interconnected by a vas-
238
P. BURIGHEL ET AL.
cular network running throughout the common tunic and
grown through regular cycles of blastogenic generations.
The two species differ greatly in the size and morphology
of the larva, which, in Botrylloides violaceus, has a trunk
about 1 mm in length with about 30 –32 ampullae and, in
Botryllus schlosseri, a trunk of about 450 ␮m, with eight
ampullae. In both species the larva metamorphoses into a
sessile oozooid, the founder of the colony, which grows
through a series of blastogenic generations. The blastozooids of Botrylloides violaceus are arranged in doublerow systems parallel to one another, while the blastozooids of Botryllus schlosseri are grouped in star-shaped
systems around the common cloacal siphon.
Whole-mount preparation
Colonies were anesthetized with MS 222, fixed in
Bouin’s fluid, washed in 50% ethyl alcohol, rehydrated,
and stained with Mayer’s hematoxylin. After washing in
distilled water, they were dehydrated in alcohol and
mounted with balsam.
Transmission electron microscopy
More than 30 adult blastozooids selected from different
colonies were anesthetized with MS222 and 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 embedded in Araldite.
Thick sections (1 ␮m) 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 tentacles were cut and analyzed in order to
verify the presence of axonal processes extending from the
basal surface of the sensory cells. Micrographs were taken
with a Hitachi H-600 electron microscope operated at 80
kV. To reveal acetylcholinesterase (AChE), tissue was
treated following the method of Gautron (1982), prefixing
the specimens in 1.5% glutaraldehyde buffered with 0.1 M
sodium cacodylate, pH 7.4, containing 5% sucrose and
using acetylthiocholine iodide (Sigma, St. Louis, MO) as
substrate. Specimens were then postfixed in 1% OsO4 in
phosphate-buffered-sucrose, dehydrated, and embedded
in Araldite. Ultrathin sections were stained with uranyl
acetate and lead citrate. Controls were treated as described above, but without the substrate.
Scanning electron microscopy
Pieces of colony containing adult blastozooids were fixed
as described for transmission electron microscopy. After
dehydration, dissection was performed in order to expose
the oral siphon lumen with the velum and tentacles. Specimens were critical-point dried, sputter-coated with goldpalladium, and observed under a Cambridge Stereoscan
260.
Axonal tracing technique
In order to verify if sensory cells are primary or secondary, the lipophilic membrane dye 1,1⬘-dioctadecyl3,3,3⬘,3⬘-tetramethylindocarbocyanine perchlorate (DiI)
(Aldrich, Milwaukee, WI) was applied on the surface of
velum and tentacle cells using a capillary tube attached to
a micromanipulator. The rationale was that, following an
extracellular application, the DiI readily diffuses over the
whole membrane of the cell, rather than remaining in and
diffusing through the aqueous cytoplasmic component or
extracellular spaces; thus, if the cells under consideration
are primary neurons, applying the dye on the cell bodies
makes it possible to follow the pathway of their entire
axonal projections. In contrast, if the cells are secondary
neurons then the dye marks as a spot on each cell (Clarke,
1999). A small aliquot of DiI (at the concentration of 3
mg/mL in DMF) was delivered in the interior of the oral
siphon to touch the velum and surface of the tentacles.
The dye deposit was checked with a conventional fluorescence microscope at the time of and after the application.
DiI fluoresces an intense red. The dye in excess was gently
removed putting the injected colonies into filtered seawater.
All photos were acquired with a Duoscan (Agfa), had
levels adjusted, were collated, and typeset in Corel
Draw 9.
RESULTS
General organization of the oral
and atrial siphons
Each blastozooid of Botryllus schlosseri and Botrylloides
violaceus, in every system of the colony, has its own oral
(or inhalant) siphon which opens independently at the
periphery. The atrial (or exhalant) siphon converges, together with the siphons of the other zooids, in a common
central cloacal chamber, in the case of Botryllus schlosseri
(Fig. 1A), or in a common, long cloacal channel, in the case
of Botrylloides violaceus. Following Millar (1953) and
Burighel and Cloney (1997), the “oral siphon” comprises
the area between the external rim of the siphon and the
ring of the velum from which the tentacles project (Fig.
1B–D). In the oral siphons of both species analyzed, the
crown of tentacles is formed of first- and second-order
tentacles. There are four first order tentacles symmetrically arranged: two long, lateral ones, located on the
right–left axis, and two shorter, medial (ventral and dorsal) ones. The shorter, second-order tentacles alternate
regularly with the first-order ones. In Botryllus schlosseri,
Fig. 1. Botryllus schlosseri. A: One star-shaped system of a colony
showing the oral siphons (arrows) of each blastozooid (b) and the
central, common cloacal chamber (asterisk). t, tunic. Scale bar ⫽ 1.5
mm. B: Oral siphon of a blastozooid. dt, dorsal tentacle; lt, lateral
tentacle; vt, ventral tentacle; arrowheads, second order tentacle.
Whole-mount preparation. Scale bar ⫽ 150 ␮m. C: Detail of a lateral
tentacle. Whole-mount preparation. Scale bar ⫽ 60 ␮m. D: Section of
an oral siphon. The tunic (arrowheads) covers the inner side of the
siphon and part of the velum (v). t, tentacles. Scale bar ⫽ 60 ␮m.
E: Oral siphon as seen from the inner side of the blastozooid. velum
(v), and lateral (lt), ventral (vt) and dorsal (dt) tentacles. dl, dorsal
lamina; pb, pericoronal band. Scanning electron microscopy. Scale
bar ⫽ 90 ␮m. F: Velum (v) and lateral tentacle (lt) seen from the outer
side. Note the ciliated cells (arrowheads) of coronal organ. t, tunic.
Scanning electron microscopy. Scale bar ⫽ 20 ␮m. G: Ciliated sensory
cells (arrows) of coronal organ on lateral tentacle. Arrowheads point to
the line of microvillar protrusions (enlarged in the inset) of supporting
cells. One asterisk, upper side of the tentacle; two asterisks, flank of
the tentacle. Inset: detail to show the microvillar protrusions flanking
the sensory cells. Scanning electron microscopy. Scale bar ⫽15 ␮m;
inset scale bar ⫽ 2 ␮m. H,I: Hair cells on the velum. Each bears a
short, stiff cilium (c) accompanied by a group of stereovilli (arrowheads) as visible in detail in the inset. In I, microvillar protrusions
(arrows) of supporting cells are visible. Scanning electron microscopy.
Scale bar ⫽ 3 ␮m in H; scale bar ⫽ 2 ␮m in inset; scale bar ⫽ 1 ␮m in I.
SECONDARY SENSORY CELLS IN ASCIDIANS
239
Figure 1
240
P. BURIGHEL ET AL.
extends into the siphon as far as the atrial velum; the
epidermis is continuous with the atrial epithelium.
On the epidermis of the atrial siphons of both species,
we did not find any sensory structure. In particular, there
were no cupular or capsular organs like those described
from the atrial siphon of Ciona intestinalis (Bone and
Ryan, 1978) and Chelyosoma productum (Mackie and Singla, 2003), respectively. However, on the outer border of
the tentacles and velum of the oral siphons of both species
we found a row of previously undescribed sensory cells,
which are completely exposed to the inhalant current of
the seawater and are arranged in such a way as to constitute a sensory organ. This novel organ, which forms a
continuous sensory line bordering the crown of velum and
tentacles (Fig. 1F,G), will be referred to here as the coronal organ. We did not find any other sensory structures in
the oral siphons of the two species examined.
The histological organization of the oral siphon in Botryllus schlosseri and Botrylloides violaceus is similar —
the data used here document the organization of the novel
sensory organ was obtained mainly from observations on
Botryllus schlosseri.
Coronal organ
Fig. 2. A: View of the inner side of the siphon to show the coronal
organ bordering the anterior face of the velum and tentacles. The
tunic covering the epidermis is not indicated. B: Sketch showing the
zigzag arrangement of hair cells in the coronal organ.
the lateral tentacles are ⬃80 ␮m long, the dorsal and
ventral ones are ⬃55 ␮m long, and the second-order tentacles are ⬃33 ␮m long (Fig. 1E,F). The epidermis extends
onto the inner surface of the oral siphon and is continuous
with the pharyngeal epithelium. The velum is small and
circular and extends between, and is continuous with, the
tentacles themselves. The tunic covers the epidermis on
the inner side of the siphon and gets thinner in the proximal area of the tentacles and the velum (Fig. 1D). In
normal conditions, when the tentacles are raised, they are
extended on the same plane of the velum and form a sort
of filter at the base of the siphon (Fig. 1B). In relaxed
conditions the tentacles bend down toward the branchial
chamber (Fig. 1D). As they lack their own musculature,
their movements are due to a combination of different
factors, such as the pressure of blood and the activity of
siphon muscles.
We define “atrial siphon” in Botryllus schlosseri and
Botrylloides violaceus as the region between the rim of the
atrial siphon and the atrial velum, which marks the outer
border of the atrial chamber. It is noteworthy that the
atrial siphon, although somewhat different in the two
species, in both cases extends an epidermal languet on its
dorsal side which comes together, with the languets of
adjacent zooids, to form the roof of the common cloacal
chamber/channel. Like the oral siphon, in the atrial siphon the epidermis is accompanied by the tunic, which
When observed under the scanning electron microscope
(Fig. 1E,F), the tentacles and the velum of the oral siphon
exhibit a smooth surface and no particular specialization
of cell apexes can be recognizable, except for the sensory
cell line of the coronal organ (Figs. 1G–I, 2). We called
these sensory cells “hair cells” because each one is characterized by tufts of stereovilli and a single cilium. The
cilium is about 4 ␮m in length and 280 nm wide; stereovilli
are long and rod-like, 2–2.4 ␮m in length and about 110
nm in diameter. The hair cells have a zigzag arrangement
in a row bordering the apex of the velum and the upper
side (facing the external siphonal rim) of tentacles (Fig.
2B). More precisely, the file of hair cells runs along the
margin of the velum and the upper side of each tentacle in
Fig. 3. Botryllus schlosseri. Transmission electron microscopy.
A: Longitudinal section of the dorsal tentacle showing the tunic (t)
partially covering it. Numerous blood cells (bc) are inside the tentacle.
Scale bar ⫽ 8 ␮m. B: Transverse section of a tentacle. Hair cells (hc) are
located at the border of anterior side (top) of the tentacle. Some nerve
fibers (arrowheads) are visible adjacent to the basal plasmalemmata of
hair cells. sc, supporting cell. Scale bar ⫽ 4 ␮m. C: A hair cell (hc)
accompanied by the supporting cells (sc). Hair cell possesses a basal
nucleus (n), rough endoplasmic reticulum cisternae (rer), a supranuclear Golgi apparatus (G), and a hair bundle composed of a cilium (c)
accompanied by numerous stereovilli (s). The apical plasmalemma of
supporting cells present a thick glycocalyx (arrowheads) and microvillar
protrusions (arrows). bl, basal lamina; cr, ciliary rootlets; mb, multivesicular body; nf, nerve fibers. Scale bar ⫽ 2.2 ␮m. D: Detail of the apical
area of a hair cell. Note that the cell presents an eccentric cilium (c), as
demonstrated by its insertion close to the lateral plasmamembrane.
Arrow, centriole; arrowheads, ciliary rootlets; bb, basal body of the cilium; G, Golgi apparatus; m, mitochondrion; s, stereocilium; sc, supporting cells; tj, tight junction; za, zonula adherens. Scale bar ⫽ 0.3 ␮m.
E: Longitudinal section of hair cells. Note the cylindrical shape of the
cells. Arrowheads, sensory cilia; s, stereovilli; n, nucleus. Scale bar ⫽ 2
␮m. F,G: Tangential sections of the hair bundle showing the zigzag
arrangement of hair cells, some of which bear an eccentric cilium (arrows). The cilium (c) is accompanied by 90 –95 stereovilli (s). G: Enlargement of F and shows in detail in inset the fibrils (arrowheads) extending
radially between the cilium and the adjacent stereovilli. Scale bar ⫽ 2
␮m in F; 0.35 ␮m in G; 0.14 ␮m in inset.
SECONDARY SENSORY CELLS IN ASCIDIANS
241
Figure 3
242
P. BURIGHEL ET AL.
such a way as to form a continuous ring at the base of the
siphon, exposed to the inhalant seawater flow (Fig. 2A). In
a lateral tentacle of Botryllus schlosseri it is possible to
find about 150 –200 hair cells and the whole organ is
constituted of about 2,000 hair cells.
In sections (Fig. 3A,B), the epithelium of both the velum
and the tentacles is generally flat and becomes thicker at
the level of hair cells. Each tentacle contains nerve fibers
(we were able to count 15–20 neurites in sections of the
proximal part of the lateral tentacles), often located at the
base of hair cells. Blood cells were also present, but not
muscle cells, although the latter are present in the mantle
at the base of the tentacles.
Supporting cells
The file of sensory cells is flanked on both sides by
supporting cells whose lateral surface contacts the hair
cells (Fig. 3C). Supporting cells are similar to adjacent
epithelial cells in cytoplasmic characteristics, but differ in
position and shape. They are C-shaped cells and actually
enclose the sensory cells, so as to isolate them. Apically,
supporting cells elongate until they reach the collar of
stereovilli; their lateral membrane follows the lateral
membrane of the hair cells; basally, they partially cover
the sensory cells reducing their area available to contact
the nerve fibers. Apically, in proximity to the sensory cells,
they raise to form small, aligned microvillar protrusions
which, in their whole, delimit a sort of canal in which the
sensorial cilia and stereovilli are found (Figs. 1G,I, 2B,
3C). The supporting cells possess an apical, thick, and
very dense glycocalyx, which is also present on contiguous
epithelial cells but not hair cells. All the cells of the velum
and tentacles, both sensory and nonsensory, are joined
apico-laterally to each other by dense, tight junctions;
often, below them a zonula adherens can be recognized
(Fig. 3D).
Sensory cells
The sensory cells are cylindrical (Fig. 3E) and are joined
to the adjacent cells by tight junctions, whereas gap junctions were never observed on their lateral surfaces. Sensory cells have a large basal nucleus and scattered mitochondria and RER cisternae. The Golgi complex lies above
the nucleus and is composed of a few stacks of cisternae
and associated vesicles (Fig. 3C).
The hair cells bear an apical structure (Fig. 3D–G) composed of a stiff cilium accompanied by about 90 –95 long
stereovilli. The position of the cilium varies: it may be
found central to the corolla of stereovilli, or may be completely eccentric, where it inserts with its basal body close
to the lateral cell membrane. In the latter case, the tuft of
stereovilli occupies the remainder of the apical membrane.
The distribution of the two subtypes of hair cells (with
centric or eccentric cilium) appears random in the coronal
organ. Each cilium has a conventional 9⫹2 microtubular
arrangement, with a dense, short basal body connected to
poorly developed ciliary rootlets; a secondary centriole is
sometimes recognizable, lying perpendicularly to the first
(Fig. 3D). The stereovilli are stiff, unbranched, all the
same length, and are filled with microfilaments that are
continuous with the cytoskeletal filaments of the apical
cytoplasm. Both the ciliary and stereovilli membranes are
covered by a fibrillar fuzzy coat (Fig. 3G, inset). Several of
the coat fibrils, up to 40 nm long, extend radially to estab-
lish connections between adjacent stereovilli and between
the stereovilli and the ciliary shaft.
Synaptic connectivity
The basal plasmalemma of each hair cell lies on a basal
lamina, which forms a continuous fibrous layer supporting
both sensory and the other epithelial cells (Fig. 3C). At
certain sites the basal plasmalemma of sensory cells exhibits infoldings in the form of one or two grooves, which
are also bounded by extensions of supporting cells (Fig.
4A). In these grooves the typical extracellular matrix is
absent (Figs. 4A–D, 5). The grooves contain numerous
neurites showing a strict relationship with the sensory
cell membrane. The basal lamina of the sensory cells
intermingles with the fibrous matrix enveloping the nerve
fibers. A few nerve fibers are also grouped in the central
blood sinus of the tentacles and the velum. Frequently, in
longitudinal sections of sensory cells a small group of
nerve fibers can be seen to pass from one hair cell to the
adjacent ones running inside their basal groove; we occasionally found branching fibers or neurites entering the
basal groove coming from the central nerve fibers.
A number of conventional axo-somatic synapses were
seen on the sensory cells and axo-axonic synapses were
also recognized (Figs. 4, 5) in the vicinity of the sensory
cells. Synaptic contacts were identified by paired, thickened, electron-dense plasma membranes lying in parallel
20 –25 nm apart, with regularly arranged filaments traversing this cleft and small vesicles attached to the membrane dense material on one or both sides of the cleft. The
dense material consisted of an amorphous layer close to
the cytoplasmic membrane face, being more pronounced
on the postsynaptic side. This configuration corresponds
to the synapses described in the cerebral ganglion of Botryllus schlosseri (Burighel et al., 1998; Manni et al., 1999).
In some cases the same presynaptic area, furnished with
numerous vesicles, was seen to make synaptic contacts
with several different neurites, reaching a width of 1.3 ␮m
as in the case of the synapses shown in Figure 4A. Besides
the unidirectional synapses, which were predominant,
synapses were found that had vesicles on both sides.
Within the active zone we could distinguish synaptic vesicles with a homogeneous, moderately electron-dense content, or synaptic vesicles containing a dense core surrounded by a pale halo (Fig. 4C).
The system of neurite-soma connections was mainly
represented by afferent synapses (Figs. 4A–C), recognizable in that the presynaptic membrane, associated with
the synaptic vesicles, belongs to the hair cell. These synapses are involved in transferring the sensorial information from the hair cells to a dendrite of a neuron, whose
soma is in the cerebral ganglion. Frequently we observed
the same hair cell to form 2–3 afferent synapses with
different neurites.
Possible efferent synapses were occasionally recognized.
In these, the presynaptic membrane belongs to an axon of
a neuron whose soma is located in the cerebral ganglion,
while the postsynaptic membrane belongs to a hair cell
(Fig. 4D).
Moreover, we observed axo-dendritic synapses (Figs.
4D, 5) in the basal groove created by hair cells, or outside
the groove, at the base of sensory cells, as well as synapses
between neurites and supporting cells (Figs. 4A, 5).
Sections treated to reveal the enzyme AChE activity
showed a reaction product, in the form of minute, electron-
SECONDARY SENSORY CELLS IN ASCIDIANS
243
Fig. 4. Transmission electron microscopy of synaptic connectivity
of the coronal organ of Botryllus schlosseri. A: Numerous nerve fibers
(nf) are located in grooves formed by the basal plasmalemma of the
hair cell (hc) and the supporting cell (sc) and delimited by the basal
lamina (bl). On the left, an extended synaptic area, marked by numerous presynaptic vesicles (arrowheads) close to the hair cell plasmalemma is visible. Arrow, synapses between a nerve fiber and a
supporting cell; n, nucleus. Scale bar ⫽ 0.3 ␮m. B,C: Afferent synapsis
(white arrows) at the base of two adjacent hair cells (hc). The afferent
synapses are recognizable for the presence of presynaptic vesicles
close to the basal plasmalemma of hair cell and a pronounced density
on the membrane of the dendrite (d). C: An enlargement of the
squared area of B, close to the hair cell plasmalemma, presynaptic
vesicles with homogeneous, moderately electron-dense content (black
arrow) and synaptic vesicles containing a dense core surrounded by a
pale halo (white arrowhead). bl, basal lamina; m, mitochondrion in
dendrite; nf, nerve fiber. Scale bar ⫽ 0.4 ␮m in B; 0.15 ␮m in C.
D: Efferent axo-somatic synapsis (white arrows): the presynaptic
membrane belongs to the axon (a), while the postsynaptic membrane
belongs to the hair cell (hc), as shown by the density of the plasmamembrane. An axo-dendritic synapsis (arrowheads) is also visible
in tangential section. bl, basal lamina; nf, nerve fiber. Scale bar ⫽
0.25 ␮m.
dense granules, which marked the profile of nerve fibers
and was concentrated either in their membrane close to
sensory cells, or was free in the narrow, basal intercellular
spaces between adjacent sensory cells (Fig. 6A, B). The
product was never found in the cytoplasm of hair cells.
Although the basal membrane of hair cells often elongate to form protrusions that partially embrace the nerve
fibers, long cellular processes, recalling axonal fibers typical of primary sensory cells, were never encountered. In
this respect, when applied in vivo at the base of the oral
siphon, the lipophilic dye DiI marked the coronal organ
cells but never revealed the presence of cellular projections arising from sensory cells and reaching the cerebral
ganglion (Fig. 6C).
The main characteristics of sensory cells are schematically represented in Figure 7.
DISCUSSION
Sensory organs in urochordates
Our data demonstrate the presence of a novel sensory
organ, the coronal organ, in the oral siphon of Botryllus
schlosseri and Botrylloides violaceus, formed from specialized cells located in the upper side of the velum and
tentacles exposed to the inhalant seawater flow. These
sensory receptor cells are flanked by supporting cells and
bear apically a cilium accompanied by numerous, long,
244
Fig. 5. Schematic drawing of synaptic connectivity between the
basal plasmalemma of a hair cell and nerve fibers. One synapsis,
whose direction is not indicated, is between a supporting cell and a
nerve fiber.
stiffened stereovilli, while basally they form invaginations
into which nerve fibers run. Numerous unequivocal synapses were found between the basal membrane of these
sensory cells and the underlying nerve fibers, proving that
these receptor cells are secondary sensory cells.
The presence of possible sensory cells in the tentacles of
ascidians was mentioned by Seeliger (1893) in the species
Ciona intestinalis, but ignored by subsequent authors.
More recently, Burighel and Cloney (1997) suggested, in
the tentacles of the oral siphon of Botryllus schlosseri,
presumptive secondary sensory cells, a report now confirmed by our data.
Urochordates possess a variety of receptor systems for
different kinds of stimuli (Bone and Mackie, 1982; Bone,
1998; Mackie and Singla, 2003). As generally occurs in
invertebrates (Budelmann, 1989), receptor cells are simple primary sensory cells, scattered or grouped, usually
bearing cilia that are modified in some way. An exception
is found in the appendicularians, where particular secondary sensory cells form electrical synapses (gap junctions)
with neurites derived from the central nervous system
(Bone and Ryan, 1979; Bollner et al., 1986; Bone, 1998).
However, this seems to represent an unusual modification
that, together with other features, both morphological (Fenaux, 1998; Burighel et al., 2001) and genomic (Wada,
1998), render the appendicularians distinct from the other
urochordates.
Features and function of the coronal organ
The morphology of the sensory coronal cells provides
some information regarding coronal organ function, suggesting that it might be a hydrodynamic receptor system,
i.e., a responsive mechanoreceptor system sensitive to any
kind of motion in the water surrounding it or to particles
in its aquatic environment (Budelmann, 1989).
The ascidian larva has both a well-developed photoreceptor and a balance organ as navigational aids for exploring the environment and selecting the best substrate on
which to fix and metamorphose (Burighel and Cloney,
P. BURIGHEL ET AL.
1997; Sorrentino et al., 2000; Lacalli, 2001); in contrast,
the adult has a sessile mode of life and relatively little
demand is placed on its sensory system (Goodbody, 1974).
Almost all the adult’s sensory organs are concerned with
protection or defense and, among them, hydrodynamic
receptors play an important role in behavioral reactions to
water movements, which occur during feeding or in response to touch. It was previously supposed that the atrial
cupular organ of Ciona intestinalis (Bone and Ryan, 1978)
and the capsular organ of Chelyosoma productum (Mackie
and Singla, 2003) were mechanoreceptors involved in
monitoring and regulating the water current through the
atrial cavity. In Botryllus schlosseri and Botrylloides violaceus, we did not find sense organs in the atrial siphons;
nevertheless, animals of both species are able to respond
to vibrations, both transmitted through the solid base of
their attachment and from the seawater (unpubl. data). In
the absence of cupular sense organs, the hydrodynamic
receptors, if present, must be located elsewhere. Indeed,
botryllid zooids do not have their atrial siphon opening
directly to the exterior, as occurs in solitary species such
as Ciona intestinalis and Chelyosoma productum, but into
a common cloacal chamber/channel. A more logical location for hydrodynamic receptors might therefore be in the
oral siphon, rather than the atrial siphon.
Physiological studies on solitary ascidians demonstrated that the tentacles are sensitive to mechanical and
chemical stimuli and are a useful device in filtering out
any large particles from the inhalant water before they
reach the pharynx (Hecht, 1918a, b; Day, 1919). For its
location and its morphological features, the coronal organ
represents a good candidate for a hydrodynamic receptor
system in botryllids, having receptor cells exposed to the
inhalant current and bearing a cilium accompanied by
stereovilli. In most invertebrate phyla, epithelial mechanoreceptor cells sensitive to water movements carry a
concentric hair bundle of a single cilium surrounded by a
stiff cylinder of interconnected stereovilli (collar configuration). This configuration is already known to exist in
cnidarian polyps and medusae and is also the standard for
the epithelial mechanoreceptors in the lower chordates
(Budelmann, 1989, 1992; Thurm et al., 1998). In this
respect, it is equivalent to the lateral line receptors of fish
and amphibians, although these latter have an eccentric
configuration of the cilium and stereovilli arranged in
diminishing order. In botryllids, the cilium of hair cells
was found either to be central to the corolla of stereovilli,
as normally occurs in ascidian sensory structures, or to be
completely eccentric. This is of particular interest, since
the collar configuration is considered to be the predecessor
of the eccentric hair bundle found in the otic and lateral
line receptors of vertebrates (Thurm et al., 1998).
In sensory coronal cells the mechanoreceptor role is also
supported by the features of the hair bundle. We observed
that, although the cilium has a typical internal 9⫹2 configuration and a pair of centrioles at its base, it is short,
relative, for example, to stigmatal cilia (Martinucci et al.,
1992); it has short ciliary roots and appears stiff in that it
was never found bent. These features indicate that it is
not capable of active beating, so it cannot be involved in
generating current flow or moving particles. On the other
hand, the hair cells do not have the features of chemoreceptors because the latter usually have long (sometimes
branched) microvilli and lengthy cilia embedded within
the mucous secretion of accessory gland cells (Altner and
SECONDARY SENSORY CELLS IN ASCIDIANS
245
Prillinger, 1980; Finger, 1997). The supporting coronal
cells do not show any particular signs of secretory activity
at the apex, where they have a thick apical glycocalyx and
microvillar protrusions arranged to create a sort of channel for collecting the hair bundles of the sensory cells.
Moreover, the cilium is connected to the surrounding
stereovilli by extracellular radial filaments. It is possible
that the fibrillar links represent the structural device for
transduction of the stimulatory force acting on the cilium
to the site of mechanosensitive ion channels, as demonstrated in other mechanoreceptor systems (Gillespie,
1995; Thurm et al., 1998).
The presence of numerous and well-developed organelles involved in protein synthetic processes, such as
numerous mitochondria, well-developed rough endoplasmic reticulum, and Golgi apparatus, suggests that sensory
cells are very active, possibly involved in the synthesis of
neurotransmitters. Hair cells possess tight junctions
which help in maintaining distinction of the apical from
the basolateral plasmalemmata domains. However, in
comparison with other ascidian epithelia (Lane et al.,
1986) they have no gap junctions, the absence of which
indicates that the sensory cells are metabolically and electrically isolated from the adjacent ones.
All the above suggests that the coronal organ could both
detect the impact of small particles and/or respond to
vibration; in the latter role, the velum might act like a sort
of a basilar membrane, vibrating in response to waterborne vibrations in the near field and transmitting these
movements to the hair cells.
Structure of the synaptic contacts
Neurites run in grooves at the base of sensory cells,
establishing synaptic contacts of various categories. The
basal lamina, delimiting these grooves, isolates neurites
from the blood compartment and favors the strict spatial
relation between them and the sensory cells.
The evidence of synapses was critical in order to classify
the hair cells as secondary receptors. This conclusion was
corroborated by the absence of axonal processes emanating from the receptors directed towards the cerebral ganglion. Although we analyzed many specimens, including
some serially sectioned for observation with the electron
microscope, none were found. Moreover, the experiments
performed in vivo with the lipophilic dye DiI on Botryllus
schlosseri confirmed that hair cells are secondary receptors. This dye is often used to visualize the pathway of
neurites (Clarke, 1999). When applied to the neuronal
soma, it spreads along its protrusions permitting their
tracing; vice versa, when applied to the peripheral nerve
fibers it permits the localization of their soma. Our experiments demonstrated that a discontinuity existed between
each sensory cell and the underlying nerve fibers because
Fig. 6. Botryllus schlosseri. A,B: Specimens treated to reveal
AChE reaction product. Electron-dense granules (arrowheads) are
located at the base of hair cells (hc) on the membranes of nerve fibers
(nf) and in intercellular spaces (white arrowhead). B: Enlargement of
the squared area of A. bc, blood cell; c, cilium; s, stereocilium; sc,
supporting cell. Transmission electron microcopy. Scale bar ⫽ 1.5 ␮m
in A; 0.5 ␮m in B. C: The DiI marks the hair cells on the velum (v) and
tentacles (t). No neurites can be seen arising from hair cells and
projecting toward the cerebral ganglion. Fluorescence microscopy.
Scale bar ⫽ 70 ␮m.
246
P. BURIGHEL ET AL.
Fig. 7.
Schematic drawing of a hair cell accompanied by supporting cells.
no axons were marked when the DiI was applied inside
the siphon. It is also of note that, probably because of the
presence of the thick glycocalyx and tunic, the dye only
marked the sensory cells of the coronal organs, while other
cells of the tentacles and velum and cells of the siphonal
epithelium did not become stained.
Hair coronal cells are contacted by two principal sets of
neurons, both with their nucleus located in the cerebral
ganglion: the afferent neurons, transmitting information
from the hair cells to the ganglion, and the efferent neurons, providing input to the hair cell and/or sometimes to
the hair cells’ afferents. This situation is reminiscent of
what is known for the hair cells of the inner ear and the
lateral line of vertebrates. Where synapses were recognized it was possible to classify the fibers as afferent
(dendritic) and efferent (axonal) ones: afferent synapses
were mainly found, although axo-somatic and axodendritic efferent synapses were also encountered.
In ascidians, synapses between peripheral fibers were
never recognized. However, in consideration that experimentally deganglionated animals maintain a neural net
able to guarantee responses to tactile and electrical stimulation, Mackie and Wyeth (2000) hypothesized the presence of synaptic interconnections in its periphery. Our
data show that synapses occur between peripheral fibers
and thus provide a possible explanation for how deganglionated animals may regulate their activities.
AChE histochemically labeled nerve fibers occur in the
basal grooves of sensory coronal cells; its reaction product,
analogous to the situation in the neuromuscular junctions
of ascidians (Burighel et al., 2001), selectively marked the
plasmalemmata and the intercellular spaces in these
sites. This enzyme is known to label the vertebrate octavolateralis efferent neurons (Hellmann and Fritzsch, 1996),
which, on the basis of the spatial and developmental associations with facial motor neurons, as well as their cholinergic nature, are thought to be rerouted motor neurons
(Manley and Koppl, 1998). The AChE method had been
successfully used in ascidians for revealing the motor system (Arkett et al., 1989) and positive nerve fibers were
described in the tentacles of Botryllus schlosseri (Burighel
et al., 2001; Zaniolo et al., 2002). If such nerves could play
an efferent role the parallelism with the efferent system of
vertebrates would be very striking. Thus, the morphological diversity and pattern of innervation of hair cells suggests the possibility of a different role for the two sets of
coronal mechanosensory cells (with centric and eccentric
cilium) and the possibility of fine control (as modulation or
amplification of the mechanical signal) from the brain, as
occurs in vertebrates (Hudspeth, 1997; Manley and Koppl,
1998; Fay and Popper, 2000; Smotherman and Narins,
2000).
In Botryllus schlosseri we estimated that about 150 –200
hair cells and no more than 20 nerve fibers exist for each
SECONDARY SENSORY CELLS IN ASCIDIANS
lateral tentacle, and each hair cell forms 2–3 afferent
synapses and at least one efferent synapse. This means
that each nerve fiber has to form dozens of synapses in
order to ensure that the innervation reaches all the sensory cells. Then, considering that the whole coronal organ
contains about 2,000 hair cells, and that in the cerebral
ganglion there are no more than 1,000 neurons (unpubl.
data), and that branched nerve fibers underlying the sensory cells were rarely encountered, we hypothesize that a
single dendrite, or axon, has to contact many different
hair cells en passant. In many animals the complement of
receptor cells far exceeds the number of central neurons
that process sensory information (Derby and Steullet,
2001); in particular, the high degree of convergence of
afferent fibers and divergence of efferent fibers found in
the coronal organ shows similarities with the common
lateral line and inner ear efferent system of Xenopus laevis (Hellmann and Fritzsch, 1996).
It is also a remarkable feature of the coronal organ that
some neurites synapse with the supporting cells. A similar
situation was described in birds (Takasaka and Smith,
1971; Koppl, 2001). A particular case is in the secondary
receptors (Langherans cells) of appendicularian oikopleurids (Bone and Ryan, 1979), in which the axon passes to
the receptor cell synapses (via gap junctions) with the
adjacent accessory cells and so receive input from two
entirely different sources. In all the above-mentioned
cases, comprising the botryllids, the meaning of synaptic
contacts with the supporting cells have not been definitely
clarified, but it is possible that the supporting cells play a
role in the control of sensory cell activity.
Coronal organ and the vertebrate
acoustico-lateralis system
As regards its morphological features, the coronal organ
can be compared with the ear and lateral line organ of
vertebrates (Bone et al., 1995). Both the coronal and the
lateral line organs have mechanoreceptors: 1) in linear
and symmetrical structures; 2) exposed to the seawater
current; 3) located in a channel (in botryllids delimited by
microvillar extensions of supporting cells); 4) possessing a
hair bundle composed of straight stereovilli accompanying
an eccentric cilium; 5) with a nonmotile cilium; 6) made up
of secondary sensory cells; and 7) in relation with both
afferent and efferent fibers, comprising a cholinergic system.
There are, of course, differences between the two organs, notably because the coronal organ also possesses
sensory cells with a centric cilium and lacks both stereovilli disposed in diminishing arrays and a gelatinous
cupula secreted by the supporting cells. A gelatinous cupula is instead present in the cupular organ of Ciona
intestinalis that Bone and Ryan (1978) proposed as a
suitable structural starting point for the evolution of the
acoustico-lateralis system of vertebrates. Cupular organs
differentiate in the atrium, a chamber which develops
from a pair of dorsal ectodermal invaginations which were
present in the fossil mitrates considered to be the ancestors to extant chordates (Jefferies, 2001; Dominguez et al.
2002). Atrial invaginations are comparable to the otic
placodes of vertebrates because both have a similar position in the embryo, are formed by ectodermal tissue, and
express members of the Pax-2/5/8 gene family (Bone and
Ryan, 1978; Katz, 1983; Wada et al., 1998). Nevertheless,
as a marked difference, cupular hair cells are primary
247
sensory cells, whereas vertebrate hair cells are secondary
sensory cells; on this basis, Mackie and Singla (2003)
produce considerable doubts that hair cells of vertebrates
and those of the cupular organ are homologous.
As regards the genetic data, some authors (Kozmik et
al., 1999; Holland and Holland, 2001) considered the possibility that the expression of Pax 2/5/8 gene has a role in
formation of the gill slits, which occurs through perforation of both pharyngeal and atrial epithelia (Manni et al.,
2002). On the other hand, other authors (Wada et al.,
1998) interpret the expression of Pax 2/5/8 to support the
homology between ascidian atria and otic placodes. It is
noteworthy that in Halocynthia roretzi (a species of the
same family of botryllids), Pax2/5/8 gene expression was
found at the level of atrial primordia, but also in the
primordium of the “pharynx,” considered to be homologous
to the vertebrate stomodeum (Wada et al., 1998). These
authors hypothesized that, in ascidians, this gene has
been coopted into pharyngeal development, perhaps in
connection with the evolution of similar cellular properties
in the pharynx and atria. In addition, recent data on gene
expression in early amphibian and chick embryos demonstrate that the potential to form otic derivatives is not
exclusive to the ectodermal region close to the hindbrain,
but that the anterior epiblast is also competent to form an
otic placode, if taken at a sufficiently early age (Gallagher
et al., 1996; Groves and Bronner-Fraser, 2000). Since the
oral siphon, and then the coronal organ, derive from the
anterior ectoderm of the embryo (Manni et al., 1999), all
these genetic data suggest that the ancestor of chordates
had a broad capacity to form otic derivatives, in particular, secondary mechanoreceptor cells. This ability would
now be restricted to the posterior epiblast of the head in
vertebrates, while it is also maintained in the anterior
ectoderm in urochordates. Thus, the morphology, embryological origins, and developmental gene expression all
support our idea that the coronal organ and the acousticolateralis system are homologous. This idea would be consistent also with the interpretation of Jefferies (2001) that
the possible ancestors of chordates, the fossil calcichordates, possessed the precursor of the acoustico-lateralis
system of extant craniates.
The cephalochordates lack otic vesicles, possibly present
in their mitrate ancestors (Jefferies, 2001). Secondary sensory cells have been described in amphioxus (Bone and
Best, 1978; Lacalli and Hou, 1999; Lacalli et al., 1999).
They are scattered in the rostral epithelium of the juvenile
and classified, on the basis of their architecture, as probable chemoreceptors. Moreover, other secondary sensory
cells (oral spine cells) are in the spines of the mouth of the
amphioxus larvae (Lacalli et al., 1999). Although they are
mechanoreceptors bearing a cilium and in synaptic contact with peripheral neurons, as vertebrate hair cells are,
they are grouped in clusters and do not possess a hair
bundle of stereovilli. Moreover, Lacalli et al. (1999) proposed that they are homologous to the vertebrate taste
bud cells which are not of placodal origin. Thus, at the
moment, available data on amphioxus are not able to
contribute to the discussion of the evolution of secondary
hair cells and the acoustico-lateralis system of vertebrates.
Among invertebrates the cephalopods possess sophisticated movement receptor systems with ciliated sensory
cells, which are reminiscent of those of vertebrates: these
include an angular acceleration receptor system composed
248
P. BURIGHEL ET AL.
of primary and secondary sensory hair cells and a system
analogous to the vertebrate lateral line formed by primary
sensory cells (Budelmann and Bleckmann, 1988; Budelmann, 1989, 1996). These sensorial systems display a high
degree of similarity with the vertebrate inner ear; however, they represent a convergent solution that serves the
same function and are certainly not homologous to the
vertebrate sensory system (Budelmann, 1992).
Since vertebrate secondary sensory cells develop from
placodes, any evolutionary hypothesis about sensory cells
in chordates has to consider the possibility that placodal
structures were present in the chordate ancestor. This
issue is therefore a subject for debates and the idea that
placodes are not exclusive to vertebrates is currently being
taken into consideration (Baker and Bronner-Fraser,
1997; Burighel et al., 1998; Wada et al., 1998; Manni et al.,
1999, 2001; Graham, 2000; Shimeld and Holland, 2000;
Begbie and Graham, 2001). Baker and Bronner-Fraser
(1997) suggested a possible scenario about the lineage
relationships of the primary and secondary cells in vertebrates. They assumed that the ancestral ciliary receptors
were concentrated in patches on the head; later, some cells
formed specialized receptor cells that lacked central projections, while others formed the neurons that innervated
them (as we can see in the acoustico-lateralis system);
finally, the receptors might have been lost from certain
placodes but the sensory neurons retained and coopted
into the innervation of nonplacodal receptors. This hypothesis is supported by data on the development of mechanosensors considered to be, from a genetic point of view,
homologous across phyla (Fritzsch and Beisel, 2001).
Considering the above data, we believe that the results
of the present work represent additional evidence in favor
of the hypothesis that the ancestor of chordates had the
ability to differentiate secondary sensory cells and that
the coronal organ of urochordates is homologous to the
acoustico-lateralis, or lateral line, system of vertebrates.
Thus, a more complete scenario can be envisaged regarding the origin of placodes in chordates in that the chordate
ancestors not only possessed the olfactory and adenohypophyseal placodes maintained in the extant tunicates
(Burighel et al., 1998; Manni et al., 1999), cephalochordates (see Holland and Holland, 2001), and vertebrates,
but also possessed the placodes of the otic area and the
genetic and cytological machinery to differentiate the
acoustic-lateralis system, including the secondary receptor hair cells which are today present in both vertebrates
and ascidians.
ACKNOWLEDGMENTS
We thank Mr. C. Friso for the drawings.
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