Hearing Research 273 (2011) 14e24
Contents lists available at ScienceDirect
Hearing Research
journal homepage: www.elsevier.com/locate/heares
Hair cells in non-vertebrate models: Lower chordates and molluscs
P. Burighel*, F. Caicci, L. Manni
Department of Biology, University of Padova, Via U. Bassi 58/B, 35131 Padova, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 23 December 2009
Received in revised form
11 March 2010
Accepted 15 March 2010
Available online 27 April 2010
The study of hair cells in invertebrates is important, because it can shed light on the debated question
about the evolutionary origin of vertebrate hair cells. Here, we review the morphology and significance
of hair cells in two groups of invertebrates, the lower chordates (tunicates and cephalochordates) and the
molluscs. These taxa possess complex mechanoreceptor organs based on both primary (sensory neurons)
and/or secondary, axonless, sensory cells, bearing various apical specializations. Compared with vertebrates, these taxa show interesting examples of convergent evolution and possible homologies of sensory
systems. For example, the “lateral line organ” of Octopoda and Decapoda, composed of primary sensory
cells aligned on the arms and the head, is considered a classic example of convergent evolution to
mechanoreception. Similarly, in ascidians, the cupular organ, formed of primary sensory cells embedded
in a gelatinous cupula, is seen as an analog of neuromasts in vertebrates. However, the coronal organ of
the oral siphon of ascidians, represented by a line of secondary sensory cells with a hair bundle also
comprising graded stereovilli, is currently the best candidate for tracing the evolutionary origin of the
vertebrate octavo-lateralis system. Several features, such as embryological origin, position, gene
expression and morphology, support this hypothesis.
Ó 2010 Elsevier B.V. All rights reserved.
1. Introduction
Hair cell may be defined as mechanoreceptors with a tuft of
stereovilli (rigid, actin-filled microvilli) and a single modified
cilium, involved in the ability of the cells to respond to
mechanical stress to the membrane. In vertebrates, hair cells
possess unique features, being located in the acoustico-lateralis
system, and being secondary sensory cells (epithelial cells contacted at their base by axons belonging to neurons located elsewhere), typically possessing a cilium (kinocilium) accompanied
by stereovilli graded in length. In non-vertebrates, mechanorecptors with a comparable apical apparatus are widespread
(Fig. 1) and have been called “hair cells”(Budelmann et al., 1997;
Burighel et al., 2003). The latter may appear in the form of both
primary sensory cells, i.e., specialized neurons, with their own
axon or secondary sensory cells.
Abbreviations: AmphiTlx, amphioxus tailless gene; as, atrial siphon; bl, basal
lamina; c, cilia/kinocilia; CNS, central nervous system; co, coronal organ; e.g.,
exempli gratia (Latin) for example; FGF, fibroblast growth factor; hc, hair cell; hz,
hertz; m, microvilli; n, nucleus; nf, nerve fiber; NS, nervous system; os, oral siphon;
Pax, paired box gene; RER, rough endoplasmic reticulum; s, stereovilli; sc, supporting cell; Tlx, tailless gene.
* Corresponding author. Tel.: þ39 049 8276185; fax: þ39 049 8276199;
E-mail addresses: [email protected] (P. Burighel), [email protected]
(F. Caicci), [email protected] (L. Manni).
0378-5955/$ e see front matter Ó 2010 Elsevier B.V. All rights reserved.
doi:10.1016/j.heares.2010.03.087
Although the hair cells of vertebrates exhibit variability in
morphology among taxa, there is little doubt that they share the
same phylogenetic origin (Manley and Ladher, 2008). Instead, there
is wide-ranging discussion about their possible homology with the
ciliated mechanoreceptor cells of non-vertebrate chordates, i.e.,
tunicates and cephalochordates and, in a wider extension to
convergence, with the mechanoreceptor cells of non-chordate
invertebrates, such as arthropods and molluscs (Fig. 1A and B).
Considering the phylum of Chordata, the search for possible
homology between the mechanosensory cells of vertebrates and
non-vertebrates has raised a debate which has now extended to
include other related fields of biology. In particular, attention
should be paid to the evolution of the neural crest and neural
placodes which, according to the “new head hypothesis”, are
structures thought to be unique to vertebrates and crucial for
vertebrate evolution (Northcutt and Gans, 1983). Indeed, in vertebrates, both sensory cells and sensory neurons are derived from the
neural crest or placodes. However, as an alternative hypothesis,
various data have recently been published reporting that cell
populations with the characteristics of placodes or neural crests
occur not only in vertebrates, but also in tunicates and cephalochordates (see Hall, 2009). This leads us to reconsider morphological data showing that lower chordates possess neuroblasts able
to migrate from the placode-like region during their development
and also that sensory cells exhibit features recalling vertebrate hair
cells.
P. Burighel et al. / Hearing Research 273 (2011) 14e24
15
Fig. 1. (A) Schematic drawing of a possible scenario of hair cell evolution. The probably simplest condition of hair cells (represented by jellyfish) consists of a primary sensory cell
possessing a cilium (kinocilium) surrounded by a corolla of microvilli. From this condition, three evolutionary lines derived: (a) the line of lophotrochozoans (here represented by
the Octopus vulgaris, the best known case, in which both primary and secondary hair cells are present), with many kinocilia but without stereovilli; (b) the line to the ecdysozoans
(here represented by an insect), characterized by primary sensory cells having a kinocilium; and (c) the line of chordates, represented by ascidians with primary and secondary
sensory cells of various morphology (also with graded stereovilli), by a fish, with a typical vertebrate hair cell with a kinocilium accompanied by graded stereovilli, and by a bird,
where the kinocilium tends to degenerate during ontogenesis and the hair bundle is only formed of graded stereovilli (a condition shared with mammals). (B) Presumed
evolutionary pathway of mechanosensory organs based on primary sensory cells in ascidians. (a) Starting point represented by scattered, single or grouped in small clusters, ciliated
sensory cells (sensory neurons). These cells occur in epidermis. (bed) Organs of the atrial chamber: (b) cupular organ; (c) cupular strand; (d) capsular organ. It has been suggested
that the cupular strand evolved from the cupular organ, and the capsular organ from the first (modified from Mackie and Burighel, 2005).
In this paper, we review the morphology of sensory cells in
tunicates and cephalochordates, but also in cephalopod molluscs,
since reference to these animal groups can provide information
about the characteristics of hair cells in complex mechanoreceptor
organs. Lower chordates are of course of interest due to their direct
phylogenetic relationship with vertebrates, and can give some
information on the evolutionary history of hair cells in the ear and
lateral line of vertebrates. Molluscs are the largest group in
Lophotrochozoa and are also the most diverse. They range from
chitons (Polyplacophora), snails and slugs (Gastropoda) and
bivalved animals such as oysters (Bivalvia) to octopuses, squids and
Nautilus (Cephalopoda). We focus here on the ciliated mechanoreceptors of cephalopods, which are perhaps the most advanced of
invertebrate mechanoreceptors and have several aspects comparable to those of vertebrates. Data from the literature indicate that
both lower chordates and molluscs show interesting examples of
convergent evolution with respect to vertebrates; moreover, they
support the view of possible homology between hair cells in tunicates and vertebrates.
with its sac-like body, differs markedly from that of a vertebrate,
whereas the cephalochordate, represented by the amphioxus
(Branchiostoma lanceolatum and Branchiostoma floridae) resembles
a small, simplified fish. For this reason, cephalochordates were
classically believed to be close to vertebrates e a view which has
recently changed, because several morphological and molecular
data (Manni et al., 2001; Bassham and Postlethwait, 2005; Delsuc
et al., 2006; Dufour et al., 2006; Jeffery, 2006, 2007) indicate that
in fact the tunicates are the sister group of vertebrates. Since the
two groups are the extant animals closest to the basal stem of
chordates, they may be useful models for studying the characteristics of the chordate ancestor. They may also be of interest for
studying the origin of vertebrates and the evolution of structures
(such as the mechanoreceptor organs), which achieved their
greatest complexity in the vertebrates. In particular, they might tell
us whether traces of derivatives of neural placodes and of the
neural crest are recognizable in lower chordates and whether the
ear and lateral line of vertebrates with their hair cells originated de
novo in vertebrates or from ectodermic placode-like regions in the
common ancestor of all the chordates.
2. The lower chordates
2.1. Tunicates
The non-vertebrate chordates include filtering marine animals
which, during their embryonic development, like vertebrates, have
a chordate plane with an axial notochord and a central nervous
system (CNS) derived from a neural plate. They comprise the
tunicates and the cephalochordates. The adult form of a tunicate,
The tunicates contain three classes: benthic ascidians, and
planktonic thaliaceans and appendicularians (Table 1). Appendicularians are solitary, whereas thaliaceans are colonial. Ascidians
have both solitary and colonial forms and, according to the position
16
P. Burighel et al. / Hearing Research 273 (2011) 14e24
Table 1
Classification of chordata.
Phylum
Subphylum
Chordata
Cephalochordata
Tunicata
Class
Appendicularia
Thaliacea
Ascidiacea
Order
Enterogona
Pleurogona
Vertebrata
of their gonads, are divided into two orders, Enterogona and
Pleurogona (Table 1). With their 3000 species, ascidians are the
most frequently studied tunicates, and some species, e.g., the solitary enterogonid Ciona intestinalis, (the genome of which was
recently sequenced) and the colonial pleurogonid Botryllus schlosseri, have become reference organisms for developmental and
evolutionary studies (Passamaneck and Di Gregorio, 2005; Satoh
and Levine, 2005; Manni et al., 2007).
The ascidian embryo develops into a swimming, chordate-like
tadpole larva (1 or a few mm long) with its tubular CNS extending
over the notochord and enlarged in the trunk (or head), to form
a “brain” possessing a large sensory vesicle. This CNS originates
from a neural plate which folds into a neural tube, showing anteriorly the expression of specific developmental genes following the
same regionalization as the embryonic CNS of vertebrates (Wada
et al., 1996, 1998; Shimeld & Holland, 2000; Manni et al., 2001).
The larva swims for a short time and then adheres to a substrate to
metamorphose into the sessile, filtering juvenile. There is
a dramatic change in larval components in the passage to the
juvenile form: the CNS regresses, together with all the larval tissues
used for swimming life (i.e., notochord, striated caudal muscle),
whereas the rudiments of the adult grow and differentiate to form
the filtering system (with the branchial basket, atrial chamber, oral
and atrial siphons), smooth muscle and the CNS. The latter is
formed de novo starting from a small area of pluripotential cells
located in the anterior roof of the larval nervous system (NS). The
adult CNS, located between the two siphons, consists of a cerebral
ganglion and its associated neural gland. Motor nerves extend from
the ganglion to the periphery to regulate the activity of branchial
cilia and muscle, whereas the peripheral NS is composed of sensory
cells which extend neurites to the ganglion.
An adult ascidian is shown in Fig. 2A. The body is characterized
by the presence of the two prominent siphons, oral (inhalant) and
atrial (exhalant). The oral siphon (Fig. 2B) gives access to the
branchial chamber, which has many ciliated stigmata on its lateral
walls. Ciliary beating causes a current of water to enter the oral
siphon, encounter the crown of tentacles, and flow into the branchial chamber. Then, passing through the branchial stigmata, water
flows into the atrial chamber and exits through the atrial siphon.
Water flow transports food particles that are filtered in the branchia
and then sent to the gut for nutrition.
2.1.1. Mechanosensory organs of ascidians based on primary
sensory cells
The sessile adult stage of ascidians presents a variety of
mechanosensory receptors, all based on ciliated cells (see Mackie
and Burighel, 2005). These receptors may be primary (with the
cell body at the periphery and a thin axon e about 0.3 mm in
diameter e extending to the cerebral ganglion) or secondary
sensory cells. Primary sensory cells are widespread not only in
ascidians, but also in pelagic tunicates, especially around the
mouth. In some ascidian species, primary sensory cells, solitary or
paired, are accompanied by supporting cells and form corpuscles
located in the basal part of the oral and atrial tentacular tunic
(Koyama, 2008).
It is noteworthy that primary sensory cells constitute the basic
components of various kinds of mechanoreceptor organs, such as
the cupular organs (Bone and Ryan, 1978), cupular strand and
capsular organs (Mackie and Singla, 2003, 2004), which are found
in several ascidians belonging to Enterogona (Fig. 1B). These organs
are located in the atrial chamber and are believed to be hydrodynamic sensors (see Mackie and Burighel, 2005) involved in monitoring changes in local water flow, like the neuromasts of the lateral
line in vertebrates. The cupular organs are small and isolated, with
a pad (macula) of a few neurons and supporting cells. The cupular
strand is 6e7 mm long and has a total of between 1000 and 2000
neurons in each individual (Mackie and Singla, 2004). The neurons
of the cupular organs and cupular strand have cilia projecting
respectively into a digitiform or elongated cupula of tunic-like
material. The capsular organs have a macula of 5e6 sensory cells,
with cilia projecting into a fluid-filled capsule surmounted by a tuft
of tunic-like material. The capsular organs are believed to be
responsible for the ability of the animals to perceive vibrations
generated up to about 80 cm away (Mackie and Singla, 2003). All
these sensory organs probably evolved in ascidians from isolated or
clustered simple primary sensory cells having their apical termination projecting into the tunic (Fig. 1B) (Mackie and Burighel,
2005). Because of their location, basic organization and function,
these mechanosensory neurons have engendered debate about
possible homology with the sensory cells of the lateral line/ear of
vertebrates. This hypothesis was reinforced by ontogenetic and
gene expression similarities, in that, like the otic placodes of
vertebrates, the ascidian atrium derives from a pair of ectodermic
invaginations and the atrial placodes express the Pax 2/5/8 gene
family (Bone and Ryan, 1978; Wada et al., 1998; Bassham et al.,
2008). Nevertheless, this homology has been criticized on the
basis of the fact that the atrial sensory organs have primary
neurons, whereas the hair cells of neuromasts are secondary
sensory cells (Mackie and Singla, 2003; Mackie and Burighel, 2005).
A new type of sensory organ, the coronal organ, has recently
been detected in the oral siphon of adult ascidians and is of
particular interest due to its location, embryonic origin, function
and, especially, the presence of secondary sensory cells resembling
the hair cells in vertebrates.
2.1.2. Secondary sensory cells in ascidians: the hair cells of the
coronal organ
The coronal organ is located at the base of the oral siphon and
appears as a continuous row of sensory cells running along the
border of the velum and tentacles (Fig. 2BeD). The coronal organ
was initially described in the colonial species B. schlosseri, where it
is composed of about 2000 cells (Burighel et al., 2003), but was later
found in all the ascidians belonging to both Enterogona and Pleurogona examined so far (Manni et al., 2004a, 2006; Mackie et al.,
2006; Caicci et al., 2007; Burighel et al., 2008). The organ is
composed of secondary sensory units which make both afferent
and efferent synapses with neurons, the somata of which lie centrally in the cerebral ganglion. The cells are always ciliated and in
some cases the cilia are surrounded by a cluster of stereovilli,
sometimes graded in length and thus closely resembling vertebrate
hair cells (Table 2). The position of the coronal organ at the base of
the oral siphon, its embryological origin, and the variability in its
cell ultrastructure deserve to be discussed in detail because, in view
of the position of tunicates as an offshoot within the chordate line
of evolution, the coronal organ may represent the early expression
of a system corresponding to the acoustico-lateralis system in
vertebrates (Burighel et al., 2003).
The ascidian oral siphon originates from the embryonic
stomodeal (“mouth”) area, the ectoderm of which invaginates and
fuses with the anterior region of the endodermic rudiment of the
P. Burighel et al. / Hearing Research 273 (2011) 14e24
17
Fig. 2. (A) An adult ascidian Ciona intestinalis (Enterogona), as: atrial siphon; os: oral siphon. Scale bar: 0.6 mm. (B) Internal view of oral siphon to show velum and tentacles in
a solitary Enterogona ascidian. (CeD) Detail of a tentacle of Molgula socialis (Pleurogona). Scanning electron microscopy. (C). Coronal organ (co) runs all along edge of the tentacle
and is composed of 2e4 rows of hair cells. Arrowheads: apical membrane extension of supporting cells. Scale bar: 8 mm. D. Coronal organ viewed from mid-side of tentacle. Note
sensory cells with two kinocilia (c) and a collar of graded stereovilli (s). Arrowheads: apical membrane extension of inner supporting cells. Scale bar ¼ 1.5 mm. (E) Sketch of various
kinds of ascidian hair cells. Pleurogona species usually possess sensory cells of type 1 (with one kinocilium surrounded by microvilli) and type 2 and/or type 3 (with a ring of graded
stereovilli or a crescent of graded stereovilli surrounding a pair of kinocilia, respectively). Supporting cells also differ in the apical plasmalemma extension and may be polarized
with respect to inflowing water. Enterogona species possess hair cells with a row of kinocilia, lack stereovilli but may have short microvilli. In some species, they are flanked by large
secretory cells, whose presence defines the coronal organ as “compound”. (F) Transverse ultra-thin section viewed by transmission electron microscopy of the coronal organ of
Molgula socialis. Sensory cells have basal nuclei, apical Golgi apparatus, numerous RER cisternae and dense cytoplasmic granules. Supporting cells are lengthened and curved to
receive sensory cells. c: cilia; m: microvilli; s: stereovilli; arrow: apical membrane extension of supporting cells. Scale bar in F ¼ 2 mm. (G) Scheme of F section highlighting nuclei of
18
P. Burighel et al. / Hearing Research 273 (2011) 14e24
branchial chamber (Manni et al., 2005). The stomodeal wall
secretes tunic and forms the internal epithelium of the siphon. The
tunic is the extracellular matrix rich in proteins and cellulose fibers,
produced by the epidermis which covers the body of tunicates. In
adults, the tunic extends into the siphon, terminating as a thin
sheet at the base of the velum and tentacles, leaving the coronal
cells exposed to inflowing water.
In general, the number and length of tentacles vary according to
species, body size and the age of the animal. In several species, the
tentacles may be ramified, with small tentacles branching from the
main axis. This increases the tentacle surface and, consequently, the
number of sensory cells exposed to incoming water, suggesting that
the filtering potential and the sensitivity of the system is improved
(Caicci et al., 2007). The tentacles may be relaxed, hanging down
toward the branchial cavity, or held up to project across the
opening of the siphon, forming a sort of filter. Variations in the state
of tentacles depend on variations in blood pressure, caused mainly
by the contractile activity of the velar sphincter muscle of the
siphon (Mackie et al., 2006).
According to comparative studies, two kinds of coronal organ
have been identified (Fig. 2E): simple, as exemplified in Ciona
intestinalis, consisting only of ciliated cells; and compound, as
exemplified in Chelyosoma productum, consisting of ciliated sensory
cells flanked by secretory cells (Manni et al., 2006). It is remarkable
that, in both types, C-shaped, non-ciliated supporting cells always
flank the sensory cells and secretory cells (when present). Supporting cells are easily distinguished from contiguous epithelial
cells due to their general shape. They also often develop microvillar
or laminar extensions apically, defining a long groove inhabited by
the hair bundles of sensory cells. All the cells of the velum and
tentacles, both sensory and non-sensory, are joined apico-laterally
to each other by dense tight junctions.
2.1.3. Variability in ascidian hair cells
The coronal sensory hair cells present a great degree of diversification and specialization, not only between species but even in
the same animal (Table 2). Thus, it is interesting to compare these
cells in various taxa in order to understand whether, despite this
variability, specific adaptive aspects characterizing the two evolutionary lines in ascidians can be identified. Referring to Pleurogona
and Enterogona, several data indicate that the two orders diverged
very early and that they achieved different levels of organization of
the filtering apparatus (Fiala-Medioni, 1974). Whereas Enterogona
possess multiciliated coronal cells, sometimes accompanied by
very short microvilli (Manni et al., 2006), Pleurogona have more
complex coronal cells provided with one or two cilia accompanied
by stereovilli, also graded in length and therefore more clearly
resembling vertebrate hair cells (Fig. 2E). Greater complexity is
seen in species such as Molgula socialis (Pleurogona, suborder
Stolidobranchiata, family Molgulidae) (Fig. 2F and G) (Caicci et al.,
2007), in which three types of sensory cells have been identified.
The peripheral sensory cells have one cilium central to the group of
short microvilli (type 1 sensory cells). The sensory cells with stereovilli, located on the inner side of the tentacle, show two types of
apical bundles: either ones with stereovilli lying on one side (type
2) or in the form of a ring around the two cilia (type 3); in both
cases, they are graded in length Type 2, and type 3 sensory cells are
randomly distributed in the organ. The stereovilli are stiff,
unbranched, and filled with microfilaments continuous with those
of the apical cytoplasm. Several coat fibrils extend radially, to
establish connections between adjacent stereovilli and between
stereovilli and ciliary membrane. Each cilium has a conventional
9 þ 2 microtubular arrangement, with a dense, short, basal body,
from which poorly developed ciliary rootlets extend; a second
centriole is sometimes recognisable, lying perpendicularly to the
first one. Type 2 and type 3 sensory cells are cylindrical, with a large
basal nucleus, scattered mitochondria, and Golgi and secretory
granules in the apical region. The cytoplasmic organelles are less
developed in the sensory cells of type 1, which lack secretory
granules. The basal plasmalemma of each sensory cell lies on
a basal lamina and forms a groove, which contains neurites (Fig. 2H
and I). Neural synapses, both afferent and efferent, can be seen.
Species of the family Styelidae, which belong to the same
suborder (Pleurogona, Stolidobranchiata) may have hair cells with
microvilli (type 1) and a crescent of stereovilli graded in length
(type 2), as in Styela plicata (Manni et al., 2004a). In the species B.
schlosseri, Botrylloides leachi and Botrylloides violaceus, the cells
exhibit a cilium emerging at the center or at the side of a tuft of
stereovilli of equal length (Burighel et al., 2003). This variability
probably represents adaptations to the ability to respond to
different stimuli, such as particles of various sizes and/or variations
in water flow. Coronal cells possessing secretory granules have
been found not only in both type 2 and type 3 sensory cells of
Pleurogona (Manni et al., 2004a; Caicci et al., 2007) but also in some
Enterogona species (Manni et al., 2006): it has been hypothesised
that they are secreted following the stimulation of sensory cells.
Some diversity is also seen regarding the supporting cells. Their
lateral plasmalemma often adheres to adjacent sensory cells
forming, in pleurogonids (Burighel et al., 2003, 2008; Manni et al.,
2004a) and some enterogonids (Mackie et al., 2006; Manni et al.,
2006), an apical laminar extension, defining the groove occupied
by the apical specialization of the sensory coronal apparatus.
However, other enterogonids lack this specialization. In the pleurogonid Molgula socialis (Caicci et al., 2007), the supporting cells are
polarized with respect to organ orientation, since the inner supporting cells (facing the middle of the tentacle) have an expanded,
apical lamina defining sensory bundles, whereas the outer ones
have a shorter, apical lamina bordering the sensorial microvilli of
type 1 sensory cells (Fig. 2F and G). It has been hypothesized that
supporting cells differently oriented with respect to the water flow
are a useful device to guide the flow towards the stereovilli,
favoring the formation of laminar flows over them, for better
perception of particles entering the branchial basket (Caicci et al.,
2007).
This short analysis stresses some of the differences observed
in Enterogona and Pleurogona coronal cells. The gross anatomy of
the coronal organ (cell arrangement, location on the tentacles and
velum) and its cellular organization (presence of cilia, and of
afferent and efferent innervation) remain common features of the
organ in all ascidians. Although at present nothing is known
about the functional significance of the differences in hair cells in
the various species, it is possible that they reflect the ability of
organs to respond to different types of signals and to produce
appropriate physiological responses, as has been demonstrated in
various acoustico-lateralis organs in vertebrates (Coffin et al.,
2004).
hair cells (gray), hair cell bodies (pink) and hair bundle (green), and supporting cells (blue). Basal lamina is marked in purple. (H) Transmission electron microscopy of synaptic
connectivity in the coronal organ of Botryllus schlosseri. Many nerve fibers (nf) are located in grooves formed by basal plasmalemma of hair cell (hc) and supporting cell (sc) and
delimited by basal lamina (bl). Right: note extended synaptic area, marked by many presynaptic vesicles (arrowheads) close to hair cell plasmalemma. Arrow: synapses between
a nerve fiber and a supporting cell; n, nucleus. Scale bar: 0.3 mm. (I) Diagrammatic representation of synaptic connectivity between the basal plasmalemma of a hair cell (pink) and
nerve fibers (pale yellow: afferent fiber; dark yellow: efferent fiber).
P. Burighel et al. / Hearing Research 273 (2011) 14e24
19
Table 2
A classification of mechanoreceptor organs based on secondary sensory cells in non-vertebrate chordates.
Receptor
Taxon (genus)
Organ/ location
Proposed function
Hair bundles
Coronal cell (Burighel et al.,
2003, 2008; Manni et al.,
2004a; Caicci et al., 2007)
Pleurogona ascidians
(all the examined genera)
Coronal organ
on velum and
tentacles of
oral siphon
Sensitivity to contact
of inflowing particles
Cilium central to
a corolla of
microvilli (type 1)
Coronal cell
(Burighel et al., 2003; 2008)
Pleurogona ascidians
(Botryllus, Botrylloides)
Coronal organ on
velum and
tentacles of oral
siphon
Sensitivity to contact
of inflowing particles
Cilium eccentric to
a corolla of microvilli
Coronal cell
(Manni et al., 2004a;
Caicci et al., 2007)
Pleurogona ascidians
(Molgula, Pyura, Styela)
Coronal organ
on velum and
tentacles of oral
siphon
Sensitivity to contact
of inflowing particles
Pair of cilia central
to a ring of graded
stereovilli (type 2)
Coronal cell
(Manni et al., 2004a;
Caicci et al., 2007)
Pleurogona ascidians
(Molgula, Pyura, Styela,
Polyandrocarpa)
Coronal organ
on velum and
tentacles of
oral siphon
Sensitivity to contact
of inflowing particles
Pair of cilia central
to a crescent of graded
stereovilli (type 3)
Coronal cell
(Manni et al., 2006)
Enterogona ascidians
(all the examined genera)
Coronal organ
on velum and
tentacles of
oral siphon
Sensitivity to contact
of inflowing particles
Row of cilia
Lower lip and pharynx
receptors (Bone, 1998)
Appendicularians
(Oikopleura, Fritillaria)
On lower lip
and pharynx
Monitoring particle
flow into pharynx
Row of cilia
Langerhans cell (Bone, 1998)
Appendicularians
(Oikopleura)
On posterior
trunk
Sensitivity to contact;
swimming control
Row of cilia
Oral spine cells
(Lacalli, 2004)
Cephalochordates
(Branchiostoma)
Oral spines
on larval
oral margin
Initiating cough response
after contact with debris
One cilium
Epidermal type II sensory cells
(Lacalli and Hou, 1999;
Holland and Yu, 2002)
Cephalochordates
(Branchiostoma)
Larval rostral
epidermis
Mechanoreceptor/
chemoreceptor ?
Cilium central to a corolla
of branched microvilli
Configuration of hair bundles
20
P. Burighel et al. / Hearing Research 273 (2011) 14e24
2.1.4. Function of the coronal organ
It has long been known that large particles entering the oral
siphon with the inhalant flow cause the individual to make
a rejection response. This consists of a rapid muscular contraction
of the body, accompanied by emission of the water which removes
the particles. Visual observations suggest that rejection responses
typically result from the direct impact of particles against the
upper surfaces of the tentacles where the coronal organ is located.
Thus, two obvious questions arise: are the coronal hair cells
involved in perceiving the particles and triggering the rejection
reaction? And, what is the general function of the coronal hair
cells? Mackie et al. (2006) tried to answer these questions by
studying siphonal sensory structures and their sensitivity in the
ascidian Corella inflata (Enterogona). Monitoring animal responses
to stimulation with a flow meter, the above authors showed that
both oral and atrial siphons are sensitive to touch and near-field
vibrations. However, this sensitivity does not diminish even when
the tentacles are removed surgically, suggesting that it is mainly
driven by primary ciliated mechanoreceptors scattered on the
epidermis of the siphon. Conversely, tentacles are sensitive to
contact with inflowing particles: beads of 500e600 mm diameter
were able to evoke rejection responses 88% of the time, beads of
355e425 mm 61% and those <125 mm less than 8%. Responses
triggered by beads were crossed (contraction of the atrial siphon,
while the oral siphon stays open), or squirting (all-or-none
response followed by contraction of the body, which causes
a violent ejection of water and particles from the oral siphon and
atrial water from the atrial siphon). As these responses were lost
after tentacle amputation, they were attributed to the coronal hair
cells.
2.1.5. Secondary sensory cells in appendicularians
Secondary sensory cells constitute the Langerhans receptors of
Oikopleura dioica, lying in the posterior trunk epithelium and
coupled with the adjacent epithelial cells by gap junctions.
Remarkably, the junctions between these receptor cells and the
afferent neurites are also gap junctions, not synapses. This makes
Langerhans cells difficult to accept as homologs of hair cells,
although their mechanoreceptor role (e.g., they cause escape of the
individual after touching) was demonstrated by Bone and Mackie
(1975).
However, secondary sensory cells were also found in the oral
area in some appendicularians (Table 2) (see Bone, 1998). In Oikopleura and Fritillaria, these sensory cells are ciliated and arranged in
rows in the lower lip and internal wall of the mouth. It was
hypothesized that they are involved in monitoring the particle flow
into the pharynx (Bone and Mackie, 1982). Thus, their position and
general aspect suggest that they represent a sort of coronal organ,
although this must still be further investigated.
2.2. Cephalochordates
Secondary sensory cells probably involved in mechanoreception
have been reported from several sites in cephalochordates. There
are scattered, ciliated sensory cells in the epidermis of Branchiostoma usually solitary or arranged in small clusters, and most of
them may be mechanoreceptors. Under the electron microscope,
they are distinguished into two main types (I and II) (see Lacalli and
Hou, 1999) (Table 2). In addition, there are two variants of type I: in
type Ia, primary sensory cells bear a single long, rigid cilium, which
projects straight from the cell body, and is accompanied by a ring of
thick microvilli immediately around it and by an external ring of
less specialized microvilli. The basal body and the striated rootlets
are both reduced in these cells. All these characteristics suggest that
the cilium beats infrequently, if ever.
Type Ib cells differ from type Ia because they have microvilli of
a unique type, erect and slender with rounded tips, lacking a thick
fibrillar core and forming a tuft-like cluster. The cilium is straight
and short, and the basal body is accompanied by an accessory
centriole and the striated rootlets are slender than in the type Ia
cells. Both variants of type I are considered primary sensory cells
(Lacalli and Hou, 1999), since Baatrup (1981) was able to follow the
extension of the axon to the CNS in serial sections. For this, they are
not reported in Table 2. Holland and collaborators (Kaltenbach
et al., 2009) have shown that type I sensory cells originate from
the ventral epidermis by delaminating individually into the
subepidermal space and then they extended an axon before reinsertion of the cell body into the lateral epidermis and ciliogenesis.
Despite marked structural differences with the vertebrate hair cells,
the authors considered the possible homology of these cells with
migrating lateral line hair cells of vertebrates, based especially on
similarities in developmental and genetic mechanisms. In particular, amphioxus type I cells express AmphiTlx, an homolog of
vertebrate Tlx genes involved in development of neural crestderived and placode-derived neurons. However, at the moment the
data appear not sufficient for supporting a definite homology.
Type II sensory cells are secondary ones with a long cilium
surrounded at its base by a distinctive collar of microvilli studded
with short branches. The cilium is recessed in the apical surface and
ends apically in a small bulb. Accessory centrioles and striated
rootlets have normal features. Sensory cells of type II frequently
show short processes extending at the base of the cell and establishing bouton-like synaptic terminals with nerve fibers running
beneath the cell, probably belonging to the Retzius bipolar cells
(Holland and Yu, 2002).
Other secondary sensory cells form the oral spines in the larva
(Table 2) (Lacalli, 2004). Oral spine sensory cells are uniciliated and
clustered so that 8e10 cilia together form a spine. Many spines
form a row on the outer margin of the mouth. The apices with cilia
project on the outside of the lip and are thus different from the
adjacent epithelial cells, whose cilia are reversed in orientation. The
bases of the ciliary spine cells are filled with closely packed clear
vesicles of 70e90 nm in diameter. Synapse-like junctions also occur
between the base of the cells and adjacent small dendrite-like cell
processes, demonstrating that the oral spine cells are secondary
sensory cells that synapse mainly or exclusively with the intrinsic
neurons. The spines are able to respond to contact with debris by
initiating a cough response: the pharyngeal slits close as the
pharynx contracts, which expels water out of the mouth to dislodge
the debris (Lacalli, 2004).
3. Molluscs
Among molluscs, the cephalopods (Fig. 3A) are mobile predators
which have evolved a very elaborate NS associated with complex
sensory systems (Budelmann, 1989; Budelmann et al., 1997). Some
of these (e.g., the cephalopod eye and the lateral line analog) find
equivalents in vertebrates, and are classic examples of the
convergent evolution of sensory systems. The hydrodynamic
receptor systems (i.e., mechanoreceptor systems sensitive to any
kind of motion of fluid) are of particular interest, as they are based
on hair cells. The latter, in cephalopods, can be both primary and
secondary sensory cells and respond to mechanical stress caused by
the movement of a surrounding medium, either an internal fluid
(endolymph), as in the case of statocysts, or seawater, as in the case
of the lateral line analog system.
Statocysts are essentially closed cavities located in the head,
embedded in the cartilage and located on each side of the brain.
They are lined with complicated arrangements of specialized
epithelia dotted with ciliated mechanoreceptors (hair cells) and
P. Burighel et al. / Hearing Research 273 (2011) 14e24
21
Fig. 3. (A) Octopus. The small dotted circle marks the statocyst position. (B) Diagrammatic representation of a transverse section of a crista in Octopus vulgaris. Three different kinds
of hair cells are present: large and small secondary sensory cells, and primary sensory cells, all characterized by a hair bundle composed of many kinocilia and short microvilli. Large
and small secondary sensory cells form afferent synapses with large and small first-order neurons, respectively. All hair cells and neurons receive multiple efferent synapses
(modified from Budelmann et al., 1987).
provide information about equilibrium, linear (gravitational) and
rotational acceleration. A review of previous literature on the role of
the statocysts is reported in Budelmann and collaborators
(Budelmann et al., 1997). Inside the statocyst cavity, the hair cells
are grouped in maculae and cristae and are associated with
attached suprastructures, such as statoliths, statoconia and cupulae. When these accessory structures are stimulated, they cause
small deflections of the ciliary groups which, in turn, stimulate the
hair cells.
Nautilus exhibits the simpler type of statocyst, an oval-shaped
cavity completely lined with about 100,000 hair cells and half-filled
with statoconia and endolymph. The hair cells are primary sensory
cells, heavily interdigitated to supporting cells, and are divided into
two groups (Table 3) (Neumeister and Budelmann, 1997). Type A
sensory cells lie in the ventral half of the statocyst and are associated with statoconia. They typically carry between 10 and 15
kinocilia, arranged in a single row, which is inclined toward the cell
surface. The kinocilia have a typical 9 2 þ 2 configuration. The
microtubules are connected to the basal body, which is in turn part
of an extensive basal root network. A row of shorter microvilli runs
parallel to the kinociliary row. The rows of kinocilia and microvilli
are not centered, but polarized, displaced on the periphery on the
cell. It has been suggested, on the basis of their location, association
with statoconia and polarization that they play a role in sensing
gravity and other forms of linear acceleration. Type B sensory cells
lie in the dorsal half of the statocyst, and carry up to 10 longer
kinocilia, irregularly arranged on the cell surface and in contact
with the endolymph. There are also short microvilli, more
numerous than those found in Type A mechanoreceptors. Type B
hair cells are thought to be responsible for sensing angular
acceleration.
The statocysts of Octopoda and Decapoda are more differentiated than those of Nautilus and bear a remarkable resemblance to
the complex vertebrate vestibular apparatus. In Octopoda, they are
sphere-like membranous sacs; in Decapoda, they have an irregular
shape, due to many cartilaginous lobes that protrude into the cyst
cavity. In both cases, they possess two separate receptor systems:
a macula/statolith system for detection of gravity and other linear
accelerations, and a crista/cupula system for detection of angular
accelerations. The hair cells in the statocysts of both Octopoda and
Decapoda carry 50e150 kinocilia of the same length, with an
internal 9 2 þ 2 tubules associated with microvilli (Table 3).
Unlike vertebrate vestibular hair cells, these hair cells do not bear
stereovilli. All the kinocilia of a single cell form an elongated
kinociliary group, inclined toward the surface of the cell and
epithelium. The majority of the receptor cells in the maculae and
cristae are secondary sensory cells, but primary sensory cells also
occur in the cristae (Fig. 3B). According to the basal foot structure
on the basal body, each cell appears morphologically polarized in
only one direction; this results in a specific pattern of polarizations
for the receptor epithelia. Thank to this pattern, the hair cells result
directionally sensitive to water movement from different directions
in a way similar to the responses of the hair cells of the vertebrate
vestibular and lateral line systems (Budelmann and Williamson,
1994).
In the gravity receptor system (macula/statolith), thousands of
hair cells, accompanied by supporting cells, are regularly arranged
in the epithelia. The hair cells are associated with accessory statoliths or statoconial layers and are secondary sensory cells in
synaptic contact with afferent and efferent nervous terminations. It
has recently been hypothesized that the macula/statolith complex
is responsible for detecting the displacement component of a sound
wave (Hu et al., 2009). The result has provided new discussion
material for the controversial issue which has existed since the
early 20th century about sound perception among cephalopods.
Sound detection was tested by means of the auditory brainstem
response approach, an electrophysiological far-field recording
method which was originally used in clinical evaluation of human
patients’ hearing ability and then successfully applied to fish,
crustaceans, amphibians, reptiles and birds. Two species, Sepiotheutis lessoniana and Octopus vulgaris, were able to detect sounds
ranging from 400 Hz to 1500 Hz and from 400 Hz to 1000 Hz,
respectively. It is suggested that this hearing ability is comparable
to that of fish without a mechanically coupled gas-bladder to the
inner ear, and makes these cephalopods able to detect sounds
produced by anthropogenic sources or by other animals, such as
odontocete cetaceans which are common predators feeding on
them.
The angular acceleration system (crista/cupula) contains many
hair cells arranged in the crista epithelium with a cupula attached
to it (Budelmann et al., 1987). The crista is subdivided into
segments; the number of hair cells in each crista segment varies
according with species, and ranges from a few hundreds to thousands. Here, large hair cells are arranged in two to four regular rows
22
P. Burighel et al. / Hearing Research 273 (2011) 14e24
Table 3
Mechanoreceptors in statocysts and lateral line analogue of Cephalopods. (See Budelmann et al., 1997 for references).
Receptor
Taxon
Organ
Proposed function
Sensory cell type
Hair bundle
Type A Hair cell
Nautilus
Statocyst
Maintenance of equilibrium
and linear acceleration
Primary
Row of 10e15 kinocilia
of same length and microvilli
TypeB Hair cell
Nautilus
Statocyst
Rotational acceleration
Primary
8e10 kinocilia of same
length and microvilli
Hair cell
Octopoda
Decapoda
Statocyst
(macula/statolith
system)
Gravity sensing and
sound detectiona
Secondary
Many kinocilia of same
length and microvilli
Hair cells
(large and small)
Octopoda
Decapoda
Statocyst
(crista/cupula
system)
Angular acceleration
sensing
Secondary
Many kinocilia of same
length and microvilli
Hair cell
Octopoda
Decapoda
Statocyst
(crista/cupula
system)
Angular acceleration
sensing
Primary
Many kinocilia of same
length and microvilli
Hair cell
Octopoda
Decapoda
Lateral
line analog
Local water movement
sensing
Primary
Many kinocilia
of same length
a
Configuration of hair bundles
From Hu et al. (2009).
in the middle of the crista ridge, whereas two to four rows of
smaller hair cells are on either side. Supporting cells separate hair
cells from each other. Both large and small hair cells are secondary
sensory cells and are also accompanied by primary sensory cells.
Large and small hair cells are in afferent contact with large and
small first-order neurons, respectively. All cells, both primary and
secondary, receive many efferent endings (Fig. 3B).
In Octopoda and Decapoda, sensitivity to local water movements is also due to the “lateral line system”, lines of epidermal
cells on the head and arms which form an organ analogous to the
lateral line in amphibians and fish. Cuttlefish have eight epidermal
lines symmetrically distributed on the head, four on each side (two
dorsally and two laterally); all the lines except the most lateral
continue onto the arms. Squids also have a short fifth line on the
ventral side of the head. The lines are composed of primary sensory
cells carrying cilia with an internal structure of 9 2 þ 2
microtubules. The number of cilia may vary between a few and
more than 100 kinocilia per cell. Each cell is surrounded by several
smaller supporting cells which only have microvilli; sensory cells
appear polarized in one direction, according to the orientation of
the basal feet of the cilia.
4. Concluding remarks
Vertebrate hair cells represent the sensory elements of a variety
of mechanoreceptor organs, for sensing disturbances in water, head
rotation, gravity, and hearing. They are secondary sensory cells
which make contact with the axons of the nearby ganglionic
neurons feeding information to brain centers: both hair cells and
associated neurons originate from neural placodes. Although hair
cells have been intensively analyzed in vertebrates, aspects
regarding their ancestry and phylogeny still need to be clarified and,
P. Burighel et al. / Hearing Research 273 (2011) 14e24
for this, the invertebrates are the appropriate reference, especially
in the light of the new concept of homology discussed in recent
years (e.g., Manley and Ladher, 2008). Indeed, many authors are now
abandoning the traditional all-or-nothing notion of homology, in
favour of a more flexible, factorial approach. The approach considers
homology at different levels (positional, cytological, developmental,
molecular), as homology may not necessarily be expressed at every
level of organisation of structures under examination. This allows an
easier comparison of structures also belonging to organisms of
different phyla. Importantly, at the molecular level, the conservation
of gene networks, in particular transcription factor cascades must be
considered. In the case of mechanoreceptor organs based on hair
cells, the knowledge of the genetic hierarchy governing the organ
development, could tell us if they were inherited from a common
protostome-deuterostome ancestor or evolved independently by
convergence. In this light, a series of studies (cited in Boekhoff-Falk,
2005; Fritzsch et al., 2007) begin to illuminate us, showing that the
homologues of key genes needed for specification or function of
auditory cells of Drosophila and other metazoans are also required
for specification and function of vertebrate ear. In general, some
genes which play a role in mechanoreceptor development, such as
those of Pax family, atonal, Notch and Delta, have been defined in
Drosophila, lower chordates, vertebrates and also molluscs (see
Manley and Ladher, 2008). Since these genes are involved in
mechanoreceptor development as far back as cnidarians, the
molecular mechanisms underlying the development of ciliated
sensory cells are likely to have been conserved. However, it should
be noted that these genes do not play an exclusively mechanosensory role but are found in other contexts during ontogeny.
Moreover, the molecular core of ciliated mechanoreceptor development has as yet only been studied in a few animal models
(Manley and Ladher, 2008; Holland, 2009).
As regard molluscs, while remarkable structural and functional
features of mechanoreceptors are recognizable, the absence of
features sustaining homology at positional and embryological
levels, make a direct comparison with the vertebrates difficult.
Although kinds of homology may be traceable at the level of certain
types of hair cells, the additional accessory structures, e.g. the
cephalopod “vestibular” apparatus, appear at present to be of
independent origin. However, the comparison between lower
chordates and vertebrates relies on more consistent correspondences and homologies, in virtue of various factors, such as phylogenesis, position, structure, function, embryology (placodes in
lower chordates) and genetics. In ascidians, the assignment of
homology of the ascidian atrial primordium to the vertebrate otic
placodes, as above discussed (see Section 2.1.1), rests on aspects of
embryonic development and on the similarity of combinatorial
gene expression (Mazet et al., 2005; Bassham et al., 2008), and also
on the signalling requirement (FGF signaling cascade) during
induction of the placode and its differentiation (Kourakis and
Smith, 2007). The finding of hair coronal cells in ascidians, with
their obvious similarities to vertebrate hair cells, have provided
important links concerning the origin and early evolution of these
receptors. Coronal cells from several ascidians vary in cell features,
in some ways analogous to the variability in vertebrates and suggesting that, as in vertebrates (Coffin et al., 2004), the differences
reflect specializations for responses to differing signals and to
trigger various physiological responses.
Data from several fields are consistent with the idea of
a common derivation for vertebrate and ascidian secondary
sensory cells. Comparative studies show that coronal cells lie in the
oral siphon of all ascidian species so far investigated (seven
Enterogona and five Pleurogona), suggesting that the coronal cells
represent a plesiomorphic condition of ascidians. It is noteworthy
that mechanoreceptors based on secondary sensory cells arranged
23
in rows are found in the oral area of both appendicularians (Bone,
1998) and the cephalochordate Branchiostoma (Lacalli, 2004).
Among vertebrates, in the fish, secondary sensory cells are found in
the ears, lateral line and derived electroreceptor organs, and extend
broadly over the trunk and head, also reaching the mouth. Thus,
from a positional point of view, there are resemblances in the
location of secondary sensory cells in tunicates, cephalochordates
and vertebrates. From a developmental point of view, various data
also indicate that cell populations with the characteristics of placodes or neural crests occur in lower chordates. Recent papers on
Branchiostoma show the presence of both migrating cells in the
ectoderm destined to differentiate into sensory neurons, which
mimic neural crest cells and placode-derived sensory cells, and
show homologs of most vertebrate genes involved in the putative
neural crest/placodes gene regulatory network (Benito-Gutiérrez
et al., 2005; Meulemans and Bronner-Fraser, 2007; Rasmussen
et al., 2007; Holland, 2009; Kaltenbach et al., 2009; Yu et al., 2009).
Thus, various data indicate that cell populations with characteristics of placodes or neural crest also occur in tunicates and
cephalochordates (Burighel et al., 1998; Manni et al., 2004b; Baker
and Schlosser, 2005; Mazet et al., 2005; Hall, 2009), suggesting that
the chordate ancestor possessed a gene network for specification of
a primitive anterior pan-placodal region (as suggested by Schlosser,
2008), from which secondary sensory cells also evolved over time,
becoming incorporated into group-specific structures. In particular,
it is noteworthy that, despite their long independent evolution,
amazing parallels can be now found in vertebrate and ascidian hair
cells, probably developed in response to similar selection pressures.
In conclusion, much data e positional, cellular, developmental
and moleculare suggest that secondary sensory cells were present
in the common chordate ancestor and gave rise to the differing
secondary mechanoreceptor cells in the three subphyla of extant
chordates.
Acknowledgments
This study was supported by grants from the Italian Ministero
della Università e Ricerca Scientifica e Tecnologica to P.B. and L.M.
References
Baatrup, E., 1981. Primary sensory cells in the skin of amphioxus (Brachiostoma
lanceolatum). Acta Zool. 62, 147e157.
Baker, C.V.H., Schlosser, G., 2005. Editorial: the evolutionary origin of neural crest
and placodes. J. Exp. Zool. B (Mol. Dev. Evol.) 304, 269e273.
Bassham, S., Postlethwait, J.H., 2005. The evolutionary history of placodes:
a molecular genetic investigation of the larvacean urochordate Oikopleura
dioica. Development 132, 4259e4272.
Bassham, S., Cañestro, C., Postlethwait, J.H., 2008. Evolution of developmental roles
of Pax2/5/8 paralogs after independent duplication in urochordate and vertebrate lineages. BMC Biol. 6, 35.
Benito-Gutiérrez, E., Nake, C., Llovera, M., Comella, J.X., Garcia-Fernandez, J., 2005.
The single AmphiTrk receptor highlights increased complexity of neurotrophin
signalling in vertebrates and suggests an early role in developing sensory
neuroepidermal cells. Development 132 (9), 2191e2202.
Boekhoff-Falk, G., 2005. Hearing in Drosophila: development of Johnston’s organ
and emerging parallels to vertebrate ear development. Dev. Dyn. 232, 550e558.
Bone, Q., 1998. Nervous system, sense organs, and excitable epithelia. In: Bone, Q.
(Ed.), The Biology of Pelagic Tunicates. Oxford University Press, Oxford, New
York, pp. 55e80.
Bone, Q., Mackie, G.O., 1975. Skin impulse and locomotion in Oikopleura (Tunicata:
Larvacea). Biol. Bull. 1549, 267e286.
Bone, Q., Ryan, K.P., 1978. Cupular sense organs in Ciona (Tunicata: Ascidiacea). J.
Zool. London 186, 417e429.
Bone, Q., Mackie, G.O., 1982. Urochordata. In: Shelton, G.A.B. (Ed.), Electrical
Conduction and Behaviour in “Simple” Invertebrates. Clarendon Press, Oxford,
pp. 473e535.
Budelmann, B.U., 1989. Hydrodynamic receptors system in invertebrates. In:
Coombs, S., Görner, P., Münz, H. (Eds.), Mechanosensory Lateral Line. SpringerVerlag, Berlin, pp. 607e631.
Budelmann, B.U., Sachse, M., Staudigl, M., 1987. The angular acceleration receptor
system of the statocyst of Octopus vulgaris: morphometry, ultrastructure, and
24
P. Burighel et al. / Hearing Research 273 (2011) 14e24
neuronal and synaptic organization. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 315,
305e344.
Budelmann, B.U., Williamson, R., 1994. Directional sensitivity of hair cell afferents in
the Octopus statocyst. J. Exp. Biol. 187, 245e259.
Budelmann, B.U., Schipp, R., Von Boletzky, S., 1997. Cephalopoda. In: Harrison, F.W.,
Kohn, A.J. (Eds.), Microscopic Anatomy of Invertebrates. Mollusca II, vol. 6A.
Wiley-Liss, New York, pp. 119e414.
Burighel, P., Lane, N.J., Zaniolo, G., Manni, L., 1998. Neurogenic role of the neural
gland in the development of the ascidian, Botryllus schlosseri (Tunicata, Urochordata). J. Comp. Neurol. 394 (2), 230e241.
Burighel, P., Lane, N.J., Gasparini, F., Tiozzo, S., Zaniolo, G., Carnevali, M.D., Manni, L.,
2003. Novel, secondary sensory cell organ in ascidians: in search of the ancestor
of the vertebrate lateral line. J. Comp. Neurol. 461, 236e249.
Burighel, P., Caicci, F., Zaniolo, G., Gasparini, F., Degasperi, V., Manni, L., 2008. Does
hair cell differentiation predate the vertebrate appearance? Brain Res. Bull. 75,
331e334.
Caicci, F., Burighel, P., Manni, L., 2007. Hair cells in an ascidian (Tunicata) and their
evolution in chordates. Hear. Res. 231, 63e72.
Coffin, A., Kelley, M., Manley, G.A., Popper, A.N., 2004. Evolution of sensory hair
cells. In: Manley, G.A., Popper, A.N., Fay, R.R. (Eds.), Evolution of the Vertebrate
Auditory System. Springer-Verlag, New York, pp. 55e94.
Delsuc, F., Brinkmann, H., Chourrout, D., Philippe, H., 2006. Tunicates and not
cephalochordates are the closest living relatives of vertebrates. Nature 439,
965e968.
Dufour, H.D., Chettouh, Z., Deyts, C., De Rosa, R., Goridis, C., Joly, J.S., Brunet, J.F.,
2006. Precraniate origin of cranial motoneurons. Proc. Natl. Acad. Sci. U S A 103,
8727e8732.
Fiala-Medioni, A., 1974. Ethologie alimentaire d’invertébrés benthiques filtreurs
(ascidies). II. Variations des taux de filtration et de digestion en fonction de
l’espèce. Mar. Biol. 28, 199e206.
Fritzsch, B., Beisel, K.W., Pauley, S., Soukup, G., 2007. Molecular evolution of the
vertebrate mechanosensory cell and ear. Int. J. Dev. Biol. 51, 663e678.
Hall, B.K., 2009. The neural crest and neural crest cells in vertebrate development
and evolution. In: Evolutionary Origins. Springer, New York, pp. 117e155.
Chapter 4.
Holland, L., 2009. Chordate roots of the vertebrate nervous system: expanding the
molecular toolkit. Nat. Rev. Neurol. 10, 736e746.
Holland, N.D., Yu, J.K., 2002. Epidermal receptor development and sensory pathways in vitally stained amphioxus (Branchiostoma floridae). Acta Zool 83,
309e319.
Hu, M.-Y., Yan, H.-Y., Chung, W.-S., Shiao, J.-C., Hwang P.-, P., 2009. Acoustically
evoked potentials in two cephalopods inferred using the auditory brainstem
response (ABR) approach. Comp. Biochem. Physiol. A. 153, 278e283.
Jeffery, W.R., 2007. Chordate ancestry of the neural crest: new insights from
ascidians. Semin. Cell Dev. Biol. 18, 481e491.
Jeffery, W.R., 2006. Ascidian neural crest-like cells: phylogenetic distribution,
relationship to larval complexity, and pigment cell fate. J. Exp. Zool. B. (Mol.
Dev. Evol.) 15, 470e480.
Kaltenbach, S.L., Yu, J.-K., Holland, N.D., 2009. The origin and migration of the
earliest-developing sensory neurons in the peripheral nervous system of
amphioxus. Evol. Dev. 11, 145e151.
Koyama, H., 2008. Sensory cells associated with the tentacular tunic of the ascidian
Polyandrocarpa misakiensis (Tunicata: Ascidiacea). Zool. Sci. 25, 919e930.
Kourakis, M.J., Smith, W.C., 2007. A conserved role for FGF signalling in chordate
otic/atrial placodes formation. Dev. Biol. 312, 245e257.
Lacalli, T.C., 2004. Sensory systems in amphioxus: a window on the ancestral
chordate condition. Brain Behav. Evol. 64, 148e162.
Lacalli, T.C., Hou, S., 1999. A reexamination of the epitelial sensory cells of amphioxus (Branchiostoma). Acta Zool. 80, 125e134.
Mackie, G.O., Singla, C.L., 2003. The capsular organ of Chelyosoma productum
(Ascidiacea: Corellidae): a new tunicate hydrodynamic sense organ. Brain
Behav. Evol. 61, 45e58.
Mackie, G.O., Singla, C.L., 2004. Cupular organs in two species of Corella (Tunicata:
Ascidiacea). Invert. Biol. 123, 269e281.
Mackie, G.O., Burighel, P., 2005. The nervous system in adult tunicates: current
research directions. Can. J. Zool. 83, 151e183.
Mackie, G.O., Burighel, P., Caicci, F., Manni, L., 2006. Innervation of ascidian siphons
and their resposes to stimulation. Can. J. Zool. 84, 1146e1162.
Manley, G.A., Ladher, R., 2008. Phylogeny and evolution of ciliated mechanoreceptor cells. In: Dallos, P., Oertel, D. (Eds.), The Senses: a Comprehensive
Reference. Audition, vol. 3. Academic press, San Diego, pp. 1e34.
Manni, L., Lane, N.J., Burighel, P., Zaniolo, G., 2001. Are neural crest and placodes
exclusive to vertebrates? Evol. Dev. 3, 287e298.
Manni, L., Caicci, F., Gasparini, F., Zaniolo, G., Burighel, P., 2004a. Hair cells in
ascidians and the evolution of lateral line placodes. Evol. Dev. 6, 379e381.
Manni, L., Lane, N.J., Joly, J.S., Gasparini, F., Tiozzo, S., Caicci, F., Zaniolo, G.,
Burighel, P., 2004b. Neurogenic and non-neurogenic placodes in ascidians. J.
Exp. Zool. B. (Mol. Dev. Evol.) 302, 483e504.
Manni, L., Agnoletto, A., Zaniolo, G., Burighel, P., 2005. Stomodeal and neurohypophysial placodes in Ciona intestinalis: insights into the origin of the pituitary
gland. J. Exp. Zool. B. (Mol. Dev. Evol.) 304, 324e339.
Manni, L., Mackie, G.O., Caicci, F., Zaniolo, G., Burighel, P., 2006. Coronal organ of
ascidians and the evolutionary significance of secondary sensory cells in
chordates. J. Comp. Neurol. 495, 363e373.
Manni, L., Zaniolo, G., Cima, F., Burighel, P., Ballarin, L., 2007. Botryllus schlosseri:
a model ascidian for the study of asexual reproduction. Dev. Dyn 236, 335e352.
Mazet, F., Hutt, J.A., Milloz, J., Millard, J., Graham, A., Shimeld, S.M., 2005. Molecular
evidence from Ciona intestinalis for the evolutionary origin of vertebrate
sensory placodes. Dev. Biol. 282, 494e508.
Meulemans, D., Bronner-Fraser, M., 2007. The amphioxus SoxB family: implications
for the evolution of vertebrate placodes. Int. J. Biol. Sci. 3 (6), 356e364.
Neumeister, H., Budelmann, B.U., 1997. Structure and function of the Nautilus
statocyst. Philos. Trans. R. Soc. Lond. B. Biol. Sci. 352, 1565e1588.
Northcutt, R.G., Gans, C., 1983. The genesis of neural crest and epidermal placodes:
a reinterpretation of vertebrate origins. Q. Rev. Biol. 58 (1), 1e28.
Passamaneck, Y.J., Di Gregorio, A., 2005. Ciona intestinalis: Chordate development
made simple. Dev. Dyn. 233, 1e19.
Rasmussen, S.L., Holland, L.Z., Schubert, M., Beaster-Jones, L., Holland, N.D., 2007.
Amphioxus AmphiDelta: evolution of Delta protein structure, segmentation,
and neurogenesis. Genesis 45, 113e122.
Satoh, N., Levine, M., 2005. Surfing with the tunicates into the post-genome era.
Genes Dev. 19, 2407e2411.
Schlosser, G., 2008. Do vertebrate neural crest and cranial placodes have a common
evolutionary origin? BioEssays 30, 659e672.
Shimeld, S.M., Holland, P.W.H., 2000. Vertebrate innovations. Proc. Natl. Acad. Sci. U
S A 97, 4449e4452.
Yu, J.K., Meulemans, D., McKeown, S.J., Bronner-Fraser, M., 2009. Insights from the
amphioxus genome on the origin of vertebrate neural crest. Gen. Res. 18,
1127e1132.
Wada, H., Holland, P.W.H., Satoh, N., 1996. Origin of patterning in neural tubes.
Nature 384, 123.
Wada, H., Saiga, H., Satoh, N., Holland, P.W.H., 1998. Tripartite organization of the
ancestral chordate brain and the antiquity of placodes: insights from ascidian
Pax-2/5/8, Hox and Otx genes. Development 125, 1113e1122.