393
Studies on the receptors in the cerebral vesicle of the
ascidian tadpole, i. The otolith
By P. N. DILLY
(From the Department of Anatomy, University College, London)
With 2 plates (figs, z and 3)
Summary
Electron microscope observations of the gravity receptor of the tadpole larva of Ciona
intestinalis show that the otolith is unicellular. The nucleus of the otolith persists
for the entire life of the tadpole. The pigment mass of the otolith is intracellular, and
it appears to be built up by fusion of granules. The otolith cell has a free part within
the cavity of the cerebral vesicle, and a foot part which is contained within a mound
of cells on the ventral wall of the cerebral vesicle. The junction between these two
parts is probably the transducer region.
The transducer region is a complex system of folded cell membrane. Distortion of
this system during rotation of the tadpole while swimming probably evokes changes in
the neurones which surround the foot process of the otolith cell. One of these neurones
is connected to the cerebral ganglion by a process which may be an axon. Fibrils
extend from the transducer region of the otolith cell to the basement membrane, and
probably serve to resist distortion of the transducer region.
The foot process of the otolith cell is connected to the surrounding cells by specializations of the cell membrane similar to attachment plaques.
The observations suggest that the otolith has been evolved from a cilium.
Introduction
T H E free-swimming tadpole larvae of ascidians react to gravity in a manner
which ensures the wide dispersal of the sessile adult stage of the life-cycle.
For about 2 h from the moment of hatching the tadpole is negatively geotropic.
Then, for the major part of its free-swimming life (11 h) it is indifferent in its
reaction to gravity. During the last 2 h or so of the free-swimming period it
becomes positively geotropic. The tadpole swims by paired myotomal bands
on either side of the notochord. These are probably controlled by a strand
of nerve-fibres that leave the hind end of the cerebral ganglion. The receptor
sensitive to gravity is a unicellular otolith situated within the cavity of the
cerebral vesicle (fig. 1). The ocellus, the photoreceptor, is made up from a few
cells, the organization of which will be discussed in a forthcoming paper.
Material and methods
Specimens of tadpole larvae from various stages of the free-swimming
life of Ciona intestinalis were fixed in buffered 1 % OsO4 for 1 to 3 h, washed
in sea-water, dehydrated through graded acetones, and 'stained' with 1%
phosphotungstic acid in absolute acetone for 1 h. The tadpoles were then
[Quart. J. micr. Sci., Vol. 103, pt. 3, pp. 393-8, 1962.]
394
Ditty—The otolith of the ascidian tadpole
embedded in Araldite, sectioned with a Porter Blum microtome using glass
knives, and examined in a Siemens Elmiskop i.
Observations
Tadpoles were taken from a selection of times during the free-swimming
period in the hope that the changes in response to gravity might be correlated
with some observable morphological change in the receptor.
FIG. I . Diagram of head region of the tadpole showing the receptors in the cerebral
vesicle. Only approximately to scale. The ocellus as well as the otolith is shown together with
the nerve-bundle to the tail-muscles, e.g., cerebral ganglion; c.v., cavity of the cerebral
vesicle; I.e., lens cell; l.v., lens vesicle; m.c., muscle-cell; m.f., myofibril; m.n., muscle-nerve;
n.c, notochord; n.c.p., nerve-cell process; n.t., neural tube; o.t., otolith; p.c, pigment cell;
p.m., piled photoreceptor membrane; r.c, retinal cell; r.c.p., retinal cell process.
The otolith protrudes into the cavity of the cerebral vesicle from a small
group of cells that forms an elevated mound in the ventral wall (fig. i). It is
a unicellular receptor consisting of an intracellular dense black mass of pigment material, a narrow stalk, and an L-shaped foot. The foot is situated in
the group of cells forming the mound. The narrow neck and bulbous pigment mass project into the cavity of the vesicle (fig. 2, A).
FIG. 2 (plate), A, otolith. Low-power micrograph to show the organization of the gravity
receptor. Part of the otolith cell projects into the cavity of the cerebral vesicle, and is free
to move within the cavity during rotation of the tadpole while swimming, b.m., basement
membrane; c, collar of the transducer region; C.V., cavity of the cerebral vesicle; ft., foot
process of the otolith cell; n., neck of the transducer region; n.c, nerve-cell; n.c.p., nervecell process, probably an axon; o.c, otolith cell-body; o.w., otolith weight.
B, transverse section through apex of developing otolith cell, showing pigment granules and
the developing pigment masses, e.r., endoplasmic reticulum; in., membrane bounding the
pigment masses; p.g., pigment granules; p.m., pigment mass; y., yolk droplet.
c, section of neck region of the otolith cell showing the anchoring fibrils, b.m., basement
membrane; c, collar of transducer region;/., anchoring fibrils;/*., foot region of the otolith
cell; n., neck of the transducer region; n.c, nerve-cell; nu., nucleus; o.c, otolith cell, free part;
y., yolk droplet.
FIG.
2
P. N. DTLLY
Dilly—The otolith of the ascidian tadpole
395
Pigment mass
In the free-swimming larva the pigment is solid, approximately spherical
in shape, and some 15 to 25 /x in diameter (fig. 2, A). However, during
development, before the tadpole hatches, the cell that becomes the otolith
can be seen to contain several smaller pigment masses, varying in diameter
between 1 and 5 ju. (fig. 2, B). The masses have usually fused together to form
the single large mass before the tadpole hatches.
The first sign of the future pigment mass is of small spherical granules
some 15 to 30 m/x in diameter (fig. 2, B). At an early stage these granules fuse
together to form the larger spherical masses. These larger masses are membrane-bound and are probably contained within vesicular extensions of the
endoplasmic reticulum, as are most cellular secretions (Palay, 19586). The
membrane relations of the smallest granules (15 to 30 mfi) have not been clearly
seen, but the granules are often seen near the larger masses in the membrane-bound spaces (fig. 2, B). The granules which form the larger masses
do not seem to be firmly joined together, since in damaged sections they are
often seen dispersed around the edges of the larger masses. The granules are
not just lightly packed together, but are in some way fused, since otherwise
spaces would appear between the granules in very thin sections. The pigment
masses probably increase in size by the addition of the granules to the surface
of the masses. The final spherical pigment mass is formed by the fusion of the
smaller (1 to 5 fj.) pigment masses. This probably takes place by break-down
of the membranes separating the masses, allowing them to fuse. The amount
of the cell taken up by the final mass is so large that at the distal limits of the
otolith cell the only membranes that surround the pigment mass are those of
the endoplasmic reticulum, and the cell membrane (fig. 2, A).
The material of the pigment mass is probably a melanin (Minganti, 1957).
The material has a higher specific gravity than ordinary tadpole tissue. This
was shown by digesting some tadpoles enzymatically and separating the pigment masses by washing and centrifugation. Some of this material was then
mixed with tadpoles from the same batch, which had been killed with 20%
urethane solution in sea-water, and allowed to settle in a centrifuge tube.
The majority of the material at the bottom of the tube was pigment.
Cytoplasmic contents of the remaining free part of the otolith cell
The remaining part of the otolith cell that is free within the cavity of the
cerebral vesicle is conical in shape. It contains the normal cytoplasmic
materials displaced by the expanded pigment mass. The nucleus is an irregular oval shape. There are several mitochondria and membrane-bound yolk
FIG. 3 (plate). A selection from a series of serial sections through the junction of fixed
and free parts of the otolith cell. OsO4 fixed, P.T.A. 'stained', a.p., attachment plaque; c,
collar; C.V., cavity of the cerebral vesicle;/., anchoring fibrils; f.p., free part of the otolith
ce\[;fx.p. fixed part of the otolith cell; n., neck; y., yolk droplets.
396
Dilly—The otolith of the ascidian tadpole
droplets in the cytoplasm. The remainder of the endoplasmic reticulum does
not appear to be organized in any special manner (fig. 2, A).
The junction region
The junction between the free and fixed portion of the otolith is organized
in a complex manner (Dilly, 1961*2). Serial sections (fig. 3) reveal that the
cell membrane is folded back upon itself to form a collar around the stalk of
the cell. The cytoplasm is filled with distended vesicles of the endoplasmic
reticulum. The collar is roughly cylindrical in its outside shape, whereas the
inner membrane slopes inwards conically from a wide base towards a narrow
neck below. The diameter of this neck is only about 075 /x. It seems
remarkably thin to support the mass of the otolith.
Foot region of the otolith cell
The foot region of the otolith cell (fig. 2, A, and c), that is the region within
the mound of cells on the ventral wall of the cerebral vesicle, extends at right
angles to the long axis of the remainder of the otolith cell to give the whole
structure an L-shape. The right angle of the L is occupied by a cell that is
probably a neuron (fig. 2, A). The oval shape of this cell is such that the vertical arm of the L is conical, whereas the horizontal arm is narrow in the
middle and wider at each end. The outer side of the vertical section of the
foot is in contact with another cell of the mound (fig. 2, A), which extends
alongside it as far as the basement membrane that separates the cerebral
vesicle from the rest of the tissues of the tadpole. Both sides of the vertical
conical region of the foot are attached to the surrounding cells by specialization of the containing membranes similar to the attachment plaques described
by Odland (1958) between the cells of the human epidermis. The plaques
are prominent at the neck of the fixed region of the otolith cell. These attachment plaques do not have the associated masses of tonofibrils that are seen
in glial cells of the goldfish (Dilly, 1961&). They are simply densities some
20 to 30 m/x thick and about 1 /A long associated symmetrically across the
containing membranes. The intermembrane gap increases to some 25 mju.
between the densities. There are no external compound membranes limiting
the attachment zones (Dilly, 1961b).
There are no other obvious modifications of the cell membrane of the
otolith cell except that along the entire length of the membrane that is enclosed within the cell mound there are small spherical electron dense granules
some 15 m/x in diameter attached to the membrane. Extending and curving
slightly from the membrane folds of the fixed-free junction through the cytoplasm of the foot to the innermost membrane of the basement membrane
complex are some 5 to 10 fibrils (fig. 2, c). At both ends the fibrils and membranes are thickened slightly at their points of contact. The fibres are solid,
some 7-5 to 15 m/j. in diameter, and have no obvious cross-striations. The
fibrils do not branch or join together but run singly through the cytoplasm.
The foot region of the otolith cell contains a large number of mitochondria,
Billy—The otolith of the ascidian tadpole
397
together with several yolk droplets and the usual vesicles and channels of the
endoplasmic reticulum: vesicular profiles are found randomly amongst the
other cytoplasmic contents.
Discussion
No significant morphological changes were observed in the structure of
the otolith that could be correlated with the various states of physiological
activity of the free-swimming tadpole. It may well be that these changes
occur elsewhere in the nervous system, or that they occur at a molecular level.
The fact that the pigment mass is heavier than the remainder of the otolith
cell, together with the narrowness of the neck at the fixed-free junction,
suggests that as the tadpole rotates on its long axis, while swimming, movements of this weight distort the otolith cell. Since the melanin of the pigment
mass is opaque to light it may be that the otolith, during its movements, acts
as a shutter, interrupting the light falling on the photosensitive endings of the
ocellus and thus, besides being a gravity receptor, acts also as a device which
ensures intermittent stimulation of the photoreceptors. An interesting observation which supports this hypothesis is that when the tadpoles are inactive
and quiescent it is possible to rouse them to active swimming by rapidly
interrupting the light falling upon them, whereas slow variation of the light
intensity through a similar range has little or no effect.
Since the neck is so narrow, and situated at the fixed-free junction, it is
reasonable to suppose that this is the point where maximum distortion will
occur as the otolith moves. The membrane relations of this junction are
complex, and may well perform a specialized function as a transducer region
that converts the mechanical displacement of the otolith during rotation of the
tadpole into nerve-signals.
The cell-body that lies in the right angle of the L-shaped foot of the otolith
cell is probably a neurone. This cell has a long process that is probably an
axon running towards the cerebral ganglion. The constant position and presence of this cell in the angle of the L, together with its process, suggest that
it is probably the neurone that transmits the impulses generated by the otolith. This may be effected by generator potentials in the junctional region,
causing action potentials to be generated in the neurone. Or the neurone may
be distorted mechanically by movements of the otolith. It may be that the
position of the otolith cell is signalled both by this neurone and by the cell
which bounds the vertical arm of the L-shaped foot region since they are both
attached to the otolith cell by plaques. It is unlikely that these attachment
plaques are synapses, since few if any vesicles are found in association with
them, and they do not exhibit the polarity which is one of the criteria established by Palay (1956 and 1958a) and Gray (1959) for the identification of
synapses. These plaques probably serve to attach the otolith cell firmly to
the surrounding cells. It is not possible at present to say what connexions
this group of cells make with the nerve-cord that leads to the tail-muscles
(fig- 0-
398
Ditty—The otolith of the ascidian tadpole
The fibrils that join the lowermost part of the neck of the fixed-free junction to the innermost membrane of the basement membrane probably serve
to anchor the neck region so that it cannot be 'pulled out' or excessively distorted during rotation of the tadpole. The anchoring fibrils may well be the
modified remains of the $• pairs of tubules with the ninefold axis of symmetry
found in-cilia (Fawcett and Porter, 1954). It is probable that the ascidian
otolith is a modified cilium. In many of the lower vertebrates there are motile
kinocilia in the lining of the cerebral canal and the cristae of the inner ear,
and it has been shown that rods and cones are derived from.the ciliated
epithelium of the neurenteric canal. These anchoring fibrils may well represent a trace of the structure of the cilium that has persisted in the evolutionary
change from a ciliary effector to a gravity receptor.
I wish to thank Professor J. Z. Young, F.R.S., and Drs. E. G. Gray and
J. D. Robertson for their advice, criticism, and encouragement. Miss J. de
Vere gave much help with the illustrations.
I am indebted to the University of London, part of this work having been
carried out during the tenure of a University of London Postgraduate Studentship. The research has been sponsored in part by the United States Air
Force Office of Scientific Research.
References
DILLY, N., 1961a. Nature, Lond., 191, 786.
19616. 'A comparison between glial and neuronal interconnections in the C.N.S. of
the goldfish', in Cytology of nervous tissue, Proc. anat. Soc, 92.
FAWCETT, D. W., and PORTER, K. R., 1954.
J. Morph., 94, 221.
GRAY, E. G., 1959. J. Anat. Lond., 93, 420.
MINGANTI, A., 1957. Acta Embryol. Morph. exp., i, 37.
ODLAND, G. F., 1958. J. biophys. biochem. Cytol., 4, 529.
PALAY, S. L., 1956. Ibid., suppl., 2, 193.
1958a. Exp. Cell Res., suppl.; 5, 275.
19586. 'The morphology of secretion', in Frontiers in cytology, ed. Palay, S. L., Yale,