/ . Embryol exp. Morph. Vol. 24, 3, pp. 511-524, 1970
Printed in Great Britain
The lateral line system at metamorphosis
in Xenopus laevis (Daudin)
By PETER M. J. SHELTON 1
From the Gatty Marine Laboratory and Department of Natural History,
St Andrews University, Scotland
SUMMARY
Marked changes in the anatomy of the lateral line system occur during the metamorphosis
of Xenopus. The distribution of rows differs in larva and adult and the orientation and number
of organs are modified at metamorphosis.
Larval plaques are functional, as shown by recording from their nerves.
Two classes of cells with polarized cilia are present in the tadpole well before the orientation
of individual organ plaques is rearranged at metamorphosis.
The topography of the skin surface around individual plaques changes at metamorphosis.
This change may reduce the directional sensitivity of organs.
Myelinated inhibitory axons in the lateralis nerve are found only when the tadpole matures.
This change takes place at a time when the adult method of locomotion is developed.
INTRODUCTION
Just how sense organs of developing animals are adapted to the concurrent
changes of behaviour is unknown. The Amphibia are especially suitable for
studies in this field because, wherever metamorphosis occurs, drastic changes in
anatomy and mode of life take place in a short time. In Xenopus laevis the larval
lateral line system develops smoothly into that of the adult. This paper describes
some major changes in this system during development and tentatively correlates
them with changes in behaviour.
The whole function of the lateral line systems of fishes and Amphibia is
subject to debate but some features are known. For example, animals possessing
lateral line organs can accurately locate objects in their environment (Dijkgraaf,
1963). Characteristics of the system which help to make this function possible
are: (1) organs are spread in rows over large parts of the surface of the animals;
(2) individual organs are maximally sensitive to water currents in one plane only
(Flock, 1965); and (3) different organs are orientated so that their planes of
maximal sensitivity are in different directions.
During the metamorphosis of X. laevis the supra-orbital and post-orbital
lines migrate to a new position around the orbit of the eye (Paterson, 1939).
1
Author's address: Research School of Biological Sciences, P.O. Box 475, The Australian
National University, Canberra City, A.C.T. 2601, Australia.
512
P. M. J. SHELTON
This movement involves changes in the orientation and location of organs so
that the plane in which they show maximal sensitivity to vibration changes at
metamorphosis. Therefore, the system as a whole must compensate for the new
distribution of individual organs. The present account shows that this conclusion
applies generally to the whole lateral line system.
A group of lateral line organs of adult X. laevis is called a plaque (Murray,
1955) within which the organs are separated from each other by raised tactile
organs (Calabresi, 1924). The latter, by virtue of their position, direct water
currents on to the lateral line organs (Gorner, 1961) and may have a protective
function. Rows of plaques make up each lateral line and a common nerve trunk
supplies each row with branches to each plaque.
Each organ contains ciliated receptor cells (neuromasts) of two classes. These
are sensitive to water displacements in opposite directions as shown by electrophysiological distinction between units (Gorner, 1963). The maximum sensitivity of the plaque is approximately at right angles to its long axis although
there is some variation in the orientation of organs in one plaque (Gorner, 1961,
1963). Each neuromast cell has a single kinocilium projecting from its apical
surface. The kinocilium possesses the standard '9 + 2' arrangement of ciliary
filaments. In addition, neuromast cells have a group of stereocilia to one side of
the kinocilium. These stereocilia are about two-thirds of the diameter of kinocilia and have no '9 + 2' pattern of filaments. They do contain fine fibrils but
there is no obvious order in their arrangement. The directional sensitivity of
organs is correlated with an anatomical orientation of stereocilia to the single
kinocilium of each neuromast cell; half of the cells have the kinocilium on one
side of the stereocilia and half have the kinocilium on the opposite side (Gorner,
1963). In adult X. laevis the nerve running from each plaque to the main nerve
trunk always contains (a) two large myelinated nerve fibres (8-10 JLC in diameter);
(b) a bundle of non-myelinated fibres, and (c) often but not always, one or more
small myelinated fibres (0-5-1-0 /i in diameter). The two large fibres are sensory
{Gorner, 1961) and synaptic-type contact between the unmyelinated ends of
sensory fibres and receptor cells has been shown in the lateral line system of
Lota vulgaris (Flock, 1965) although the exact pattern of innervation is unknown.
The small myelinated fibres are efferent and inhibitory (Russell, 1968) and
inhibitory fibres have been shown to make synaptic-type contact in L. vulgaris
(Flock, 1965). The function and destination of the non-myelinated fibres is
unknown.
The position of the lateral line rows in larval X. laevis has been described most
accurately by Nieuwkoop & Faber (1967), and in the adult by Escher (1925),
Horst (1934), Paterson (1939) and Murray (1955). The anterior organs are supplied by the anterior lateralis nerve and the posterior ones by the posterior
lateralis nerve. Peripherally the anterior lateralis nerve supplies three lateralis
rows, the supra-orbital, the infra-orbital and the hyomandibular, each of which
has further subdivisions. The posterior lateralis nerve divides peripherally to
Lateral line system
513
supply five rows, the occipital, the aortic (previously undescribed), the lower
lateral, the middle lateral and the upper lateral lines. The lower lateral line has
several subdivisions. In addition to the rows there are anterior and posterior
auditory groups. All these parts of the system have been examined during
metamorphosis.
MATERIALS AND METHODS
Developmental stages of laboratory bred animals were classified according to
the system of Nieuwkoop & Faber (1967). They were anaesthetized in a
1:10000 solution of MS 222 (Sandoz) in distilled water and then pithed. All
material for examination was fixed at 0 °C in 1% osmium tetroxide buffered to
pH 7-4 with veronal acetate and made up to a concentration of 300 m-osmoles
with sucrose (after Palade, 1952). Fixative was pipetted under and on top of the
skin. Material being prepared for whole mounts was allowed to darken noticeably before being removed and dehydrated in acetone. Skins were laid out on
microscope slides and covered with small pieces of glass to keep them flat
during dehydration. They were finally mounted in Araldite under cover slips on
microscope slides. The osmophilic properties of the organs and nerves made
them stand out against the paler background. Whole mounts of skin were
photographed and the distribution of organs and their rows was transferred to
diagrams of the complete system. Five adult animals and five stage 55 tadpoles
were examined and combined data concerning each lateral line row were tabulated. Material for electron microscopy was removed after 10 min in situ fixation and fixed for a further 20 min in covered watch-glasses. After dehydration
in acetone, material was embedded in Araldite. Blocks of tissue were cut on an
L.K.B. ultramicrotome and sections were stained with lead citrate and uranyl
acetate for examination with an A.E.I. EM 6B electron microscope. For light
microscopy 1 ft sections were stained for 1 min with toluidine blue. For conventional electrophysiological recording tadpoles were held down and bathed
with Ringer solution (see Russell, 1968) and nerve trunks were picked up on
silver wire hook electrodes.
RESULTS
The changes in position, number and orientation of the lateral line
organs at metamorphosis
The distribution of lateral line rows in larva and adult is shown in Figs. 1 and 2
respectively. The names of the subdivisions of the rows are listed in Table 1
together with data regarding the position of the rows, the number of plaques
found in each row and the angles at which plaques are orientated with respect to
an antero-posterior median axis.
The number of plaques in many of the rows is approximately the same in
larva and adult, in others there is a drastic reduction in numbers. The anterior
auditory groups, the aortic row and the tail portions of the middle lateral and
P. M. J. SHELTON
514
'.'at.
oc.
p.a.
a.
a.a.
p.o.
s.o.
hy.
mid.lat.
-0°
90°
med.v.
p.l.lat
a.l./at
a.
po.
in.
Lateral line system
515
upper lateral lines are lost completely. Numbers of plaques in the hyomandibular, anterior lower lateral and caudal rows are severely reduced (see Table 1).
In certain rows there appears to be an increase in plaque number at metamorphosis, this apparent increase is probably due to confusion in knowing
where one row begins and another ends and does not represent formation of
new organs (there is, for instance, a large reduction in organs of the anterior
lower lateral line and an increase in the posterior lower lateral).
Lines which are dorsal, lateral or ventral in the larva often maintain these
positions in the adult; however, the infra-orbital, tentacular, aortic, anterior
lower lateral, posterior lower lateral, caudal and posterior auditory rows show
distinct changes of position at metamorphosis (see Table 1).
The orientation of the long axis of plaques with respect to the animal's median
antero-posterior axis was measured in a number of larvae and adults. For each
row the range of angles to the reference axis was ascertained and the extremes
noted. While plaques in certain lines approximately maintain their larval
orientation, many do not. These include the post-orbital, hyomandibular, occipital, anterior lower lateral, median ventral and caudal lines (see Table 1).
In the anterior lower lateral and caudal lines there are changes in position,
number and orientation of organs. In the hyomandibular and posterior lower
lateral lines there are changes in two of these parameters. In all lines there is
some change in at least one of the three features examined.
The sensory role of the lateral line nerve trunks in the larva
While it was found impossible to record nervous activity from single plaques,
recordings from whole nerve trunks were made using silver wire hook electrodes.
All recording was done from the middle lateral nerve trunk as a relatively long
length of nerve was available. Spontaneous afferent nervous activity was
recorded from proximally cut larval nerves as early as stage 54, at least 20 days
before the functional loss of the tail. The activity showed a clear increase when
a vibrating rod was introduced into the water (Fig. 3).
Fig. 1. The distribution of sense organs in a stage 55 larva. (A) Dorsal view,
(B) ventral view, (C) lateral view; a. = aortic lateral line row; a.a. = anterior auditory lateral line group; a.l.lat. = anterior lower lateral line; an. = anal lateral line;
c. = caudal lateral line; hy. = hyomandibular lateral line; in.o. = infra-orbital
lateral line; l.lat. = lower lateral line; man. = mandibular lateral line; max. = maxillary lateral line; med.v. = median ventral lateral line; mid.lat. = middle lateral
Jine;oc. = occipital lateral line; p. = parietal lateral line;p.a. = posterior auditory
group; p.l.lat. = posterior lower lateral line; p.o. = post-orbital lateral line;
pr.o. = pre-orbital lateral line; s.o. = supra-orbital lateral line; t. = tentacular
lateral line group; u.lat. = upper lateral line.
516
P. M. J. SHELTON
Table 1. Changes in the lateral line rows at metamorphosis
Stage 66 adults
Stage 55 larvae
Lateral line row
Supra-orbital complex
(a) Parietal
(b) Supra-orbital
Infra-orbital complex
(a) Post-orbital
(b) Infra-orbital
Position
of row
Dorsal
Dorsal
Number Angle to
of organs reference
axis
in row
3-4
10-15
4-6
Dorsal
5-6
Lateral
and ventral
Hyomandibular complex
3-5
(a) Tentacular
Ventral
3-6
(b) Pre-orbital
Ventral
(c) Mandibular
9-12
Ventral
(d) Hyomandibular
Ventral . 9-15
Anterior auditory
Dorsal
5-8
Dorsal
7-10
Occipital
Dorsal,
7-9
Aortic
lateral
and ventral
Lower lateral complex
(a) Anterior l.lat.
Lateral
12-18
and ventral
(b) Posterior l.lat.
Latefial
8-12
and ventral
Lateral
(c) Median ventral
3-4
Lateral
(d) Caudal or anal
15-20
Middle lateral
Dorsal
(a) Trunk region
25-30
and lateral
(b) Tail region
Dorsal
Many
Upper lateral
(a) Trunk region
Dorsal
18-22
and lateral
(b) Tail region
Dorsal
Many
Dorsal
Posterior auditory
7-9
Position
of row
Number Angle to
of organs reference
in row
axis
150-180
70-90
Dorsal
Dorsal
3-5
12
120-140
0-170
60-70
90-160
Dorsal
Dorsal
3-4
4-5
130-170
10-40
15-45
60-140
100-160
30-120
70-120
20-150
-10-30
Lateral
Ventral
Ventral
Ventral
Absent
Dorsal
Absent
4
4-5
8-9
3-4
10-70
50-140
140-160
140-160
10-120
Ventral
4-5
0- 30
70-170
Ventral
20-22
30-140
80-90
15-20
Ventral
Ventral
3-5
160-180
0-10
60-120
Dorsal
26-34
and lateral
Absent
80-90
20-150
170-180
70-130
-
-
_
8-10
_
4-5
Dorsal
16-24
and lateral
Absent
Lateral
5-7
.150-170
_ _
50-120
-
0-140
-
100-110
Table 1 shows the differences in distribution of lateral line rows in larvae and adults.
Information is gathered from an examination of five larvae and five adults. The position of
each row and the numbers of plaques in them are recorded. The orientation of plaques was
determined by extrapolating lines through their long axes and measuring the angles subtended with the median antero-posterior axis. Reference axes for the measurement of angles
are shown in Fig. 1A and B.
Fig. 2. The location of organs in adult Xenopus (stage 66). (A) Dorsal view,
(B) ventral view, (C) lateral view, (D) arrangement of organs around the orbit.
Terminology is the same as in Fig. 1.
Lateral line system
p.l.ht
mid.lat.
/.'at.
517
518
P. M. J. SHELTON
The orientation of stereocilia with respect to the kinocilium and the orientation
of the ciliary groups with respect to the long axis of the plaque
Sections of the sensory hairs of larval organs at stage 54 reveal a clearly
orientated arrangement of groups of stereo- and kinocilia similar to that found
in the adult. There are two populations of sense cells identified by the relative
position of the kinocilium to the stereocilia in each group. Some cells have their
kinocilium on one side of the stereocilia, the others have their kinocilium on the
opposite side (Fig. 4). Ciliary groups are arranged so that the plane of maximum
sensitivity would be approximately at right angles to the plaque's long axis.
II
II
I
1 I I
I IK
>t.
1 sec
Fig. 3. Electrophysiological recording from a stage 54 tadpole showing afferent
activity in the middle lateral line nerve trunk. The four traces represent consecutive
parts of the same recording. Spontaneous nervous activity is apparent in the early
part of the record. Increased activity occurs when a vibrating rod is introduced into
the environment. The arrow marks the start of stimulation.
The surface structure of larval and adult organs
At metamorphosis the skin thickens, the distance between the basement
membrane and the surface increasing from 30 JLL in the larva to 100 fi in the adult.
The lateral line organs in the larva are about 50 /.i deep and in consequence their
outer surface protrudes above the level of the surrounding skin (Figs. 5, 7). The
adult organs increase in size to 80 JLI deep but sink down into the skin. At the
same time tactile organs develop between the organs in each plaque (Figs. 6, 7),
clearly separating neighbouring organs. The result is that the larval sensory
Lateral line system
519
hairs are situated well above the skin surface while the adult ones are partly
below it. These changes in morphology of the organ plaque may have functional
significance.
0
0
0
OoO 0°
QOouo_oo0o
oo
00
0-
Fig. 4. This diagram is taken from an electron micrograph. It shows the relative
positions and the orientation of cilia from three adjacent cells in an organ from the
supra-orbital lateral line at stage 54. Two classes of cell are apparent. Two cells have
their kinocilium (k) on one side of the stereocilia (s) and the other has the opposite
arrangement. The orientation of the two cell types occurs in the adult and gives
the organ its directional sensitivity; a = anterior; / = long axis of plaque;
p - posterior.
Fig. 5
Fig. 6
Fig. 5. An illustration of the position of ciliary groups (c) in larval Xenopus showing
organs protruding above the animal's surface.
Fig. 6. This drawing shows how the adult organs sink below the surface and the
ciliary groups (c) are separated by tactile organs (033
E M B 24
520
P. M. J. SHELTON
A
Fig. 7. (A) A transverse section of a larval plaque at stage 58; it shows the profile
of the surface and the neuromast cells (n). (B) Longitudinal section of an adult organ
plaque, showing a lateral line organ between two tactile organs (jt). The channel (c)
formed by the tactile organs is said to direct currents on to the sensory hairs of the
neuromast cells (n). The material was embedded in Araldite and stained with
toluidine blue.
Lateral line system
521
v
o
Fig. 8. (A) An electron micrograph showing a pair of myelinated fibres (a) and
a group of non-myelinated fibres (n) in the nerve bundle innervating a single tadpole
plaque. (B) An additional class of fibres is present in the adult. As well as the two
myelinated afferent fibres (a), small myelinated efferent fibres (e) are often found;
n = non-myelinated fibres.
33-2
522
P. M. J. SHELTON
The inner vat ion of larval sense organs
Examination of the nerve bundles innervating single organ plaques in the
larva consistently reveals two morphologically distinct groups of fibre; two
myelinated fibres approximately 2-4 fi in diameter and a number of unmyelinated fibres up to 0-5 fi in diameter (Fig. 8 A). At stage 60 another class of smaller
myelinated fibres is present occasionally. At later stages the additional class of
small myelinated fibre is found more frequently, and at stage 66 (adult) is nearly
always present (see Table 2, Fig. 8B). This small myelinated fibre is known to
be of efferent function in the adult, the pair of larger myelinated fibres is of
known afferent function.
Table 2. Plaque innervation at different stages of development
Stage
Number of
innervations
studied
52
54
57
60
66
4
6
15
9
11
Number with
myelinated
Number with
two afferent efferent fibre(s)
present
fibres present
4
6
15
9
11
0
0
0
2
10
Table 2 shows that myelinated efferent fibres are absent in larvae prior to metamorphosis,
they are first seen at stage 60. Ten innervations at stage 57 were examined using the electron
microscope, the rest were examined using toluidine blue-stained, thin Araldite sections and
an oil immersion light microscope.
DISCUSSION
The larval lateral line system is clearly functional at least by stage 54. Myelination of the nerve pairs running to plaques is complete by stage 52 and afferent
activity has been recorded from tadpole nerve trunks as early as stage 54. The
orientation of cilia at the apex of the sensory cells suggests that organs have the
same type of directional sensitivity in the larva as has been shown for the adult
(Gorner, 1963). The plane of maximum sensitivity may be less clearly defined,
however, because larval ciliary groups do not lie in channels but are raised above
the skin surface. An orientated arrangement of cilia has been noted in Rana
pipiens tadpoles (Jande, 1966), an animal which loses its lateral line system at
metamorphosis. It would seem, therefore, that the orientated cilia in larvae are
of functional significance to the tadpole rather than just a stage in the development of the adult.
The redistribution of organ plaques and their changes of orientation must
result in changed planes of maximum sensitivity at metamorphosis. If the tadpole lateral line system is used as in the adult to locate objects in the environment, the peripheral modifications necessitate changes in the central nervous
Lateral line system
523
system. It is possible that the larval system is not used as a 'distant touch'
receptor at all and is merely a generalized vibration receptor used in conjunction
with the Mauthner cells in rapid escape movements. This seems unlikely when
an obviously orientated arrangement of hair cells occurs in both R. pipiens and
Xenopus tadpoles.
The small myelinated inhibitory class of fibres may be present in larvae in
a non-myelinated form or it may grow out as a completely new element of the
nervous system at metamorphosis. Its maturation corresponds to a stage in
development when there is a distinct change in locomotory behaviour. The
tadpole swims in a fish-like manner and the adult moves using a characteristic
hind-leg kick. It has already been noted that the level of efferent activity sharply
increases when adults attempt to move (Gorner, 1967). It may be that the adult
animal develops the myelinated inhibitory system to 'switch off' peripherally
the lateral line organs during swimming movements. This peripheral control by
a fast conducting myelinated system may not be necessary in Xenopus tadpoles
where locomotion depends upon a sinusoidal tail movement.
RESUME
Le systeme de la ligne laterale chez Xenopus laevis (Daudiri) an moment
de la metamorphose
Des changements prononces surviennent au cours de la metamorphose de Xenopus, dans
Panatomie du systeme de la ligne laterale. La repartition des canaux differe chez la larve et
chez Padulte, en outre l'orientation et le nombre des organes sont modifies a la metamorphose.
Des plaques larvaires sont fonctionnelles, ainsi que l'a montre 1'enregistrement de leur
activite nerveuse.
Deux especes cellulaires, avec cils polarises, sont presentes chez le tetard bien avant que
l'orientation des plaques individuelles d'organes se soit modifiee a la metamorphose.
La topographie de la surface cutanee autour des plaques individuelles change a la metamorphose. Cette modification peut reduire la sensibilite directionnelle des organes.
On trouve des axones inhibiteurs myelinises dans le nerf lateral seulement lorsque le tetard
devient mature. Ce changement a lieu au moment ou le mode de locomotion adulte se
developpe.
This work was supported by a scholarship from the Medical Research Council. Thanks are
due to Dr David Sandeman for help during the work and for his valuable suggestions.
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{Manuscript received 20 January 1970)