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First published online March 12, 2009
Journal of Experimental Biology 212, 914-921 (2009)
Published by The Company of Biologists 2009
doi: 10.1242/jeb.027250
Responses of hatchling Xenopus tadpoles to water currents: first function of lateral line receptors without cupulae
School of Biological Sciences, University of Bristol, Bristol, UK
* Author for correspondence (e-mail: a.roberts{at}bristol.ac.uk)
Accepted 13 January 2009
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Summary |
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 TOP Summary INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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Key words: lateral line, neuromast, Xenopus laevis
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INTRODUCTION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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;
Bleckmann, 1994;
Coombs et al., 1989;
Dijkgraaf, 1963;
Montgomery et al., 1995) The
sensory receptors responsible lie in the skin and the simplest type, found in
amphibians, are free neuromasts. These are easily visible, for example around
the eye in adult Xenopus, and form rows called stitches. Each
neuromast contains a group of receptor hair cells with kino- and stereo-cilia
embedded in a jelly-like cupula extending out from the skin surface
(Gorner, 1963). It is well
established that water currents can deflect the cupula of free neuromasts,
move the kinocilia in its base and excite the hair cells, which then release
transmitter to excite the cranial nerves which innervate them. Within each
neuromast, the hair cells are arranged in different orientations in order to
be able to detect cupula deflections in different directions.
The developmental origin of the lateral line neuromasts from neurogenic
placodes lying underneath the skin and their innervation by cranial nerves has
been studied in some detail in Xenopus
(Schlosser and Northcutt,
2000; Winklbauer,
1989). There have also been studies using scanning electron
microscopy and other methods to define the development of lateral line
neuromasts and their receptive hair cells in the axolotl
(Northcutt et al., 1994), the
flounder (Otsuka, 2003) and
the eel (Okamura et al.,
2002). However, there have been few studies on the development of
lateral-line neuromast function (Blaxter,
1987). In later larval stages both Amblystoma and
Xenopus use their lateral line receptors to detect water currents and
have been shown to turn towards a water current from a small pipette
(Scharrer, 1932;
Shelton, 1971) and to orient
towards the source of water current in a flow chamber
(Simmons et al., 2004). When
does this capability first develop and do the newly formed neuromasts work in
the same way when they first function?
After only 2 days of development and even before they hatch from the egg
Xenopus embryos are capable of swimming when touched anywhere on the
body (van Mier et al., 1989).
However, responses to water currents do not appear to have been examined at
these early stages of life. We have therefore investigated developing
Xenopus embryos and larvae to see when they first respond to water
currents, whether these responses depend on lateral line neuromasts, and how
these newly formed neuromasts operate. We suggest that, unlike the mature
neuromasts which have a jelly-like cupula to amplify water movements, the
early responses of lateral-line hair cells may depend on the direct
stimulation of hair cell kinocilia by water movements.
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MATERIALS AND METHODS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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).
Embryos were removed from their egg membranes before testing. Responses to
water currents were studied initially in a 300 mmx300 mm tank containing
dechlorinated tap water 20 mm deep at 17 to 21°C. A circular target was
placed under the bottom of the tank and individual tadpoles placed over the
centre of the target lying on their left side with their head facing in the
`forwards' direction (Fig. 1A).
The target was divided into four equal 90° quadrants and the stimulating
pipette was placed in the centre of one quadrant. This quadrant was called
`towards' and the other three were labelled as shown in
Fig. 1A. When a tadpole swam,
it was simple to see which quadrant they entered. This was used to judge their
direction of swimming. A brief jet of water was directed at the ventral side
of the tadpole from a rigidly mounted 50µm glass nozzle 10 mm from the
tadpole and connected to a water bottle (water head 500 mm) by opening a
solenoid valve for 10 ms. Suction stimuli were applied via a rigidly
mounted 5 mm diameter glass tube 10 mm from the tadpole connected to a pipette
bulb that could be rapidly released to draw in water.
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Responses of animals at different developmental stages were also tested using similar methods, but were performed in a 90 mm diameter plastic Petri dish with water 7 mm deep. Smaller dishes were used in later experiments as it was easier to reach in to manoeuvre the animals prior to testing, and when they swam, they quickly hit the sides of the dish and stopped swimming in the segment they had entered. This made recording the direction of swimming very simple. In these tests suction was applied using a plastic Pasteur pipette. For lesion studies, tadpoles were anaesthetised in MS-222 in saline (see below) for 1 min, then transferred to a dish of saline and held down with crossed pins in a V-shaped groove in the Sylgard base of the dish. Under a dissecting microscope a tungsten needle was ground to a chisel shape about 100 µm wide. This was used to abraid the skin in a restricted region. The tadpole was then transferred to 50% saline for 1 h before testing. Control tadpoles received the same treatment but were not abraded. Tests on abraded and neomycin-treated animals were conducted in a double-blind fashion so that the person doing the tests did not know which were treated or control animals.
Electrophysiology
Tadpoles at stage 37–38, were used at 20±2°C in saline:
115 mmol l–1 NaCl, 3 mmol l–1 KCl, 2 mmol
l–1 CaCl2, 2.4 mmol l–1
NaHCO3, 1 mmol l–1 MgCl2, 10 mmol
l–1 Hepes; adjusted to pH 7.4 with 5 mol l–1
NaOH. Dissection and lesioning was carried out using finely etched tungsten
needles and forceps. The methods for making recordings from the motor nerves
have been described previously (e.g.
Lambert et al., 2004).
Briefly, tadpoles were anaesthetised with 0.1% MS-222 in saline for
20–30 s, then pinned onto a rotatable Sylgard block in a bath of saline.
Tadpoles were slit along the dorsal fin and transferred to
-bungarotoxin (10 µmol l–1 in saline, Sigma, St
Louis, MO, USA) for up to 20 min. After immobilisation, tadpoles were returned
to the bath and re-pinned with their right side up. Some skin overlying the
dorsal half of the body on the right side was removed to expose the myotomes.
To record ventral root activity and see swimming responses, a suction
electrode (60 µm tip diameter) was placed over an intermyotomal cleft.
Display techniques were conventional, recording were viewed on an oscilloscope
and permanent records made on a chart recorder.
Observations of living hair cells
Tadpoles were transferred to 50% physiological saline and decapitated just
caudal to the gills. The head was then transferred to a 55 mm diameter plastic
Petri dish containing 50% saline with a glass coverslip glued over a 10 mm
diameter hole in its base. A 25 µm diameter tungsten pin was inserted
rostrally so that it passed longitudinally through the isolated head and into
the side of a small block of Sylgard fixed to the coverslip. The head was then
rotated about its longitudinal axis so that dorsal was up and the head was
secured with another tungsten pin. The dish was moved to the fixed stage of an
Olympus BX50WI microscope. Observations were made with a x40 water
immersion lens using bright field or differential interference contrast (DIC)
optics. Photomicrographs were taken using a Nikon Coolpix 990 camera. To
stimulate the neuromasts, saline was ejected from the 3–4 µm diameter
tip of a glass pipette mounted in a holder and connected to a flexible tube
that allowed pressure to be applied using a 1 ml syringe. To manipulate the
kinocilia, a fine glass probe or a fine tungsten needle with its end bent to
point vertically was used. All probes were positioned and moved using a
Huxley-type micromanipulator.
Scanning electron microscopy
Embryos and larvae were washed in 0.1 mol l–1 phosphate
buffer; fixed for 2 h in 2% gluteraldehyde in phosphate buffer; washed in
buffer and then dehydrated through a graded ethanol series before
critical-point drying. The dried specimens were glued to aluminium stubs,
sputter coated with gold, and examined using scanning electron microscopy
(SEM) in a Philips 501B microscope.
All the procedures used have had University of Bristol ethical committee approval. Statistical tests were performed using Excel or Minitab and all means are given with their standard errors.
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RESULTS |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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). We first evaluated spontaneous swimming in unstimulated
tadpoles. Individuals were placed on the target, we waited until they swam,
recorded the direction, and then tested another tadpole. Spontaneous swimming
was mainly forwards (N=40: towards 8, away 2, forward 26, back 4;
2=15.24; d.f.=3, P=0.002). In response to a 10 ms
water jet from a 50 µm pipette they mainly swam towards the jet
(N=50, towards 34, away 2, forward 6, back 0, no response 8;
2=29.39, d.f.=3, P<0.001, whereas they swam away
from 50 ms of suction from a 5 mm pipette (N=50, towards 2, away 23,
forward 3, back 0, no response 22; 2=22.82; d.f.=3,
P<0.001). These tests indicate that hatchling tadpoles respond to
water currents by swimming against the direction of flow.
In Xenopus, effective swimming (powerful enough to move the animal
through the water) first appears at about embryonic stage 31/32
(van Mier et al., 1989). No
responses to water currents were seen prior to stage 31/32. We therefore
examined the development of swimming responses to water currents from nearly a
day before hatching, at stage 31, to stage 41, which is about 1 day after
hatching. For each stage of development we tested 10 animals once and recorded
the number swimming towards a water jet or away from suction; swimming in
other directions and not responding (as above). Swimming responses to both
stimuli were seen from the first stage tested but the proportion of tested
animals responding to stimulation with swimming increased with age up to the
time of hatching (stage 37/38; Fig.
2; regression analysis of proportion swimming against age: for
water jet R2=0.75, P=0.026 and for suction:
R2=0.94, P=0.001). From stages 31 to 41 swimming
was predominantly towards a water jet (81%) or away from suction (74%).
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). All
observations were made on stage 37/38 tadpoles as these are most convenient
for motor nerve recording. In preliminary observations we used tadpoles
immobilised in -bungarotoxin and made recordings from the motor nerves
to the trunk muscles (Fig. 3A).
To establish that recording was successful we touched the skin with a fine
hair and checked that fictive swimming activity was evoked with motor bursts
at intervals within the normal swimming range (40–100 ms;
Fig. 3B)
(Kahn et al., 1982). We then
tested water jet stimulation with a narrow, 10 ms jet of water from a 20 µm
pipette 20 mm from the ventral side of the tadpole. This stimulation was
tested over the whole body surface but was only found to be effective in
evoking fictive swimming if directed at the head
(Fig. 3B).
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Lesions were then used to find what cranial pathways were necessary for such fictive swimming responses. After all lesions, 5 min were allowed for recovery, the recording electrodes were put in place, and the trunk skin was stroked with a fine hair to check that this stimulus evoked fictive swimming. We found that water jet stimulation still evoked swimming after removal of the midbrain and forebrain (N=8/8) and removal of the otic capsules on both sides (N=8/8). Since lateral line neuromasts are innervated by cranial nerves, the next operation was to cut vertically along each side of the hindbrain to sever all hindbrain sensory cranial nerves (V to XI). After this lesion, swimming responses to water jets were dramatically reduced (N=1/9).
To follow up observations on immobilised tadpoles we tested the effects of
cranial nerve lesions on the behaviour of tadpoles. In 30 tadpoles at stage
37/38 short vertical cuts (
0.1 mm long) were made rostral and caudal to
the otic capsule on both sides. When each tadpole was tested once to water jet
stimulation, lesioned tadpoles swam (11/30) significantly less than controls
(26/30; contingency table analysis, d.f.=1; P<0.005).
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), we
used a narrow jet (40µm) to give a very local stimulus to tadpoles lying on
their side on the bottom of a dish (N=70 individuals, five repeats
each = 350 trials). Current just caudal to the eye resulted in significantly
more swims (153) than current rostral to the eye (83; Mann-Whitney
W=5740.0 N=70, P=0.008). To locate the receptors we
then abraded the skin just rostral or caudal to the eyes with a small,
chisel-shaped needle (Fig. 3A,
see grey shaded areas) and then tested responses to the water jet from a 1 mm
pipette 15 mm from the tadpole. For each treatment five tadpoles were each
tested 40 times and the number of swimming responses out of 40 trials was
determined. In the control group, without abrasions, the number of swimming
responses (24.2±3.35; mean ± s.e.m.) was similar to tadpoles
with abrasions rostral to both eyes (21.8±4.97; P=0.65) but
the number of swimming responses was significantly reduced after abrasions
caudal to both eyes (7.8±4.15; Fig.
3C) when compared with both the operated and unoperated controls
(P<0.001 in each case; ANOVA with Tukey's pairwise comparisons,
F=6.48, d.f.=6).
The antibiotic neomycin is known to produce loss of function in sensory
hair cells in the mammallian auditory system
(Gale et al., 2001) and in the
amphibian (mudpuppy) lateral line system
(Shiozawa and Yanagisawa,
1979). We therefore looked at the effects of a 30 min wash in 10
µmol l–1 neomycin sulphate (pH 7.4). After 60 min in
normal tap water, we tested the responses of ten treated and ten control
tadpoles to the water jet. As in the previous abrasion tests each tadpole was
tested 40 times. We found a significant reduction in the number of swimming
responses to the water jet in neomycin-sulphate-treated tadpoles
(Fig. 3D; 9.6±6.2
treated and 19.6±7.3 control tadpoles; t-test:
t=3.31, P=0.004, d.f.=18).
Features and development of lateral-line neuromasts
The evidence from electrical recording and behavioural tests suggested that
the receptors involved in detection of water currents could be lateral-line
neuromasts lying just caudal to the eye and innervated by cranial nerves. To
investigate when and where lateral line neuromasts first appear and how they
then develop from stage 29/30 to 42 we used scanning electron microscopy to
examine the outer surface of the skin in gluteraldehyde-fixed and
critical-point dried specimens (at least four at each stage). The head skin
has three types of cells: normal skin cells with a fairly smooth surface
(longest dimension often >20 µm), ciliated cells which have very large
numbers of short cilia and are distributed over the whole body surface
(longest dimension 10 µm), and mucus cells with a small exposed
surface (longest dimension <10µm) usually with clear holes
(Fig. 4). Neuromasts were found
at stage 37/38 just caudal to the eye (Fig.
4B–D) and were easily distinguished from ciliated skin cells
as their exposed surface was smaller (longest dimension <10 µm) and they
had fewer, longer cilia (two to eight).
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Neuromasts were not found at stage 29/30 but by stage 32 a short row of three to six had formed just caudal to the eye (Fig. 6A). By stage 37/38 the number of neuromasts in this first row (infra orbital) had increased and a second row had appeared just dorsal to the gills (Fig. 6B). At stage 42 the number of neuromasts was similar (Fig. 6C).
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Observations on living hair cells
The SEM images showed that early neuromasts were present from stage 32 when
embryos first respond to water currents. However, the images suggested that
the kinocilia projecting from lateral line hair cells were not embedded in a
jelly cupula. Since the cupulae may have been dissolved or destroyed during
preparation for SEM, we made direct observations of living head skin in 11
tadpoles at stage 37/38 in 50% physiological saline using a x40 water
immersion lens. When viewed from the dorsal side using bright-field or DIC
optics, two or three groups of straight, static, kinocilia about 20 µm long
were seen at different dorsoventral positions projecting from the skin just
caudal to the eyes (Figs 7 and
8). There were from three to 11
kinocilia in each group. The groups of kinocilia lay in the positions where
neuromasts had been found in the SEM investigation (see above). In two cases a
jet of 50% saline was directed at the group of kinocilia from a glass pipette
with 5 µm diameter tip opening. When the current flowed, the kinocilia
remained straight along most of their length, but pivoted around their bases
to become deflected by the current (Fig.
7).
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Surprisingly, no cupulae could be seen in the 30 neuromasts examined, using
either bright-field or DIC optics. To test if cupulae were present but
invisible, fine glass or tungsten probes were manipulated into or around the
kinocilia. The preparation and rationale of these two tests is illustrated in
Fig. 8A,B which shows a diagram
of a typical excised fish neuromast where the kinocilia are embedded in a
jelly-like cupula (Van Trump and McHenry,
2008). In the first test, probe 1 is moved in towards the
kinocilia and it should contact the cupula first. By moving the cupula, the
kinocilia inside should all move with the cupula and to the same extent. In
the second test, probe 2 is moved over the ends of the kinocilia. If a cupula
is present extending beyond the ends of the kinocilia this should move and
take the kinocilia with it.
The first test was performed on 11 tadpole neuromasts. When a probe was moved in towards the sides of the kinocilia, there was no indication that they moved before being contacted by the probe (Fig. 8C,D). Furthermore, when the first kinocilium was moved by the probe, the others in the same neuromast did not move (Fig. 8E,F). During these tests it was noticed that, after contacting the kinocilia, the probe could often move them as it was moved away and without apparent contact. The simplest explanation of this is that the kinocilia are covered in mucus which becomes attached to the probe and can then pull on the kinocilia.
In the second test on nine neuromasts, a probe was moved backwards and forwards past groups of kinocilia less than 20 µm from their tips. No movements of the kinocilia were seen (Fig. 8G–L). This would not be expected if there was a jelly-like cupula extending beyond the kinocilia.
Similar observations were made on four neuromasts from two older tadpoles at stages 41 and 44. As expected from the SEM observations, older neuromasts had more kinocilia but probe manipulations showed that kinocilia could still be moved independently and moving the probe over the ends of the kinocilia did not displace them. These observations suggest that cupulae are not present at these later stages.
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DISCUSSION |
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TOPSummaryINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONReferences |
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), embryos are able to respond to water currents even though
there may be as few as three or four neuromasts on each side of the head. Over
the next 10 h or so of further development, more neuromasts appear and a
higher proportion of tested animals respond to water currents. When they
detect a water current the embryos and young tadpoles start to swim and this
swimming is directed into the water current. This type of directed turning
towards a water current had been shown in much older Ambystoma and
Xenopus larvae (Scharrer,
1932; Shelton,
1971). In Ambystoma this response was interpreted as a
feeding response allowing the larvae to detect small prey that produced local
water movements. Since the embryo and young larval Xenopus tadpoles
do not yet have a mouth, and do not feed, a role in prey detection seems
unlikely at these early stages.
What are the other possible roles of the young tadpole's directional
swimming response to water currents? Rheotaxis is a behavioural orientation to
water currents that is common in fish. Swimming into water currents may help
them to maintain their position in a stream and avoid being swept away by
currents. In fish (Montgomery et al.,
1997) and larval Xenopus
(Simmons et al., 2004) this
behaviour has been shown to depend in part on the superficial, free neuromasts
of the lateral-line system. Unfortunately, in the Xenopus study
although 18 tadpoles were tested at stages 37 to 45, there is no information
on how many were tested at each different stage. Furthermore, the animals were
not viewed from above so orientation angles of swimming into the water current
in the horizontal plane were not accurately measured. This makes it difficult
to assess the behaviour of the younger tadpoles in the sample. Rheotaxis is
therefore well established in Xenopus tadpoles but may be more
important in later free swimming stages [after stage 43 but see Shelton
(Shelton, 1971)] than at the
earlier stages we have examined (up to stage 40) when tadpoles are very
inactive. They spend most of their time (99%) hanging from a strand of mucus
secreted by the cement gland on their head
(Jamieson and Roberts, 2000).
Another role was suggested by the tadpoles responses to suction
(Fig. 1D). In many fish
(Wainwright et al., 2007) and
aquatic amphibians (Lauder and Shaffer,
1986) suction feeding is the main method to catch prey. An ability
to initiate swimming and then swim against a water current could help young
tadpoles avoid being sucked up by fish or older amphibian larvae or
adults.
Development of neuromasts and operation without cupulae
Lateral line kinocilia and neuromasts were seen well before hatching in SEM
images of stage 32 embryos. About 5 h earlier at stage 29/30, close
examination of the skin caudal to the eye showed some small depressions where
neuromasts might be about to erupt but no sensory kinocilia. In a few hours a
functional lateral-line sensory system develops that allows the embryos to
respond to water currents by swimming. This implies that within this period
the hair cells become capable of detecting kinocilium deflection and releasing
transmitter, and that the sensory neurons that innervate them are excited and
carry an impulse discharge into the hindbrain to synaptically activate the
circuits that drive swimming (Li et al.,
2006). These conclusions do not rule out a role for saccular hair
cells in detecting movements of the whole body resulting from strong water
currents.
As they develop, the neuromasts form lines or rows. The first to form by
stage 32 is the infra-orbital line just caudal to the eye and this then
extends ventrally. The second aortic line forms by stage 37/38, slightly more
caudally and dorsal to the gills. The pattern of extension of neuromast lines
is very much as expected from the development of the underlying neurogenic
placodes in Xenopus (Schlosser
and Northcutt, 2000) and is similar to that found in the axolotl
(Northcutt et al., 1994). Many
features of the earliest neuromasts of axolotl are similar in SEM images to
our findings in Xenopus. However, in both the axolotl and teleosts
evidence of cupulae have been detected (including by SEM) at very early stages
of neuromast development (Blaxter,
1984; Otsuka,
2003). By contrast, we found no evidence for the presence of a
gelatinous cupula in Xenopus neuromasts from embryonic stage 32 to
the tadpole, 1.5 days after hatching, at stage 44.
Do the early neuromasts in newly hatched Xenopus tadpoles work
without a gelatinous cupula? Since the preparative methods for SEM examination
might have destroyed cupulae, we made observations in living tadpoles.
Neuromast kinocilia could be seen using a x40 water immersion lens but
there were no signs of cupulae. Rather than trying to see cupulae, for example
by coating their surface with reflective polystyrene microspheres
(Van Trump and McHenry, 2008),
we chose to use mechanical tests to seek evidence for their existence by
moving probes into and around groups of neuromast kinocilia. In the
developmental stages that we examined, there was no indication of any
invisible structure surrounding kinocilia or linking them together. We looked
at the oldest tadpoles permitted by British Home Office regulations (stage 44)
and still found no evidence for cupulae. However, direct observations also
showed that living kinocilia could be deflected by local water currents. Taken
together, our evidence indicates that young Xenopus tadpoles detect
water currents which directly move the kinocilia protruding some 20 µm from
the surface of neuromasts forming two rows just caudal to the eyes. The young
Xenopus tadpole, therefore, provides an opportunity to investigate
the properties of naked kinocilia.
What is the significance of neuromasts without cupulae? If early neuromasts
operate without cupulae at early stages of development then one can guess that
they will have reduced sensitivity as there is good evidence that sensitivity
relates to cupula length (McHenry et al.,
2008; Van Trump and McHenry,
2008). Cells in the tadpole skin secrete mucus and this is driven
caudally over the body surface by the numerous ciliated skin cells. Our
observations that probes which have touched kinocilia can move them when
pulled away, suggests that the kinocilia are coated with mucus. If the
kinocilia can move independently, then they could give independent directional
information. We looked at the orientation of kinocilia and stereocilia but in
many cases clear measurement of orientation angle was not possible because a
tangle of kinocilia lay over the surface of the neuromast, which was not
viewed orthogonally. Our impression was that there was a range of orientations
rather than the strictly opposed orientation found in mature neuromasts
(Bleckmann, 1994;
Coombs et al., 1989;
Montgomery et al., 1995).
However, we rarely saw a neuromast with only a single kinocilium which may
support the idea that hairs cells develop in pairs
(Rouse and Pickles, 1991).
Finally, the lateral-line neuromasts in the young Xenopus tadpole
appear to be the simplest so far described. Even if Haeckel's idea that
ontogeny recapitulates phylogeny (Haeckel,
1992) is no longer accepted, the young Xenopus tadpole
appears to give us an insight into the simplest possible vertebrate sensory
system to detect water movements over the body surface.
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Acknowledgments |
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