J. Embryol. exp. Morph. Vol. 65, pp. 149-163, 1981
Printed in Great Britain © Company of Biologists Limited 1981
149
Selective bilateral motor innervation in Xenopus
tadpoles with one hind limb
By ALAN H. LAMB 1
From the Department of Pathology, University of Western Australia
SUMMARY
Bilateral innervation of a single hindlimb bud was induced by amputating the other limb
bud and disrupting the barriers between the two sides. Though the routes of the crossed
nerves were necessarily abnormal, the motor projections that developed subsequently were
normal as determined by horseradish peroxidase tracing. The limb therefore appears to be
innervated selectively, each region being invaded and/or synapsed with only by motoneurones
at particular locations. The numbers of motoneurones surviving after metamorphosis were
almost normal on both sides provided the operation was done before motor invasion of the
limb bud begins. From this it is argued that the axons were probably guided actively to their
correct destinations. Without such guidance, axons would probably not have been able to find
their correct termination sites and motoneurone survival would therefore have been depressed.
The normal motoneurone numbers also imply that the single limb was supporting twice its
usual quota of motoneurones. The hypothesis that motoneurones compete in the limb for
survival is therefore not supported.
1
INTRODUCTION
There seem to be two schools of thought on how the motor projections to the
limb develop in the embryo. The passive innervation school maintains that
axons follow paths of least resistance like water down a gully. Complexity of
surrounding anatomy divides the flow into branches and it is fortuitous which
motoneurones invade given muscles. By implication, synaptogenesis is not
selective, any motoneurone may connect with any muscle. Projections vary
little because the anatomy creating the pathways is common to all (Horder,
1978). The opposing school claims that there must be some selectivity to ensure
that muscles always receive innervation from motoneurones at particular
locations in the spinal cord. Selectivity may take the form of active axonal
guidance bringing the motoneurones into contact with their correct muscles
and/or selective synaptogenesis such that their motoneurones cannot synapse
with incorrect muscles but only with their own (Landmesser, 1980).
Both schools are supported by recent experiments in the chick embryo. In
support of the passive, rotated limb buds and grafted supernumerary limb buds
1
Author's address: Department of Pathology, University of Western Australia, Nedlands
6009 Perth, Western Australia.
150
A. H. LAMB
receive abnormal projections apparently because motor axons continue to
follow their usual courses, finding foreign sites where their correct sites should
be (Hollyday, Hamburger & Farris, 1977; Morris, 1978; Stirling & Summerbell,
1979, 1980). In support of selective innervation, spinal cords rotated in the
rostrocaudal axis send their projections to correct sites, undeterred by having
started from the wrong end (Lance-Jones & Landmesser, 19806).
In this paper I describe an experiment further supporting selective innervation.
Tadpoles were reared with one hindlimb bud removed. The spinal nerves were
induced to innervate the opposite limb bud. It was found that despite the
abnormal routes taken by the spinal nerves the motor projections were normal.
The experiment also contains a test of a hypothesis about the control of
motoneurone numbers. Up to 75 % of developing motoneurones die in normal
embryos (Prestige, 1974; Hamburger, 1975) and it is hypothesized that they are
the losers in a competition for an unknown but essential limb factor. Bilateral
innervation effectively doubles the number of motoneurones competing in the
one limb which ought to lower the proportion of survivors. Contrary to predictions, it was found in this study that the number surviving to maturity was
close to normal on both sides so that the limb was supporting twice its normal
complement of motoneurones. This result argues strongly against peripheral
competition. Preliminary results have been reported previously (Lamb, 1980).
METHODS
Operations were carried out on Xenopus laevis tadpoles in a solution containing 1 gram of sea salt, 4 grams of NaCl, and 9 grams of D-glucose per litre
plus 1:5000 MS222 (Sandoz). The left hindlimb bud was removed from 10- to
15-day-old tadpoles at Stages 49 to 51. (Nieuwkoop & Faber, 1967), spanning
the stage at which motor axon invasion of the limb bud begins (Stage 50; Lamb,
1976). The other limb bud was left undisturbed and not moved into the midline
as described previously (Lamb, 1980). Midline barriers dorsal to the anus which
normally confine the spinal nerves to their own sides were disrupted with a
needle. Tadpoles recovered in saline (5 gm/1). During the next few stages of
development some tadpoles regenerated new limb buds which were removed
immediately. None were longer than 0-3 mm on removal. One to 6 weeks after
completion of metamorphosis frogs were given injections of horseradish peroxidase solution (HRP. Sigma Type VI, 30 % in phosphate buffer, pH 7-0) into
one of five different muscles to label motoneurone pools. Since the object was to
determine the extent of a pool and not to label all the neurones within it, small
injections were given in about half the animals (approx. 0-01 /d). Though few
motoneurones were labelled, small injections had the advantage that any
ectopic labelled motoneurones could be more confidently ascribed to abnormal
projections rather than spread of HRP to other muscles. Other animals
were given larger injections (up to 0-5 fi\) to allow comparison with previous
Selective bilateral motor innervation
151
V
Fig. 1. Monopodal frog compared to a normal frog. The remaining limb seems
normal in all respects. Movements of the limb were visually observed, unassisted,
and were indistinguishable from normal. Though subtle abnormalities may not be
detected without electromyography, it is most improbable that such abnormalities
could affect motoneurone survival should they be present. Increased survival is
associated only with gross motor impairment bordering on paralysis (Pittman &
Oppenheim, 1979; Olek, 1980).
projection studies in juvenile frogs (Lamb, 1976). The frogs were killed 2 days later
for motoneurone counts and mapping of HRP-labelled motoneurones from
serial transverse sections. Diaminobenzidine was used for the HRP histochemistry. Full descriptions of the methods are given elsewhere (Lamb, 1976).
Motoneurones were identified using the criteria of Prestige (1967) and those
with nucleoli counted in every third section without correction for nucleolar
splitting. Labelled motoneurones were mapped in the rostrocaudal and mediolateral axes.
RESULTS
Seventeen frogs were studied, three of which were used for cell counts in an
earlier study (Lamb, 1980). All developed only one hind limb; there was no
trace of the other. The hind limbs were normal in every respect including size
and function (Fig. 1). Movements in particular were normal as judged by eye,
and the animals, once accustomed to having only one hind leg, were able to
swim and feed with alacrity.
152
A. H. LAMB
Table 1. Motoneurone counts in monopodal frogs: left limb bud removed
Stage at
operation
49
49
49
49
49
49*
49
49
50
50
50*
50*
50
51
51
51
51
Left
Right
Total
1320
1521
1401
1569
1371
1389
1299
1200
1431
1436
1155
1119
591
870
732
453
1326
1545
1449
1620
1449
1476
1455
1635
1575
1662
1353
1617
1668
1629
1716
1356
998
2646
3066
2850
3189
2820
2865
2754
2835
3006
3098
2508
2736
2259
2499
2448
1809
2439
441
Lef t/right
(%)
100
98
97
97
95
94
89
73
91
89
85
69
35
53
43
33
22
* From Lamb (1980).
Motoneurone numbers were almost normal on the operated sides in the
majority of cases (Table 1). In tadpoles operated at later maturity the numbers
tended to be lower.
On the amputated side the spinal nerves which normally innervate the limb
(principally segmental nerves 9 and 10 with a variable contribution from 8)
fused into a single well-defined nerve trunk which crossed dorsal to the anus
and joined the opposite sciatic nerve at the base of the limb (Fig. 2a). At the
junction, the two nerve trunks intermingled with fibres from each interweaving
to all parts of the common sciatic nerve. However, there was a tendency for
fibres from the amputated side to lie more peripherally in the common nerve
(Fig. 2 b). Stripping apart of the two trunks inevitably caused tearing of many
of the axons but nevertheless fascicles from each trunk could be followed into
both the anterior and posterior tibial nerves. No detectable abnormalities were
found of the routes of the main branches of the sciatic nerve or tibial nerves. A
formal study of the paths of axons from each side during development and at
maturity is in progress and will be reported later.
Distributions of HRP-labelled motoneurones were all normal (Figs. 3-8).
Labelled motoneurone pools on both sides were correctly situated in the
rostrocaudal and mediolateral axes. The correctness of the mediolateral representation is especially significant since the limb and the contralateral ventral
horn must have been 180° out of register from their normal axial alignment. To
Selective bilateral motor innervation
153
c
\
u >
•••"
'
r-'J''"'
(a)
Fig. 2. Junction of right and left sciatic nerves at the base of the limb (L). Each
sciatic nerve is itself formed by the fusion of spinal nerves 8, 9 and 10. In this animal
spinal nerve 11 also made a small contribution, (a) Dorsal view after removal of the
verebral column which normally overlies the entire plexus. The stumps of spinal
nerves visible in this photograph are numbered according to Gaupp (1896). The
crural nerve (CN) on the unoperated side leaves the plexus proximal to the junctions
of spinal nerves 9 and 10 and after the junction of 8 and 9 as in normal animals.
No crural nerve was ever seen on the operated side x 25. (b) The common sciatic
nerve dissected free to the division into anterior and posterior tibial nerves. The two
contributing sciatic nerves have been pulled apart to show more clearly the manner
of their fasciculation. The uncrossed sciatic nerve (U) was partly ensheathed by
the crossed nerve (C) though some fibres from each were intermingled. Both tibial
branches received fascicles from each sciatic nerve, x 50.
conclude from these results that motor projections were normal it must be
assumed that any motoneurones projecting inappropriately would have been
labelled. This seems safe since several experiments have shown that inappropriately projecting motoneurones can be labelled (e.g. Lamb, 1976; Hollyday
et al. 1977; Morris, 1978; Stirling & Summerbell, 1980), while no evidence has
been found to show that such motoneurones cannot be labelled.
154
Fig, 3. Horseradish-peroxidase-labelled motoneurones (arrows) in a transverse
section of spinal cord. Low power x 130 high power x 340, phase contrast.
155
Selective bilateral motor innervation
Knee flexor: semimembranosus
Stage of operation
49
H.R.P. volume (AII)
001
50
001
•
•
•
•
•
•
•
•
•
•
•
•
•
•
#
•
•
•
•
•
•
•
•
•
•
•
•
•
Motoneurone number 1299
Labelled motoneurones 18
1455
25
1436
15
1662
18
Scale
• 1
•
5-8
• 2
•
1 9-16
#3-4
i
| 17-32
Fig. 4. Distributions of motoneurones projecting to semimembranosus in monopodal frogs. Each pair of rectangles represents right and left ventral horns of one
animal. Rostral is to the top and medial to the right of each rectangle. The left limb
hud was removed at the stage of development shown above each pair. Motoneurones
were labelled by retrograde axonal transport of HRP injected into semimembranosus
2 days before the frogs were killed. The volume injected is given above each pair.
Scale gives the numbers of labelled motoneurones. Total labelled motoneurones in
each ventral horn is given below as is the total motoneurone count (labelled plus
unlabelled).
156
A. H. LAMB
DISCUSSION
Selective innervation
Motor axons from the left ventral horn were induced to innervate the right
limb bud, the left having been amputated earlier. The resulting crossed innervation is functional; stimulation of right or left spinal nerves causes equivalent
movements of the limb (Denton, C , Lamb, A. and Wilson, P., unpublished
results). To reach the limb bud the axons needed to take very abnormal routes,
crossing the midline dorsal to the anus before meeting up with ipsilateral axons
at the base of the limb bud. Despite this, and the 180° misalignment that must
exist between the transverse axes of the ventral horn and the contralateral limb,
all motor projections tested were normal. The results do not support the hypothesis that motoneurones grow into and synapse with the limb in a non-selective
manner or that the characteristic adult motor projections are fortuitous.
Selectivity must have operated at some point to give the normal projections.
Either the axons must have been actively guided, or synaptogenesis must have
been selective, or both. Guidance of axons is found in normal tadpoles (Lamb,
1976), but whether it is active or passive is not clear. Selective synaptogenesis is
strongly supported by experiments in tadpoles with partially ablated limb buds.
Proximal or distal segments of the limb bud were removed before being innervated. Using HRP the corresponding motoneurones were subsequently found
to terminate in the remaining inappropriate part of the limb. Later still all these
cells died leaving alive only motoneurones appropriate for the remaining limb
segment (Lamb, 1981a).
Though selective synaptogenesis on its own would seem sufficient to account
for the normal projections in this study, the numbers of surviving motoneurones
suggest that active guidance was also partly if not entirely responsible. Before
showing how this is so, it should be noted that contralateral motoneurone
numbers were almost normal in most of the tadpoles operated at Stage 49.
Poorer survival rates were strongly correlated with greater maturity at operation.
The most probable reason is that axons have greater difficulty finding the
contralateral limb bud as distances increase. In addition, many axons must have
been severed in older tadpoles since proximal limb bud becomes innervated
during stage 50. Although motor axons severed before the onset of cell death can
regenerate, axotomy nevertheless depresses survival rates (Lamb, 19816).
The fact that motoneurone survival was approximately normal when the
operations were done early enough means it is unlikely that axons grew passively
or randomly into the limb bud. This is because without active guidance, axons
would first have had difficulty finding the remaining limb bud and then, those
that succeeded, finding their appropriate regions within it. As a result few if any
motoneurones would have survived in contrast to the normal numbers found. It
is therefore reasonable to interpret the results in favour of active guidance. A
theoretical alternative to active guidance is that axons could successfully find
Selective bilateral motor innenation
157
their correct sites by branching non-selectively to all sites and then eliminating
incorrect branches. However, developing motor axons appear not to branch to
any significant degree except for their terminal ramifications (Lamb, 1976;
Landmesser, 1978; Lance-Jones & Landmesser, 1980a). Studies of projections
at intermediate stages are needed to resolve these points. It is important to note
that even if active guidance is confirmed, passive guidance is not denied. Passive
forces such as contact guidance undoubtedly play an important role, but active
forces seem necessary to give at least minimal directives to the growth cone.
A question arises whether contralateral axons used ipsilateral axons as guides.
That is not to suggest that the contralateral axons were passively guided for they
would still have had to choose which ipsilateral axons to follow. Though this
may have involved fasciculation, usually considered passive, the fasciculation
would have to have been selective requiring active recognition between compatible axons. Selective fasciculation was first proposed by Weiss 40 years ago
(e.g. Weiss, 1941), but it has received little attention because it is difficult to
examine experimentally. However, the concept is attractive since all that is
required is for axons to respond selectively to information transmitted by
predecessors already at the target. Retrograde chemical or electrical signals are
not hard to envisage. The difficulty is to devise tests which can show unambiguously that axons make choices on the basis of that information and not
in response to non-axonal cues. There is hope that monopodal tadpoles may
provide a suitable model and this is to be investigated.
Other theories of axonal guidance have been discussed in recent reviews
(Constantine-Paton, 1979; Landmesser, 1980).
The results of this experiment complement two recent studies. In the first
(Beazley & Lamb, 1979), optic nerves induced to take abnormal routes in
Xenopus tadpoles, were found to make normal retinotopic projections onto the
tectum. In contrast to the present study, neuronal loss (in the retina) was
probably increased judging from the small size of the mature optic nerves, and
the authors were unable to decide whether the axons were actively guided. However, the experiment showed that non-selective synaptogenesis could not be
induced simply by making axons enter the wrong part of the tectum. In the
other study, in chick embryo, spinal cords reversed in the rostrocaudal axis
were nevertheless able to establish their normal motor projections, providing
excellent evidence for active guidance (Lance-Jones & Landmesser, 1980£).
A serious discrepancy with several earlier experiments is raised. Limb buds
rotated or grafted as supernumeraries alongside normal limb buds have been
found to receive systematically abnormal projections (Hollyday et al. 1977;
Morris, 1978; Stirling & Summerbell, 1979, 1980). In particular, rotated wing
buds receive the projections that would be predicted from passive axonal
guidance and non-selective synaptogenesis (Stirling & Summerbell, 1979, 1980).
The implications for specificity of neuromuscular synaptogenesis have been
discussed elsewhere (Lamb, 1981a). Lance-Jones & Landmesser (1980Z>) have
6
EMB 65
158
A. H. LAMB
Knee extensors: cruralis and gluteus magnus
Stage of operation
H.R.P. volume (MD
49
001
Motoneurone number
1401
14
Labelled motoneurones
1449
22
1431
62
1575
82
51
0-5
Fig. 5. Motoneurones projecting to knee extensors.
tried to reconcile the results in terms of active guidance. (See also Landmesser,
1980, review). They suggested that normal projections result after cord rotation,
but not limb rotation because axons leaving the rotated cord immediately
encounter tissues of incongruous polarity and have time to adjust before
reaching the limb. Axons approaching a rotated limb have no such opportunity
and find themselves caught irretrievably in the wrong part of the plexus. Unfortunately, this very plausible explanation is weakened by the present study.
Assuming the motor axons were actively guided, those on the contralateral side
could have had no direct knowledge of abnormality until near the amputation
site and no axial confusion until they had crossed behind the anus. Unless
Selective bilateral motor innervation
159
Ankle plantar flexor: gastrocnemius
Stage of operation
49
H.R.P. volume (pi)
001
Motoneurone number
Labelled motoneurones
Fig. 6. Motoneurones projecting to gastrocnemius.
knowledge about the limb field was obtained indirectly, directional adjustments
must have been made at the last moment either at the base of the limb bud or
within the limb bud itself. For the moment it seems that the discrepancies
cannot be resolved.
Competition hypothesis
An important result of this study is the confirmation of an earlier report that
the limb is able to support twice its usual complement of motoneurones (Lamb,
1980). It was argued that the result refutes the hypothesis that motoneurone
survival is determined by competition for limited target factors. However,
6-2
160
A. H. LAMB
Toe dorsiflexors
Stage of operation
H.R.P. volume (ji\)
Motoneurone number
Labelled motoneurones
51
01
49
01
#
870
27
1629
57
1320
38
•
1326
47
Fig. 7. Motoneurones projecting to toe dorsiflexors.
Purves (1980) has speculated that the initial bilateral innervation may somehow
induce a greater supporting capacity within the limb (as opposed to the operation itself doing so; see Lamb, 1980). Since there appears to be no way of convincingly disproving the suggestion, peripheral competition among motoneurones remains a possibility.
A more recent study reinforces this need for caution. Removal of parts of the
motoneurone pools to certain muscles of the chick hindlimb appeared to
reduce the number of dying motoneurones in the remaining portions (LanceJones & Landmesser, 1980«). This evidence is by far the best in support of
Selective bilateral motor innervation
161
Intrinsic toe plantar flexors
Stage of operation
H.R.P. volume Oil)
Motoneurone number 1200
Labelled motoneurones 9
Fig. 8. Motoneurones projecting to intrinsic plantar flexors of the toes.
competition. It is of interest that cell rescue seemed possible for motoneurone
pools of only some muscles. If it is shown that these are in the minority it may
explain the rather puzzling observation that supernumerary limbs are able to
rescue some cells from death but not the majority (Hollyday & Hamburger,
1976; Lamb, 1979c).
A similar experiment in Xenopus produced no evidence for competition. Part
of the ventral horn supplying the knee flexors was removed on one side only in
young tadpoles and the animals were examined well after cessation of cell death.
Total cell numbers in the remaining segments were not increased. It was also
162
A. H. LAMB
found that at least some motoneurone deaths are definitely not due to competition (Lamb, 19796).
It seems the evidence at hand cannot decide whether competition controls
motoneurone survival. Though it should be borne in mind that competition
probably controls survival elsewhere in the nervous system (e.g. Pilar, Landmesser & Burstein, 1980), that does not constitute an argument for competition
among motoneurones. In any case, whether they compete or not, it is almost
certain that competition on its own is insufficient to account for all naturally
occurring motoneurone deaths. Searches for additional causes therefore seem
justified.
I thank Dr. L. D. Beazley for her comments and Mr. S. B. Baker for technical assistance.
Support was provided by the National Health and Medical Research Council of Australia
and the Muscular Dystrophy Research Association of Western Australia.
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