/ . Embryol. exp. Morph. Vol. 72, pp. 269-286, 1982
Printed in Great Britain © Company of Biologists Limited 1982
269
Motor projection patterns to the hind limb of
normal and paralysed chick embryos
By N. G. LAING 1
From the Department of Physiology, University of Edinburgh
SUMMARY
Counts were made of the number of motoneurons innervating the hind limbs of 10-day
normal and paralysed chick embryos whose right hind limb buds had been subjected to
varying degrees of amputation prior to innervation. The number of motoneurons on the intact
sides of the paralysed embryos was found to be similar to the number present in normal
embryos prior to the major period of motoneuron death. Since it has previously been shown
that paralysis does not increase the number of motoneurons generated, this means that normal
motoneuron death was largely prevented in the paralysed embryos.
There were differences in the distributions of motoneurons in the rostrocaudal axis of the
spinal cord between normal and paralysed embryos. Therefore, cell death does not eliminate
a uniform fraction of motoneurons throughout the rostrocaudal extent of the chick embryo
lumbar lateral motor column. It is also argued that there are differences in the relative
contribution of the various lumbosacral levels to different parts of the limb, e.g. the shank,
before and after the period of cell death.
In both normal and paralysed embryos there was a linear relationship between the volume
of limb muscle which developed after amputation and the number of motoneurons surviving
in the spinal cord. There was no evidence of a 'compression' of motoneurons into the
remaining muscle either after amputation alone or after amputation combined with paralysis.
Motoneurons are therefore rigidly specified for certain parts of the limb.
The relationship between motoneuron number and muscle volume on the amputated side
differed from that of the intact side. For a similar increase in muscle volume there was a smaller
increase in motoneuron number on the intact sides. This suggested a parallel to the paradoxically small increase in motoneuron number that occurs on the addition of a supernumerary limb.
INTRODUCTION
A number of recent studies have given evidence that the motor projections to
the limb change during embryonic development. Lamb (1976, 1977) using
retrograde horseradish-peroxidase (HRP) labelling has shown that the pattern
of motor innervation of the Xenopus hind limb changes during development and
that at least some of this change comes about through motoneuron death. He
found that as the larvae developed the rostrocaudal extent of the spinal cord
innervating some limb regions became smaller: some connexions present in
early embryos were lost during the period of normal motoneuron death.
Pettigrew, Lindeman & Bennett (1979) have produced both HRP and electro1
Author's present address: Department of Pathology, University of Western Australia,
Perth, Western Australia.
270
N. G. LAING
physiological evidence for a similar change in innervation in the chick embryo
forelimb. Changing innervation has also been seen electrophysiologically in the
axolotl limb (McGrath & Bennett, 1979) and rat intercostal muscles (Harris &
Dennis, 1977). On the other hand, it has been claimed, on the basis of both
HRP and electrophysiological studies that no such change occurs in the chick
embryo hind limb (Landmesser & Morris, 1975; Landmesser, 19786) and that
here motoneuron death is not concerned with the removal of such inappropriate
connexions.
Paralysis of embryos greatly reduces the amount of motoneuron death (Creazzo
& Sohal, 1978, 1979; Laing & Prestige, 1978; Olek, 1980; Olek & Edwards,
1978; Pittman & Oppenheim, 1978, 1979). Therefore, in paralysed embryos it
should be possible to see the projection pattern present prior to motoneuron
death. Retrograde transport of HRP should be an efficient method of revealing
the projection patterns but has a serious drawback in that the whole motoneuron
population is not labelled (Lamb, 1979; Landmesser, 19786), and it is difficult
to rule out leakage of HRP to neighbouring areas. An alternative is to examine
the distribution of motoneurons in the spinal cord after partial amputation.
This will reveal the position of the entire population innervating the remaining
part of the limb. Amputation at various proximodistal levels will ultimately
reveal the pattern of innervation of the whole limb.
After partial removal of the optic tectum in the goldfish, the whole innervating
retina can form a complete topographic map on the remaining tectum: the
innervation can 'compress' into the remaining target (Gaze & Sharma, 1970;
Yoon, 1971, 1976). It was therefore possible that a similar 'compression'
could occur in the partially amputated limbs: that axons could make connexions adequate for motoneuron survival by innervating parts of the limb they
do not normally innervate.
The aims of the present work were to see if the patterns of innervation of
normal and of paralysed limbs were the same and to see if a 'compression'
could occur in the limb.
METHODS
General
White Leghorn eggs were used in all experiments. Before incubation they
were prepared for windowing by boring a hole into the airsac and drilling
an approximately 1 x 1 cm window in the shell with a carborundum disc
attached to a dental drill. The square of shell was not removed but covered with
Scotch tape and the eggs incubated at 38 °C in a forced draught incubator. The
predrilled windows were opened on the day of the first manipulation. a-Bungarotoxin (a-Bgt) (Boehringer or Miami Serpentarium) was used to produce
paralysis because the irreversible nature of its action (Chang & Lee, 1963) made
it relatively easy to obtain complete lack of movement up to day 10 of incubation.
Mo tor projections
271
The a-Bgt was dissolved at a concentration of 1 mg/ml in Hank's Balanced
Salts Solution (pH 7-4) containing 10 mM HEPES buffer, 50i.u./ml penicillin
and 50/^g/ml streptomycin (all Flow Laboratories). The solution for control
injections was the same, except for the omission of the toxin.
All solutions were passed through a 0-45 /*m Millipore filter prior to use. All
operations were carried out using instruments sterilised in alcohol.
Amputation techniques
Amputations were always of the right hind limb since the rotation of the
embryo makes the right limbs more accessible.
Radical amputations (virtually all limb tissue removed) were performed on
day 3 of incubation (stages 17 and 18 of Hamburger & Hamilton, 1951). Evan's
blue dissolved in Hank's Balanced Salts solution was injected in the yolk sac
beneath the embryo in order to increase the definition of the embryonic structures. The extraembryonic membranes were cut and the exposed limb bud was
removed by cutting around it with electrolytically sharpened tungsten needles
and pulling it off the embryo.
Graded amputations were produced in embryos at stages 20-24, when the
limb buds are more developed, using small pieces of broken razor blade attached
by Araldite (Ciba-Geigy) to fine glass rods. One was positioned deep to the limb
bud as a support while the other was used to cut through the limb bud. The cuts
were made at different proximodistal levels in order to produce graded amputations such as those described by Hampe (1959). Axons first enter the hind
limb but at stage 24 and reach the knee at stage 25 (Oppenheim & Heaton.
1975). Therefore, in order not to axotomise neurons, only distal cuts were made
in stage-24 limb buds.
Injection techniques
On days 3 and 4 injections were carried out by tearing the extraembryonic
membranes and displacing the amniotic fluid with a 100/*l loading dose of the
toxin or sham-injection solution.
On days 6 and 8, injections of 50/^1 were made intraperitoneally with a
100 fi\ Hamilton syringe fitted with a 33-gauge needle.
Embryos amputated on day 3 were injected on days 3, 6 and 8. Embryos
amputated on day 4 were injected on days 4, 6 and 8.
Tissue processing
Embryos were killed by decapitation on day 6 and day 10 of incubation and
the lumbar vertebral column dissected out. In 10-day embryos the hind limbs
were left attached to the vertebral column in order to allow determination of the
muscle volume. Tissues were fixed in Carnoy's solution and processed for
paraffin histology. Serial sections were cut at 8 [im and stained with haematoxylin
and eosin.
272
N. G. LAING
Motoneuron counts
The numbers of lateral motor column motoneurons in every tenth section of
the lumbar lateral motor column were counted blind. Only cells containing one
or more nucleoli were included in the counts. The counts thus obtained were
corrected for double counting using an Abercrombie (1946) correction factor.
Motoneuron distribution
The distribution of motoneurons within the lumbar lateral motor column was
examined by dividing the rostrocaudal extent of the column into ten 'bins' of
equal length.
Determination of muscle volume
The volume of muscle in the hind limbs of the 10-day-old embryos was
calculated from camera-lucida drawings, at x 14, in the standard manner
(Bueker, 1945; Konigsmark, 1970) except that photocopies were cut up and
weighed. In this way the original drawings were preserved for further reference.
RESULTS
Distribution of innervation within the limb
The muscle volume in the hind limb was determined for 17 out of 20 paralysed
embryos fixed at day 10 and for 16 out of 22 sham-injected controls. Motoneurons were counted in all embryos (Tables 1 and 2). The total number of
motoneurons was greater on the intact sides of paralysed embryos (16586 ± 1458,
mean±s.D.) than in the controls (10445 ±1036) (P > 0-001, Mann-Whitney
U-test). The number of motoneurons in the control embryos was not significantly
different (P > 0-4, Mann-Whitney U-test) from that of totally untreated
embryos (10229 ± 1429, n = 14).
The total number of motoneurons present in 10-day paralysed embryos was
not significantly different (P > 0-1) from that in 6-day control embryos
(17241 ± 1243; n = 5). The number of motoneurons peaks at day 6 (Hamburger,
1975) and thus the paralysis can be said to have prevented normal cell death.
Although the chronic paralysis increased motoneuron number, it reduced muscle
volume in intact limbs from 17-69 + 4-42 mm3 to 7-94± 1-59 mm3 (P < 0-001,
Mann-Whitney U-test) (Fig. 1). Degenerating muscle fibres were seen in the
paralysed embryos.
There were interesting relationships between motoneuron number and muscle
volume in the various limbs (Fig. 1). There was a significant correlation between
muscle volume and innervating motoneuron number in the intact limbs of
control embryos (r = 0-73). Thus, some of the large variation in motoneuron
number in control embryos seemed to be related to between-embryo variation in
muscle volume. A similar correlation between limb size and motoneuron number
273
Motor projections
Table 1. Motoneuron number and muscle volume of intact and amputated sides of
sham-injected embryos fixed at day 10. Values from the amputated sides are
expressed as percentages of the values from the intact side
Muscle volume (mm3)
Motoneuron number
A
Intact
Embryos
side
C148
C157
A 102
9330
8678
10276
10619
10324
9810
9467
10365
10064
10482
10736
11346
10345
8493
10660
10050
10530
10818
13199
11703
11971
10530
'A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
A
104
106
120
121
160
161
163
165
167
172
176
319
320
327
328
329
330
331
332
X
S.D.
n
10445
1036
22
Amputated
side
10290
9220
1454
727
960
5961
6538
9721
8602
6599
10132
6387
8266
2525
5941
5742
4274
5378
6126
7114
7930
3355
—
—
A
Intact
side
Amputated
side
%
110-3
106-2
14-2
6-8
9-3
60-8
69-1
93-8
85-5
630
94-4
56-3
79-9
29-7
55-7
57-1
40-6
49-7
46-4
60-8
66-2
31-9
10-26
—
20-38
—
17-38
11-66
—
191
13-44
15-3
—
18-31
10-37
18-83
23-81
18-99
21-26
22-29
—.
24-03
17-61
10-05
—
0-14
—
10-19
1013
—
17-31
8-89
15-9
—
13-93
2-69
10-18
11-89
7-70
10-51
9-76
—
15-20
5-51
98-0
—
0-7
—
58-6
86-9
—
90-6
66-1
104-1
—
76-1
25-9
54-1
49-3
40-5
49-4
43-8
—
63-3
31-3
—
—
—
17-69
4-42
16
—
—
—
—
—
—
0/
/o
was noted for Rana pipiens larvae by Decker & Kollros (1969). There was no
significant correlation (r = 0-37) for the intact sides of paralysed embryos.
In both paralysed and sham-injected embryos as the degree of amputation
increased both muscle volume and motoneuron number became reduced towards
zero. In the sham-injected embryos the regression lines for the intact and amputated sides differed (Fig. 1). This indicated a different relationship between
muscle volume and motoneurons in amputated and intact limbs: for the same
increase in muscle volume there was a greater increase in motoneuron number on
the amputated sides.
The relationship between the muscle volume and motoneuron survival after
amputation could be seen by comparing the amputated side with the contralateral
intact side. The volume of muscle in the amputated limb was expressed as a
percentage of the volume of muscle in the intact contralateral limb and the
number of motoneurons on the amputated side as a percentage of the number
274
N. G. LAING
Table 2. Motoneuron number and muscle volume of intact and amputated sides of
paralysed embryos fixed at day 10. Values from the amputated sides are expressed
as percentages of the values from the intact side
Muscle volume (mm3)
Motoneuron number
Embryo
Intact
side
Amputated
side
D267
D276
B 30
B 47
B 84
B 88
B102
B105
B106
B112
B 116
B128
B132
B135
B136
B141
B144
B 147
B149
B150
18474
13960
17287
15634
15627
17177
16766
16917
15929
16368
14879
17459
16313
16539
16615
14413
15737
18796
16697
20127
17217
12657
14900
14969
12444
14461
10921
10839
9906
X
16586
1458
20
S.D.
n
7601
5193
1530
1132
5845
12979
9041
10763
12307
12568
17459
—
—
/o
93-2
90-7
86-2
95-7
79-6
84-2
65-1
64-1
62-2
46-4
34-9
Intact
side
Amputated
side
/o
11-69
9-99
85-5
—
—
—
7-34
91-2
8-05
—
619
—
—
5-21
8-47
3-35
84-2
81-9
46-4
47-3
56-4
38-5
29-6
8-8
6-9
10-34
7-22
6-43
711
6-83
6-45
7-12
8-70
35-3
78-1
62-7
68-4
65-5
75-3
86-7
—
—
0-1
2-0
—
7-95
6-95
9-57
10-03
7-57
6-77
5-51
4-39
4-08
5-78
4-62
5-51
69-3
63-2
42-6
57-6
61-0
81-4
—
—
—
—
—
—
7-94
1-59
17
304
401
2-63
1-91
001
0-17
on the intact side. The result of plotting the values thus obtained against each
other is shown in Figure 2. There was a remarkably linear relationship between
the degree of motoneuron and muscle survival after amputation in both shaminjected (r = 0-97) and paralysed (r = 0-96) embryos. The linear relationship
indicates that there is a uniform 'innervation density' (number of innervating
motoneurons per unit of muscle volume) throughout the proximodistal extent
of the limb. This finding relates to the linear relationship between the wet weight
of a muscle and the number of motoneurons projecting to it noted by Landmesser
(1978a).
There was no sign of any major compression of the motoneuron innervation
into the limb in either the sham-injected or paralysed embryos. In neither group
was there 100% survival of motoneurons with significantly less than 100% of
normal muscle volume present. This suggested that, with or without paralysis,
there was very little possibility for motoneurons normally innervating distal
muscles to contact the remaining proximal muscles.
Motor projections
20,000
275
r
10,000-
10
20
Muscle volume (mm 3 )
Fig. 1. Comparison of muscle volume and motoneuron number in 10-day embryos.
• , treated intact sides; D, treated amputated sides. • , control intact sides;
O, control amputated sides. Solid lines are the regression lines for the intact side,
dashed lines the regression lines for the amputated sides. Each point represents
one side of one embryo.
100r
10
20
30
40 50 60 70 80 90 100
Muscle volume amputated
intact (%)
Fig. 2. Degree of motoneuron loss compared to degree of muscle loss; amputated
side value expressed as percentage of the intact side value for each embryo. Sixty
percent muscle volume corresponds approximately to a knee amputation; only
thigh remaining. Upper line regression line for treated embryos (O), lower line
regression line for control embryos (#). Each point represents one embryo.
276
N. G. LAING
3000-
2500-
p
2000-
1500-
1000-
500-
1 2
Rostral
3
4
5
6
Bin
9 10
Caudal
Fig. 3. Distribution of motoneurons within the lumbar lateral motor column on
intact sides. Rostrocaudal extent of the column divided into ten equal 'bins': mean
number of motoneurons per bin with standard error of mean. Comparison of
treated (O) and control ( • ) embryos.
The P = 0-1 confidence limits for the two regression lines in Fig. 2 overlapped
continuously indicating that the two regressions were not significantly different.
This in turn would suggest that there was a constant proportion of the innervating motoneurons dying throughout the limb. There was not a greater proportion of cell death amongst motoneurons innervating the thigh than amongst
motoneurons innervating the shank.
Distribution of motoneurons within the spinal cord
Since motoneuron death was reduced in the paralysed embryos it was of
interest to see where the excess motoneurons were within the lateral motor
column. This was investigated for the rostrocaudal axis by diving the full length
of the lateral motor column into ten equal 'bins'. Segments were not used
because the division of the spinal cord into segments during development is
arbitrary, with the result that the lateral motor column is not always present in
the same segments. Using segments adds another variable which may obscure
any effect of treatment.
Motor projections
277
Table 3. Proportion of total motoneuron number in each bin for the three groups of
embryos: untreated, control and treated. The value for each bin is the mean with
standard error of the mean for all embryos in that group and bin
Untreated
Control
(/i = 14)
(#i = 11)
\
t
Bin
X
1
2
3
4
5
6
7
8
9
10
20-
S.E.M.
5-0 + 0-40
10-2 + 0-38
13-1 ±0-24
13-2 ±0-28
13-3±O-36
13-4 ±0-30
12-3 + 0-25
101 ±0-41
6-1 ±0-36
3-4 ±0-21
{
Treated
in = 20)
S
X
X
S.E.M.
4-9 ±0-36
11-2 ±0-45
14-1 ±0-42
14-9±0-33
14-7 ±0-23
12-8 ±0-23
10-9 ±0-31
8-2 ±0-32
5-5 ±0-24
2-9±0-15
4-8 + 0-23
10-7 + 0-26
13-1+0-24
13-3 + 0-27
13-1+0-25
13-6 ±0-21
12-6 ±0-24
9-7 + 0-25
5-7±0-19
3-5±0-16
(a)
S.E.M.
Control plus
untreated (n = 36)
\ r
^
X
S.E.M.
4-8 + 0-21
10-5 ±0-22
13-1 ±0-17
13-3 ±0-20
13-2 + 0*19
13-5±0-17
12-5±0-18
9-9 + 0-22
5-8±0-18
3-5±0-ll
(b)
fe 18|
16-
e
14-
| n1 10o
B
"c3
2
6-
1 4-
2-
1 2 3 4 5 6 7 8 9
10
Rostral
Bin
Caudal
1 2 3 4 5 6 7 8 9
10
Rostral
Bin
Caudal
Fig. 4. Distribution of lumbar lateral motor column motoneurons on intact sides.
For each embryo the number of motoneurons in each bin was expressed as a percentage of the total number of motoneurons in the column. Data plotted as mean
with standard error of the mean, (a) Control embryos ( # , n = 11) compared
with untreated embryos (O,n = 14); (b) treated embryos (O,n = 20) compared
with the untreated and control groups combined together ( # , n = 36) + ,
0-05 > P > 0-01; *P < 0-001; (Mann-Whitney U-test). Data as in Table 3.
There were greater numbers of motoneurons in all bins on the intact sides of
paralysed embryos (Fig. 3). The difference between the curves in Fig. 3 represents the number of motoneurons prevented from dying or, because prevention
of motoneuron death was so good, the position of motoneurons that normally
die. To allow direct comparison of the three groups of embryos, the number of
motoneurons in each bin in each embryo was expressed as a percentage of the
278
N. G. LAING
total number of motoneurons in that particular embryo. The values for all the
embryos in each group were then pooled (Table 3). The distribution of motoneurons innervating intact limbs appeared to be the same in control and
untreated embryos (Fig. 4A); no significant difference could be detected in any
bin. However, significant differences were found in seven out of the ten bins
when the treated group was compared with the combined control and untreated
groups (Fig. 4B). The probability values were of the same order of magnitude
whether the test used was the non-parametric Mann-Whitney U-test or the
parametric f-test. The probability values were also similar whether the comparison
was with the combined control and untreated groups or with the control group
alone except that in bin 10 the level was changed from P < 0-001 to
0-01 > P > 0001.
The fact that the two distributions in Fig. 4B are different means that
motoneuron death does not affect a constant fraction of the starting population
of cells throughout the rostrocaudal extent of the lateral motor column.
Wherever the distribution for treated embryos lies below that for the combined
control group a smaller than average fraction of motoneurons die. Wherever
the treated embryo distribution lies above the control a greater than average
fraction of motoneurons die. Therefore, a greater than average loss of motoneurons occurs in bins 3,4 and 5. For some reason the population of limbinnervating motoneurons originally created in these bins is subject to a greater
neurothansasia than the rest of the population.
One measure of the shape of a curve is the coefficient of skewness (Kendall &
Stuart, 1969). The coefficient was calculated from the motoneuron counts for
each embryo prior to binning. It depends mostly on the main body of the data
and is therefore largely independent of the estimation of the ends of the lateral
motor column whereas binning depends on this. The skewness test is, then, even
more accurate than using bins. The values for the control group ranged from
-0-038 to 0-297 (mean = 0-133, n = 22), for the untreated group from -0-018
to 0-243 (mean = 0-115, n = 14) and for the treated group from 0-52 to 0-534
(mean = 0-249, n = 20). Comparing the values with the Mann-Whitney U-test,
the control and untreated groups were not significantly different (P > 0-6)
whereas the treated group was significantly different from the combined control
groups (P < 0-001). Thus, the treated group showed greater skewness than the
control group, i.e. the shapes of the curves were different.
Insight into why motoneuron death was not uniform along the length of the
lateral motor column could be obtained by examining the distribution of
motoneurons innervating the partially amputated limbs. The distribution of
motoneurons surviving various degrees of amputation may be seen in Fig. 5. In
sham-injected embryos (Fig. 5 A), as the degree of amputation became less,
more motoneurons survived the operation. A peak in the distribution arose
regularly in the rostral tenths, with a relative plateau in the caudal tenths. The
63 % distribution approximated to a knee amputation and thus to the distribution
Motor projections
279
20 r (a)
18
16
3
14
o 12
12
o
c
10
8
6
1 2
Rostral
3
4
5 6
Bin
7
9 10
Caudal
1 2 3 4
Rostral
5 6 7 8 9
10
Bin
Caudal
Fig. 5. Distribution of motoneurons within the lumbar lateral motor column after
various degrees of amputation, (a) Control; (b) treated embryos. Dotted lines are
the same data as in Fig. 4 (intact slides: 100 % distribution). Other lines represent the
distribution for the percentage motoneuron survival shown by the associated
number. Each line is synthesized by averaging the values for the number of embryos
shown in brackets.
Table 4. Proportion of motoneurons in each bin on the intact and the amputated
sides of embryos in which approximately 60 % of motoneurons survived on the
amputated side
Treated (n = 5)
Control (n = 4)
A
Intact sides
Bin
X
S.E.M.
'Thigh'
X
S.E.M.
1
4-3 ±0-46
4-4 + 0-94
2
3
4
10-7 ±0-83
13-1 ±0-32
131 ±0-59
12-7 ±0-46
10-3±0-88
12-7 ±0-66
10-9±0-53
6-1+0-72
4-6 ±0-44
4-4 ±0-74
3-1 ±0-65
3-5 ±0-56
2-9±0-18
5
6
7
8
9
10
14-0 ±0-25
12-9 ±0-60
9-9 ±0-38
5-5 ±0-29
3-8 ±0-55
Intact sides
X
S.E.M.
4-5 ±0-92
1O-5±O-81
14-1 ±0-46
14-6±0-89
14-3 ±0-59
13-2 ±0-37
11-1 ±0-34
8-8 ±0-59
5-9 ±0-46
3-1+0-32
•Thigh'
X
S.E.M.
4-0 ±0-53
8-5 ±0-44
ll-9±0-37
10-7 ±0-72
7-2 ±0-34
6-1 ±0-33
5-2 ±0-38
3-8 ±0-49
3-7±O-31
2-8 ±0-21
of motoneurons innervating the thigh musculature. A similar peak and plateau
for thigh musculature could be inferred from the horseradish peroxidase data of
Landmesser (19786). The distribution in the paralysed embryos although similar,
did not follow this pattern exactly (Fig. 5B). For example, the 64 % distribution,
approximating to knee amputation, did not correspond to the intact distribution
in bin 3, whereas the equivalent distribution (63 %) in the control embryos did.
280
N. G. LAING
20-,
(a)
18-
18-
16"
16-
14-
c
|
•
14-
I
12-
1210-
10-
0
8-
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0
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0
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:.
CD
09
42-
ib)
3
4
8
8
80
8
0
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.
jjQ
42-
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7
8
9 10
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i
i
i
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r
4
i
r T
5 6 7
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9 10
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Fig. 6. Distribution of motoneurons on the intact (.) and amputated (O) sides of
embryos in which motoneuron survival was approximately 60%. (a) Control;
(b) treated (the amputated side values averaged give the 63% and 64% lines in
Figs. 5 a and 5 b). The Mann-Whitney U-test was used to compare the values for the
two sides +,0-05 > P > 0-01; x, 0-01 > P > 0001.
To emphasize this point the data for nine embryos with approximately 60 %
motoneuron survival is shown in expanded form in Fig. 6 and Table 4. Using
the Mann-Whitney U-test, the distribution of motoneurons on the amputated
sides was found to be different from that of the intact sides in bins 5-9 in controls
(Fig. 6 A), but different in bins 3-9 in treated embryos (Fig. 6B). In controls the
probability value for bin 4 was 0-058, which is bordering on significance.
A f-test, which unlike the U-test, takes account of how far apart the values in the
two groups are, gave a probability value of 0-05 > P > 0-01. There was therefore, considering the small number of embryos involved, probably a significant
difference at bin 4 in control embryos. Now the difference between the distribution of motoneurons on the amputated and intact sides reveals the position of
the motoneurons innervating the part of the limb which had been amputated.
In Fig. 6 the differences represent the innervation of the shank since the 63 %
and 64% survival occurred with amputation approximately at the knee. In
controls the two sides were different in bins 4 to 9 (Fig. 6 A) so the shank
innervation in controls was from these bins. For treated embryos there was also
a difference at bin 3. Thus the shank received innervation from bin 3 in embryos
in which motoneuron death was prevented but not in control embryos where
motoneuron death had occurred normally. Motoneuron death thus removed
this innervation not found in the adult.
Motor projections
281
DISCUSSION
The aim of the present study was to investigate the innervation of intact and
amputated limbs in control and paralysed embryos. By this approach, the
question of whether or not the pattern of innervation changes during development could be answered. Also, information could be expected on the plasticity
of the motoneuron/limb system, answering the question of whether 'compression' can occur in the limb.
The data showed that there is an increase in motoneuron number in the a-Bgt
injected embryos and a reduction in muscle volume. Muscle atrophy in paralysed
limbs has been reported many times but usually at later stages of development
(e.g. Drachman, 1964; Giacobini et al. 1973). Pittman & Oppenheim (1979) note
that shank weight is reduced in 10-day curare and a-cobratoxin paralysed
embryos. The reduction is to 75 % of control in the a-cobratoxin paralysed
embryos which is not as great as the present reduction in muscle volume (to 45 %
of control). However, as well as muscle, shank weight will include bones, tendons,
etc. which may not be so seveiely affected by paralysis.
Paralysed embryos have greater numbers of motoneurons innervating a
smaller volume of muscle. It is probable that a trophic feedback from muscle to
nerve normally keeps embryonic motoneurons alive and this feedback is
presumably greater in the paralysed embryos. Possibly related cases are the
interesting finding of increased nerve growth factor (NGF) levels in denervated
irides (Ebendal, Olson, Seiger & Hedlund, 1980), and the more recent finding of
increased concentrations of trophic factor in denervated muscle (Hill & Bennett,
1982). The basic finding does not address the problem of whether motoneuron
death comes about normally through redundancy or rejection (Prestige, 1976).
The paralysis may be increasing the number of available contact sites, reducing
redundancy, by keeping muscle more myoblastic (Giacobini et al. 1973) or by
allowing more contacts per muscle fibre (Pittman & Oppenheim, 1979). Alternatively paralysis could block the mechanism by which axons are rejected by
muscle fibres just as it slows loss of multiple innervation (Brown, Jansen &
& Van Essen, 1976). Of course, both sorts of process could be occurring together;
with contact site number being increased and rejection being decreased.
The different relationships between muscle volume and motoneuron number
on the intact and amputated sides of sham-injected embryos may relate to the
effect of supernumerary limbs on motoneuron survival. There is a puzzlingly
small increase in motoneuron number when a supernumerary limb is grafted
onto an embryo, 15 % average in Xenopus (Hollyday & Mendell, 1976) and 17 %
average in chick (Hollyday & Hamburger, 1976). In the present experiments, for
a doubling in intact limb size through natural variation there is an increase in
motoneuron number of 21 % (Fig. 1, control intact sides 10 mm3 to 20 mm3). In
amputated limbs a doubling of muscle volume produces a much greater increase
in motoneuron number (83 % for 9 to 18 mm3: control amputated sides, Fig. 1).
282
N. G. LAING
Thus, the relationship between muscle volume and motoneuron number after
supernumerary grafting is similar to that for intact limbs and dissimilar to that
for amputated limbs.
If it is supposed that in a normal, whole limb there is a complete set of
post-synaptic targets (cf. Prestige & Willshaw, 1975), then the difference
between grafting and amputation may be stated thus: an amputated limb
contains an incomplete set of targets whereas a limb tcr which a supernumerary
has been added has a full set of targets but extra sites at each target position. It
would seem that the number of targets is more important than the number of
sites at a target in determining the number of motoneurons supported by a limb.
When there is a small amount of any one post-synaptic target present it will
maintain a relatively large number of motor units, a result possibly related to
those of bilaterally innervated limbs (Lamb, 1980).
From the comparison of the degrees of muscle and motoneuron survival on
the amputated sides (Fig. 2), it is clear that no major 'compression' of the
motoneuron innervation into the chick hind limb takes place either with, or
without, paralysis. A similar result showing lack of compression of motoneuron
innervation into non-paralysed chick embryo limb has recently been reported
(Whitelaw & Hollyday, 1980). a-Bgt allows greater numbers of motoneurons to
make connexions with each muscle, but does not apparently allow the formation
of connexions by motoneurons which normally innervate other parts of the
limb. Motoneuron number in the paralysed embryos is increased by 60 %, and
Lamb (1980) has shown that Xenopus limbs are capable of maintaining at least
twice their normal number of motoneurons. These two observations, taken
together, suggest that the supportive capacity of the limb is not fully utilised in
the paralysed embryos.
Axonal guidance (Lance-Jones & Landmesser, 1980) may prevent the formation of connexions between motoneurons destined for the deleted parts and
the surviving muscles. Alternatively the motoneurons destined for deleted parts
may connect with the surviving muscle but die because of incompatibility
(Lamb, 1981). The lack of compression observed in paralysed embryos would
then show that paralysis cannot prevent the motoneuron death resulting from
such an incompatibility.
There are several explanations for the regression lines in Fig. 2 not going
through the origin. Firstly, waiting only until day 10 may not be allowing the
effect of amputation to reach completion as in the study on the chick wing by
Hamburger (1934) where at days 8 and 9 40% of motoneurons remained when
all muscle had been removed. However the effect of early (day 3) amputation of
the hind limb is complete at day 10 (Laing, 1982, unpublished observations),
and a similar relationship was seen in embryos fixed at days 16 and 20 after
amputation at day 10 (Laing, unpublished observations). The population of cells
which survives amputation is real, but it may not be as large as suggested by the
data. The discrimination of motoneurons from large interneurons situated
Motor projections
283
laterally in the grey matter can be difficult in the absence of other motoneurons.
For this reason the number of' motoneurons' counted in the radically amputated
embryos may be inflated by the inclusion of interneurons. It is also difficult to
recognize scattered muscle fibres and take them into account in the cameralucida drawings. Therefore, the volume of muscle present following radical
amputation may be larger than indicated. Finally, some of the surviving motoneurons may be innervating the contralateral limb (Lamb, 1980). An alternative
interpretation is that the survival of some motoneurons is independent of the
presence of their target tissue. This is an uncomfortable hypothesis. Lamb (1981)
has recently found that bilateral amputation in Xenopus gives 100% loss of
motoneurons. This shows that at least in that species the survival of all motoneurons is dependent on the limb.
Distribution of motoneurons within the spinal cord
The data show differences in the distributions of motoneurons between control
and paralysed embryos. Since the paralysis prevents motoneuron death, it is
concluded that the projection pattern present prior to motoneuron death is
retained in the paralysed embryos and is different from the post-death pattern.
This conclusion is at variance with that of a study using paralysis combined
with retrograde HRP labelling (Oppenheim, 1981). The discrepancy may arise
from the different degrees of prevention of motoneuron death in the two studies.
Unlike the present study (Fig. 3), previous data on the prevention of
motoneuron death with curare and botulinum toxin (Pittman & Oppenheim,
1978, 1979) showed no effect on the rostral-most part of lateral motor column.
But, Oppenheim (personal communication) has recently found that using
cobratoxin and starting injections at day 4 gives a hypothanasia in the rostral
part of the lateral motor column. Some of the effect may be due to the use of
different neuromuscular blockeis, but the disparity is probably due to the onset
time of the paralysis since the botulinum toxin and curare were first applied on
day 6 (Pittman & Oppenheim, 1978, 1979). If this latter is true, it suggests that
the rostral motoneurons are amongst the first to die. Certainly the rostral
motoneurons are amongst the first to become 'limb-dependent' (Prestige,
1967) as shown by radical amputation (Oppenheim, Chu-Wang & Maderdrut,
1978).
The fact that the distributions of motoneurons within the lumbar lateral motor
column in paralysed and control embryos are different (Fig. 4B) means that the
proportion of motoneurons dying is not constant throughout the limbinnervating pool. In an earlier study (Landmesser, 1978£) it was concluded that
motoneuron death did not alter the rostrocaudal distribution of motoneurons
supplying various muscles and muscle groups. The difference in results may
arise from the use of segments rather than bins to denote the position of the
motoneuron pools. As mentioned in the results section the demarcation of
spinal cord segments is arbitrary and variable. The segments vary in length
284
N. G. LAING
between animals and the segmental position of the motor column also varies.
Using bins nullifies any variation from these sources.
The conclusions from the present study are that neurothanasia eliminates
a constant proportion of the motoneurons innervating each part of the limb
(Fig. 2), but not a constant proportion within the spinal cord (Fig. 4). This could
only happen through non-uniformity of death in sub-populations such as has
been shown for the shank (Fig. 6) where the innervation from bin 3 entirely
disappears after motoneuron death. Motoneuron death therefore plays a part
in producing the normal mature pattern of innervation of the limb.
This work was carried out while I was research associate to the late M. C. Prestige and
funded by the Medical Research Council of Great Britain. I should like to thank Dr R. K.
Milne of the University of Western Australia Mathematics Department for statistical advice
and Dr A. H. Lamb for his patient criticism during the preparation of the manuscript.
REFERENCES
M. (1946). Estimation of nuclear population from microtome sections. Anat.
Rec. 94, 239-247.
BROWN, M. C , JANSEN, J. K. S. & VAN ESSEN, D. (1976). Polyneuronal innervation of
skeletal muscle in new-born rats and its elimination during maturation. J. Physiol. 261,
387-422.
BUEKER, E. D. (1945). The influence of a growing limb on the differentiation of somatic motor
neurones in transplanted avian spinal cord segments. /. comp Neurol. 82, 335-361.
CHANG, C. C. & LEE, C.-Y. (1963). Isolation of neurotoxins from the venom of Bungarus
multicinctus and their modes of neuromuscular blocking action. Archs int. Pharmacodyn.
Ther. 144, 241-257.
CREAZZO, T. L. & SOHAL, G. S. (1978). Effects of alpha and beta bungarotoxin on the development of trochlear nucleus and superior oblique muscle. Soc. Neurosci., 4, 110 (Abstr.).
CREAZZO, T. L. & SOHAL, G. S. (1979). Effects of chronic injections of a-bungarotoxin on
embryonic cell death. Expl Neurol. 66, 135-145.
DECKER, R. S. & KOLLROS, J. J. (1969). The effect of cold on hind-limb growth and lateral
motor column development in Rana pipiens. /. Embryol. exp. Morph. 21, 219-233.
DRACHMAN, D. B. (1964). Atrophy of skeletal muscle in chick embryos treated with botulinum toxin. Science, N.Y. 145, 719-721.
EBENDAL, T., OLSON, L., SEIGER, A. & HEDLUND, K.-O. (1980). Nerve growth factors in the
rat iris. Nature, Lond. 286, 25-28.
GAZE, R. M. & SHARMA, S. C. (1970). Axial differences in the reinnervation of the goldfish
optic tectum by regenerating optic nerve fibres. Expl Brain Res. 10, 171-181.
GIACOBINI, G., FILOGAMO, G., WEBER, M., BOQUET, P. & CHANGEUX, J. P. (1973). Effects of
a snake a-neurotoxin on the development of innervated skeletal muscles in chick embryo.
Proc. natn. Acad. Sci., U.S.A. 70, 1708-1712.
HAMBURGER, V. (1934). The effects of wing bud extirpation on the development of the central
nervous system in chick embryos. /. exp. Zool. 68, 449-494.
HAMBURGER, V. (1975). Cell death in the development of lateral motor column of the chick
embryo. /. comp. Neurol. 160, 535-546.
HAMBURGER, V. & HAMILTON, H. L. (1951). A series of normal stages in the development of
the chick embryo. / . Morph. 88, 49-92.
HAMPE, A. (1959). Contribution of l'etude du developpement et de la regulation des deficiences
at des excedents dans la patte de l'embryon de poulet. Archs Anat. microsc. 48, 345-478.
HARRIS, A. J. & DENNIS, M. J. (1977). Deletion of 'mistakes' in nerve muscle connectivity
during development of rat embryos. Soc. Neurosci. 3, 107 (Abstr.).
ABERCROMBIE,
Motor projections
285
M. & BENNETT, M. R. (1982). Elevated growth factor(s) activity of denervated muscle.
Neurosci. Lett. (Suppl.) 8, S54.
HOLLYDAY, M. & HAMBURGER, V. (1976). Reduction of the naturally occurring motor neuron
loss by enlargement of the periphery. /. comp. Neurol. 170, 311-320.
HOLLYDAY, M. & MENDELL, L. (1976). Analysis of moving supernumerary limbs of Xenopus
laevis. Expl Neurol. 51, 316-329
KENDALL, M. G. & STUART, A. (1969). The Advanced Theory of Statistics, vol. I, 3rd ed.,
p. 85, London: Griffin.
KONIGSMARK, B. W. (1970). Methods for the counting of neurons. In Contemporary Research
Methods in Neuroanatomy (ed. W. J. H. Nauta & S. O. E. Ebbesson), pp. 315-340. New
York: Springer Verlag.
LAING, N. G. & PRESTIGE, M. C. (1978). Prevention of spontaneous motoneurone death in
chick embryos. J. Physiol. 282, 33-34P.
LAING, N. G. (1982). Timing of motoneuron death in the brachial and lumbar regions of the
chick embryo. Devi Brain Res. (in the press).
LAMB, A. H. (1976). The projection patterns of the ventral horn to the hind limb during
development. Devi Biol. 54, 82-99.
LAMB, A. H. (1977). Neuronal death in the development of the somatotopic projections of the
ventral horn in Xenopus. Brain Res. 134, 145-150.
LAMB, A. H. (1979). Evidence that some developing limb motoneurons die for reasons other
than peripheral competition. Devi Biol. 71, 8-21.
LAMB, A. H. (1980). Motoneurone counts in Xenopus frogs reared with one bilaterallyinnervated hind limb. Nature, Lond. 284, 347-350.
LAMB, A. H. (1981). Target dependency of developing motoneurons in Xenopus laevis.
J. comp. Neurol. 203, 157-172.
LANCE-JONES, C. & LANDMESSER, L. (1980). Motoneurone projection patterns of the spinal
cord. /. Physiol. 302, 581-602.
LANDMESSER, L. (1978 a). The distribution of motoneurones supplying chick hind limb
muscles. /. Physiol. 284, 371-389.
LANDMESSER, L. (19786). The development of motor projection patterns in the chick hind
limb. /. Physiol. 284, 391-414.
LANDMESSER, L. & MORRIS, D. G. (1975). The development of functional innervation in the
hind limb of the chick embryo. /. Physiol. 249, 301-326.
MCGRATH, P. A. & BENNETT, M. R. (1979). The development of synaptic connections between
different segmental motoneurones and striated muscles in the axolotl limb. Devi Biol. 69,
133-145.
OLEK, A. J. (1980). Effects of a and ft Bungarotoxin on motor neuron loss of Xenopus larvae.
Neuroscience 5, 1557-1563.
OLEK, A. J. & EDWARDS, C. (1978). Effect of alpha and beta bungarotoxin on naturally
occurring motor neurone loss in Xenopus larvae. Soc. Neurosci. 4, 122 (Abstr.).
OPPENHEIM, R. W. (1981). Cell death of motoneurons in the chick embryo spinal cord V.
Evidence on the role of cell death and neuromuscular function in the formation of specific
peripheral connections. /. Neurosci. 1, 141-151.
OPPENHEIM, R. W.,CHU-WANG, I.-W. & MADERDRUT, J. L. (1978). Cell death of motoneurons in the chick embryo spinal cord III. The differentiation of motoneurones prior
to their induced degeneration following limb-bud removal. /. ccmp. Neurol. Ill, 87-112.
OPPENHEIM, R. W. & HEATON, M. (1975). The retrograde transport of horseradish peroxidase
fiom the developing limb of the chick embryo. Brain Res. 98, 291-302.
PETTIGREW, A. G., LINDEMAN, R. & BENNETT, M. R. (1979). Development of the segmental
innervation of the chick forelimb. /. Embryol. exp Morph. 49, 115-137.
PITTMAN, R. & OPPENHEIM, R. W. (1978). Neuromuscular blockade increases motoneurone
survival during normal cell death in the chick embryo. Nature, Lond. 271, 364-365.
PITTMAN, R. & OPPENHEIM, R. W. (1979). Cell death of motoneurons in the chick embryo
spinal cord IV. Evidence that a functional neuromuscular interaction is involved in the
regulation of naturally occurring cell death and the stabilization of synapses. /. comp.
Neurol. 187, 425-446.
HILL,
IO
EMB 72
286
N. G. LAING
OPPENHEIM, R. W.
& HEATON, M. (1975). The retrograde transport of horseradish peroxidase
from the developing limb of the chick embryo. Brain Res. 98, 291-302.
PRESTIGE, M. C. (1967). The control of cell number in the lumbar ventral horns during the
development of Xenopus laevis tadpoles. /. Embryol. exp. Morph. 18, 359-387.
PRESTIGE, M. C. (1976). Evidence that at least some of the motor nerve cells that die during
development have first made peripheral connections. J. comp. Neurol. 170, 123-134.
PRESTIGE, M. C. & WILLSHAW, D. J. (1975). On a role for competition in the formation of
patterned neural connections. Proc. R. Soc. Lond. B, 190, 77-98.
WHITELAW, V., HOLLYDAY, M. (1980). Motoneurone target selectivity in chicks with deleted,
duplicated or reversed limb segments. Soc. Neurosci. 6, 647, (Abstr.).
YOON, M. G. (1971). Reorganization of retinotectal projection following surgical operations
on the optic tectum in goldfish. Expl Neurol. 33, 395-411.
YOON, M. G. (1976). Progress of topographic regulation of the visual projection in the halved
optic tectum of adult goldfish. /. Physiol. Lond. 257, 621-643.
(Received 14 June 1982, revised 12 July 1982)