/ . Embryol. exp. Morph. Vol. 36, 3, pp. 453-468, 1976
Printed in Great Britain
453
The response of the brachial ventral
horn of Xenopus laevis to forelimb amputation
during development
By JOANNE E. FORTUNE 1 AND ANTONIE W. BLACKLER 2
From the Section of Genetics, Development and Physiology,
Cornell University, New York
SUMMARY
The normal development of the brachial ventral horn of the frog Xenopus laevis and the
response of the brachial ventral horn to complete forelimb extirpation at five developmental
stages were assessed histologically. Differentiation of brachial ventral horn neurons occurred
in pre-metamorphic tadpoles between stages 52/53 and 57. Mean cell number in the brachial
ventral horn reached a peak of 2576 (S.E.M. = ± 269, n = 2) per side of the spinal cord at
stage 55 and decreased to 1070 (S.E.M. = ± 35, n = 7) by the end of metamorphosis. Cell
degeneration was presumed to be the mode of cell loss since it was most prevalent during the
period of rapid decrease in cell numbers. The response of the ventral horn to forelimb removal
varied with the stage of the animal at amputation. Following amputation at stage 52/53 or 54
the ipsilateral ventral horn neurons appeared less differentiated than those on the control
side and a rapid cell loss of about 80 % occurred on the operated side. These effects occurred
more rapidly after ablation at stage 54 than at stage 52/53. Amputation at stage 58, 61, or 66
caused chromatolysis in the ventral horn, a period of relative cell excess on the operated side,
and a delayed neuronal loss of 32-66%. It was concluded that excess cell degeneration
accounted for cell loss and that suppression of normal neuronal degeneration caused the
relative cell excess on the operated side. The data indicate that the brachial ventral horn was
indifferent to the periphery before stage 54, was quickly affected by limb removal between
stages 54 and 58, and by stage 58 had entered a phase in which a delay preceded cell death. No
forelimb regeneration occurred.
INTRODUCTION
Both autonomous and dependent differentiation play important roles in the
development of the vertebrate central nervous system. Motor and sensory
neurons have been particular targets of research efforts to distinguish between
peripheral and central influences on the developing spinal cord. The present
study investigated the relative importance of autonomous and dependent
differentiation in the development of the brachial ventral horn neurons of the
frog Xenopus laevis.
1
Author's address: Department of Animal Science, Cornell University, Ithaca, New York
14853, U.S.A.
2
Author's address: Section of Genetics, Development and Physiology, Cornell University,
Ithaca, New York 14853, U.S.A.
454
J. E. FORTUNE AND A. W. BLACKLER
Table 1. Abbreviated staging guide for Xenopus laevis {taken from
Nieuwkoop & Faber, 1956)
Stage
Approximate
age (days)
50
15
51
17
52
21
53
54
56
58
59
24
26
38
44
45
61
48
63
66
51
58
Description
Forelimb-bud enclosed within operculum,
hindlimb-bud somewhat conical
Forelimb-bud oval in lateral aspect,
hindlimb-bud has conical shape
Forelimb-bud conical in shape,
hindlimb-bud shows ankle constriction
Hind and forelimbs in paddle stage
All four fingers and all five toes indicated
Wrist and elbow clearly indicated
Forelimbs broken through operculum
Tentacles begin to shrivel
Tentacles considerably regressed, forelimbs
at level of posterior half of heart
Forelimbs at level of anterior half of heart
Tail almost completely resorbed,
metamorphosis complete
The ventral horns, or lateral motor columns, are groups of nerve cell nuclei
that are located in the spinal cord at brachial and lumbar levels and provide motor innervation to the limb and girdle muscles. In Xenopus laevis the
ventral horn is formed in larvae at stage 50-51 (see Table 1 for staging
guide) by migration of undifferentiated neuroblasts of the ventricular layer
(Prestige, 1973) and neuronal differentiation is complete by stage 57-58 (Kollros,
1956; Hughes & Tschumi, 1958). During this period of growth and differentiation cell degenerations result in a decrease in total cell number in the ventral
horn (Hughes, 1961; Prestige, 1967). This developmental pattern is similar
to that exhibited in other vertebrate species (Hamburger, 1958; Hughes, 1968 #, b;
Kollros, 1968).
Previous studies on several vertebrate classes suggested that although the
initial migration and differentiation of motor neuroblasts may be independent
of the periphery, the presence of the limb is necessary for the maintenance of
the motor neurons that innervate it (Hamburger, 1958; Hughes, 1968 a, b;
Kollros, 1968). Although nerve cell death is the final effect of limb removal,
Prestige (1967) found that the time of nerve cell body degeneration in Xenopus
lumbar ventral horns depended on the stage at which limb amputation was
performed. Prestige has suggested that lumbar motor horn neurons pass
through three temporally successive phases. Phase I neurons are insensitive to
the periphery; limb removal has no effect. When neurons enter phase II they
come under peripheral control and quickly degenerate if the limb is amputated.
Phase III neurons are not immediately affected by limb removal, but die after a
period of time. The older the animal at the time of amputation, the longer the
Forelimb amputation during development in Xenopus
455
phase III neurons survive. To account for this variation of response with time,
Prestige suggested that neurons receive and accumulate a 'maintenance
factor' from the limb and that they die only when their store of the factor is
exhausted.
In the present experiments forelimbs of Xenopus tadpoles or juveniles were
extirpated at five developmental stages and the response of the brachial ventral
horn was assessed histologically at various times after amputation. Since Hughes
(1968 a) reported that Xenopus forelimbs have extensive powers of regeneration, it was expected that regeneration would follow forelimb removal and that
a study of ventral horn nuclei reacting simultaneously to extirpation and
regeneration would further elucidate the mechanism of peripheral control of
ventral horn size.
MATERIALS AND METHODS
Experimental animals were the progeny of three matings of animals of the
Cornell Xenopus colony and were maintained at 22-24 °C in aged tap water.
Left forelimbs of animals at stages 52/53, 54, 58,61, and 66 were amputated as
close to the body wall as possible. Operations on stage-66 animals were performed on the day metamorphosis was completed. A small tear was made in
the operculum in order to gain access to the limb-bud at stages 52/53 and 54.
This wound healed within a few days.
Animals were anesthetized with M.S. 222 (tricaine methane sulfonate,
1:1000) and operations were performed in full strength Steinberg saline (Hamburger, 1960) to facilitate blood clotting. Animals were maintained in water
containing sulfadiazine sodium for several days after amputation. The loss of
the forelimb did not cause the animals any difficulties in swimming or eating
and all developed normally. Operated animals were observed closely for forelimb regeneration.
Animals were fixed in half-strength Bouin's at various times after limb
extirpation. Specimens were decalcified with Decal solution (Scientific Products)
for 3-12 h prior to dehydration and clearing. Serial sections (10 /on) of the
brachial region were cut and stained with Erlich's hematoxylin and eosin.
Motor neurons of the brachial ventral horn were counted in every third section
on both the operated and control sides of the spinal cord and the appearance of the cells was noted. The brachial region of the ventral horn is
located in the area of the second and third spinal ganglia (Kollros, 1956) and is
fairly well demarcated since organization of motor neurons into a definite
column projecting from the mantle layer occurs only at hind and forelimb
levels. Ventral horn neurons were identified by both position in the motor
column and appearance of the cells. Neurons were counted only if they met the
following criteria: nuclei paler and larger than those of mantle layer neuroblasts, presence of cytoplasm around the nucleus, and presence of a nucleolus.
Since at stages 52-54 neurons are still in the initial stages of differentiation, the
456
J. E. FORTUNE AND A. W. BLACKLER
cytological criteria could not be applied as rigorously at these stages. Presence of
a nucleolus and position in the ventral horn were the principal counting criteria at these earlier stages. The nucleolus is small in comparison to section
thickness (Prestige, 1967) and Jones (1937) concludes that if sections are
approximately 10 /im. thick, no correction need be made for split nucleoli if
only definite and distinct nucleoli are counted. Neuron counts included
chromatolytic cells. Chromatolysis is a neuronal reaction to axotomy that is
characterized by disappearance of the Nissl substance and is followed by a
recovery phase in which the nucleus is surrounded by a dense ring of basophilia (Prestige, 1967). Chromatolytic cells were identified by the basophilic
perinuclear ring.
The error introduced by the sampling procedure was estimated by complete
cell counts of three ventral horns which had 941, 457, and 51 neurons. The
average percentage error due to sampling for these counts was ±1-8 %, ± 9-0 %,
and ±15-7%, respectively. The corresponding coefficients of variation are
0-02, 0-10, and 0-18. Sample counts ranged from 2844 to 675 on the control
side and from 2557 to 46 on the operated side. On the control side the
sampling error was usually less than ±1-8% since most counts were greater
than 941. On the operated side the sampling error was less than ± 1-8% for
the largest counts and ±15-7 % for the smallest count. Although the sampling
error is large for the smallest counts, the size of the experimental change induced in these cases was much greater than the sampling error.
Degenerating neurons in the ventral horn were not classed as living cells,
but were counted separately. No sampling procedure was employed for these
counts; every section was examined for degenerating neurons.
RESULTS
Forelimb amputation and regeneration
There was no evidence of regeneration following the 128 forelimb amputations performed in the course of these experiments. Animals whose forelimbs
had been amputated at stage 58, 61, or 66 usually developed small bony stumps
at the site of amputation. In contrast, ablation at stage 52/53 or 54 resulted in a
slight indentation at the site of amputation.
Normal development of the brachial ventral horn
Normal developmental changes in size, appearance, and number of brachial
ventral horn neurons were determined by observations of control animals and
the contralateral sides of experimental animals. At stage 52/53 the ventral horn
cells were still in the neuroblast stage. The neuroblasts were grouped in a
distinct rounded mass at the lateral border of the ventral half of the mantle
layer (Fig. 1). No cytoplasm was visible around the closely packed, oval neuroblast nuclei. The nuclei had a long axis of about 11 jam, while the diameter of
Forelimb amputation during development in Xenopus
Fig. 1. A cross-section through the brachial spinal cord of a stage-52/53 tadpole. The
arrows indicate the brachial ventral horns, x 96.
Fig. 2. A cross-section through the brachial spinal cord of a stage-58 tadpole. The
arrows indicate the brachial ventral horns, x 60.
Fig. 3. A cross-section through the brachial spinal cord of an animal fixed 2 months
after forelimb amputation at stage 52/53. Note the absence of a ventral horn on the
operated side (right side of photograph), x 60.
Fig. 4. A cross-section through the brachial spinal cord of an animal fixed 3 months
after forelimb amputation at stage 58. The arrow indicates the ventral horn region
on the operated side; note the holes in the tissue in this area, x 60.
Fig. 5. A higher magnification of Fig. 4, showing the ventral horn area on the
operated side, x 378.
457
458
J. E. FORTUNE AND A. W. BLACKLER
Table 2. Normal development of the brachial ventral horn o/Xenopus laevis*
Age
Stage
(days)
52/53
22-5
54
55
56
57
58
59
61
62
63-64
66
26
32
38
41
44
45
48
49
52
58
Mean length
Number of ventral
of ventral horn region
horns
C«m)
3
5
2
3
3
5
1
5
2
2
7
680
780
830
860
950
1050
1070
950
960
960
900
Mean number of neurons
per side of spinal cordt
A
t
Living
2452 ±61
2576 ±269
2560 ±161
1768 ±283
1709 ±223
1523
1459 ±19
1402 ±27
1267 ±166
1070 ±35
Degenerating
\
l±0-7
6 ±2-4
5 ±0-5
38 ±17-0
20 ±2-1
29 ±11-6
23
19 ±3-4
16 ±20
11 ±0-5
l±0-3
* Data are based upon the ventral horns of normal animals and the contralateral horns of
experimental animals. Amputation did not affect neuronal number on the contralateral side.
t ±S.E.M.
mantle cell nuclei was about 7 /an. Nucleoli were slightly more distinct in
motor column nuclei than in mantle layer cells and were approximately 1-5/mi
in diameter.
The neuroblasts gradually developed into mature neurons between stage
52/53 and stage 57. The nuclei became round rather than oval, attaining a
final diameter of 11-14 /an, while the diameter of the mantle layer cells remained at about 7/tm. The motor cell nucleoli gradually enlarged to a diameter
of 3-4 fim, while the surrounding nucleoplasm became increasingly pale.
Cytoplasm was first observed around the nuclei at stage 55 and increased in
volume as the neurons matured. At stage 52/53 the motor column was a
rounded mass projecting from the mantle layer at an angle of about 80° from
the dorso-ventral vector of the central canal of the spinal cord (Fig. 1). By
stage 58 the neurons were arranged in a column projecting from the mantle
layer at an angle of about 45° from the dorso-ventral vector of the central
canal (Fig. 2).
Changes in the total number of neurons accompanied these changes in
size, appearance, and position of individual neurons. Table 2 and Fig. 6 A show
the average number of neurons in the brachial ventral horn at most stages from
52/53 to 66. While the length of the brachial ventral horn region increased from
680 to about 1000 jam as the forelimb developed, the number of ventral horn
neurons dropped gradually from a mean of 2576 (S.E.M. = ± 269, n = 2) at
stage 55 to 1070 (S.E.M. = ± 35, n = 7) at the completion of metamorphosis.
Counts continued to decrease slightly after stage 66 was reached and stabilized
at around 900 per side, although there was much individual variation. Thus, in
Forelimb amputation during development in Xenopus
140
120
(F) Stage 66
;oo
-5
0
+5
80
60
40
20
120
100
80
60
40
20
(E) Stage 61
120
100
80
60
40
20
(D) Stage 58
120
100
80
60
40
20
(C) Stage 54
+ 10
+5
0
-5
-10
+ 15
+ 10
+5
0
-5
-10
+ 60
+ 40
+ 20
0
-20
-40
(B) Stage 52,53 •
+ 60
+ 40
+ 20
0
-20
-40
120
100
SO
60
40
20
(A) Control
^
2500
S S 2000
t I 1500
£ c 1000
500
20
40
80
160
Age of animals (days)
320
Fig. 6. Numbers of living (
) and degenerating (
) neurons in normal
brachial ventral horns (A) and numbers of living (
) and degenerating (
)
neurons in brachial ventral horns on the side of forelimb amputation compared
to control ventral horns from the same animals (B-F). The arrows indicate the time
of forelimb amputation. Most points represent one animal; some points represent
the mean of 2-5 animals (see Tables 2-7).
459
460
J. E. FORTUNE AND A. W. BLACKLER
Table 3. Numbers of ventral horn neurons following for elimb amputation
at stage 52/53 (O = operated side, C = control side)
Number of neurons
Stage
Body length Days after
amputation
(mm)
52/53
—
0
52/53
37
1
54
38
3
54
38
5
55
43
7
56
49
9
56
46
11
57
61
14
66
22
25
66+
21
30
66 +
22
42
66+
22
60
66+
22
122
Living
O—
C—
0 —
£
O2277
C2361
O1896
C2316
O1733
C2844
O1071
C2238
O 738
C2690
O 410
C1215
O 273
C 991
O 180
C1062
O 46
C 975
O 135
C1147
O 55
C 812
\
Degenerating
2
2
28
0
11
2
19
0
65
5
10
19
40
65
11
17
1
2
0
3
1
0
0
0
0
0
noting and interpreting changes in neuron number caused by limb extirpation,
it must be remembered that the operated side is being compared to a progressively smaller control number.
The number of degenerating neurons observed in control animals was
initially very low (1-0 ± 0-7). Degenerations reached a maximum at stages 56-58
(38 ± 14-0-29 ±11-6) and then declined as metamorphosis approached (Table
2). Only a few cell degenerations were observed in normal ventral horns after
metamorphosis (Fig. 6 A).
Amputation at stage 52\53
The first quantitative difference in living cells between operated and control
sides occurred 5 days after limb amputation at stage 52/53 and by the end of the
first week the number of neurons on the operated side was 62% that of the con-
Forelimb amputation during development in Xenopus
461
trol side (Table 3; Fig. 6B). By the end of the second week only 34% of the
normal number of neurons was observed on the operated side. The changes
induced by ablation seemed to be complete by one month after the operation
when only 17% of the normal number of cells remained. During the first week
after amputation excess cell degenerations occurred on the operated side as
compared to the control side (Table 3; Fig. 6B). After the first week there were
fewer degenerating cells on the operated side as compared to the control.
Neurons on the operated side appeared less differentiated as compared to
those of the control side from the fifth day after amputation. By the eleventh day
there was no motor column as such on the operated side and this was true of all
animals killed after that time (Fig. 3). The spinal cord on the operated side was
slightly reduced in cross-sectional area by the ninth day; the size difference
between the two sides increased with the age of the animals.
Amputation at stage 54
Following limb amputation at stage 54, the first difference in numbers of
living cells was noted 3 days later when the operated side had 21 % fewer
neurons than the control ventral horn (Table 4; Fig. 6C). The relative difference
was 40 % by the end of the first week. The decrease in relative numbers was
completed within 2 weeks, when the operated side had only 18% as many
neurons as the contralateral side. For the first 5 days after amputation the
operated side exhibited a higher rate of cell degeneration than the control side;
but starting with day 7, cell degeneration was lower than on the control side
(Table 4; Fig. 6C).
Most neurons on the operated side were less mature by one or two stages
than those on the control side during the first week after limb extirpation.
After about 9 days there was no real motor column. The few cells that remained
on the side of amputation were as mature as those on the control side, but there
were not enough of them to form a column. By the twelfth day the operated
side of the spinal cord was slightly smaller in cross-sectional area in the brachial
region than the unoperated side and this difference was also seen in all animals
killed after the twelfth day.
Amputation at stage 58
The number of neurons on the operated and control sides remained approximately equal for the first 5 days following forelimb extirpation at stage 58
(Table 5; Fig. 6D). From the seventh through the thirteenth day, the number of
neurons on the amputated side was consistently higher than on the control side,
with a difference between the two sides of as much as 18%. By day 16 the
operated side had 84% as many neurons as the control side. A gradual decrease
followed and after 8 months the number of motor cells on the operated side was
36% that of the control side. From the third through the seventh day cell
degeneration on the operated side was lower than on the contralateral side;
462
J. E. FORTUNE AND A. W. BLACKLER
Table 4. Numbers of ventral horn neurons following forelimb amputation
at stage 54 (O = operated side, C = control side)
Stage
Body length Days after
amputation
(mm)
54
41
0
54
42
1
55
43
3
56
49
5
57
53
7
57
54
9
58
55
14
62
52
18
62
48
21
66 +
19
30
66 +
23
42
66 +
30
91
Number of neurons
Living
Degenerating
O2557
C2395
O2469
C2633
O1827
C2307
O1779
C2751
O 849
C2151
O 454
C1938
0 461
C2535
0 264
C1428
0 267
C1375
0 203
C1020
0 164
C1021
0 146
C 1115
7
5
30
14
47
4
37
29
12
24
14
19
34
75
5
18
7
14
1
2
0
2
0
0
but from day 10 through day 16 excess degeneration occurred on the operated
side (Table 5; Fig. 6D).
Some neurons on the operated side, particularly in the more posterior regions
of the brachial ventral horn, appeared to be in a state of mild chromatolysis
between day 3 and day 16. By 25 days after amputation chromatolysis was no
longer evident, but there were holes or gaps in the spinal cord in the area of the
motor column (Figs. 4, 5). Such vacant spaces were also seen in animals killed
after 25 days and were, for the most part, confined to the posterior half of the
ventral horn. The spinal cord on the operated side was smaller in cross-sectional
area in all animals killed from day 25 on.
Amputation at stage 61
Cell numbers were maintained at a normal level ipsilaterally during the first
week after forelimb ablation at stage 61 (Table 6; Fig. 6E). By the end of the
first week there was a small relative excess on the operated side. This suppression
Forelimb amputation
during development
in Xenopus
463
Table 5. Numbers of ventral horn neurons following forelimb amputation
at stage 58 (O = operated side, C = control side)
Number of neurons
Stage
Body length Days after
amputation
(mm)
58
—
0
58
68
0
59
66
1
61
58
3
63-64
29
5
64
20
7
66
20
10
66 +
19
13
66+
20
16
66 +
20
25
66 +
21
31
66 +
27
49
66 +
23
92
66 +
36
245
Living
Degenerating
O1707
C1678
O1332
C1293
O1518
C1523
O1458
C1427
O1461
C1432
O1471
C1336
O1194
C1015
O1164
C1059
O 861
C1023
O 313
C 929
O 457
C 941
O 487
C 962
O 393
C 921
O 309
C 848
20
20
14
15
25
23
13
26
3
11
2
12
11
0
9
1
6
1
0
1
0
0
0
0
0
0
0
0
of cell loss on the operated side produced an excess of 22% by day 18. However, in the following 2 weeks cell numbers decreased sharply on the operated
side. The loss continued at a slower rate in the succeeding weeks, yielding a
final cell deficit of about 60 %. Numbers of degenerating cells were similar on
the two sides except on day 3, when the operated side had a much lower rate,
and on day 18 when there were excess degenerating cells on the operated side
(Table 6; Fig. 6E).
Chromatolytic cells were first evident at the end of the first week and persisted
through the next several weeks. By the end of the first month chromatolytic
cells were no longer present, but there were a number of holes in the ventral
horn area of the spinal cord. These gaps were visible in all animals killed after
464
J. E. FORTUNE AND A. W. BLACKLER
Table 6. Numbers of ventral horn neurons following forelimb amputation
at stage 61 (O = operated side, C = control side)
Stage
Body length Days after
amputation
(mm)
61
—
0
61
65
0
63-64
31
3
66
18
7
66 +
21
18
66 +
19
31
66 +
22
49
66 +
24
77
66 +
22
153
66 +
33
184
Number of neurons
A
Living
Degenerating
O1407
C1470
O1519
C1471
O1085
C 1101
O1177
C1121
O 1388
C 1135
O 408
C 718
0 612
C 946
0 398
C 925
0 261
C 675
0 308
C 746
24
25
11
11
1
10
0
0
8
2
0
0
0
0
0
0
0
0
0
0
one month. The chromatolytic cells and holes were confined primarily to the
posterior half of the spinal cord.
Amputation at stage 66
There was no neuronal reaction to limb extirpation at stage 66 until the end of
the first week. At that time and in the next 3 weeks the operated side of the
spinal cord contained more neurons (2 %-34 %) than the control side (Table 7;
Fig. 6F). During the second month cell loss began on the operated side and
resulted in a deficit of 32-45 %. Numbers of degenerating cells were small in
both amputated and control ventral horns (Table 7; Fig. 6F).
Chromatolysis began during the first week after amputation and was still
evident after one month. Again, the posterior sections contained most of the
chromatolytic cells. Seven weeks after the operation chromatolysis had ceased
and there were many large holes in the posterior half of the ventral horn region.
The spinal cord on the operated side was smaller in the 7-week animal, but no
difference in cross-sectional area was evident in the two animals killed thereafter (at 3 and 5 months).
Forelimb amputation during development in Xenopus
465
Table 7. Numbers of ventral horn neurons following forelimb amputation
at stage 66 (O = operated side, C = control side)
Number of neurons
Stage
Body length Days after
amputation
(mm)
66
19
0
66
19
0
66 +
20
3
66 +
18
3
66 +
19
7
66 +
19
7
66 +
21
18
66 +
23
30
66 +
24
49
66 +
30
91
66 +
22
153
Living
Degenerating
O1016
C 976
O 1212
C 1158
O 954
C 954
0 900
C 927
O1122
C 879
O1009
C 755
O1125
CHOI
O1114
C 966
O 482
C 709
O 547
C 997
O 636
C 937
1
0
1
1
0
1
0
1
0
2
1
1
0
0
2
0
0
0
0
0
1
0
DISCUSSION
In our experiments Xenopus forelimbs never regenerated after total ablation
and thus, we cannot confirm Hughes (1968 a) report of the regenerative abilities
of Xenopus forelimbs. In the present experiments the indentations that formed
at the sites of amputations performed at stage 52/53 or 54 indicate that ablation
removed part of the prospective shoulder. Perhaps regeneration did not occur
after amputation at these stages because almost all the 'regeneration territory'
of the arm and shoulder was removed (Guyenot & Ponse, 1930). At later stages
(58, 61, and 66) complete limb ablation left the shoulder girdle intact, but by
these stages the animals were apparently incapable of regeneration after total
limb removal.
The pattern of normal development of the brachial ventral horn was similar
to that previously reported for the lumbar ventral horn. As individual cells
differentiated, brachial cell number fell from as many as 2576 neurons per side
to about 900 (Table 2; Fig. 6 A). Hughes (1961) and Prestige (1967) found that
30
EMB 36
466
J. E. FORTUNE AND A. W. BLACKLER
in the lumbar ventral horn a peak number of 3000-6000 neurons per side was
reduced to 1200-1600. The smaller number of motor neurons in the brachial as
compared to the lumbar ventral horn may be related to the smaller size of the
forelimb. The data suggest that reduction in cell number during development of
the brachial motor column is caused by cell degeneration. Degenerations were
first seen in appreciable numbers just after the peak in cell numbers was reached
(Table 2; Fig. 6A). Large numbers of degenerating cells were present only
during stages 56-64, the period of rapid cell loss. The size and appearance of
brachial motor neurons during development were similar to descriptions
previously given by Hughes (1961) and Kollros (1956; 1968) for the lumbar
motor column.
The number and appearance of ventral horn neurons on opposite sides of
the spinal cord were always very similar in unoperated animals. The final result
of limb extirpation at each of the five stages studied was loss of a considerable
number of motor horn neurons on the side of operation. Thus, in Xenopus
laevis many brachial ventral horn neurons depend upon the forelimb for
maintenance.
The pattern of cell loss and the appearance of brachial motor neurons varied
with the stage at which amputation was performed. The older the animal at
amputation, the smaller was the final cell deficit (Fig. 6B-F). These differences
are probably related to the amount of prospective limb and shoulder tissue
removed at each stage, since extirpation was more complete at earlier stages.
The timing of cell loss also varied with the stage at amputation. The decrease in
cell number following ablation at stage 54 occurred from days 3-18, while
operation at stage 52/53 produced cell loss from day 5-30. Thus, although
amputation at these two stages resulted in about the same percentage final cell
loss, the response was quicker following amputation at stage 54. Amputation at
stages 58, 61, and 66 produced a period of relative excess in cell numbers on the
operated side. The excess did not result from an increase in cell numbers but
reflected a slower rate of cell loss from the operated side as compared to the
control side which continued to lose cells at the normal rate. The older the
animal at amputation the longer was the period of relative excess: stage 58 until day 13; stage 61 -until day 18; stage 66-until day 30 following amputation. In these three experimental series the period of relative excess was
followed by a period of cell loss on the operated side. The length of the period
of cell loss increased as the age of the animal at operation increased. Cell loss
following amputation at stages 58, 61, and 66 was complete by days 25, 31, and
49, respectively (Tables 5, 6, 7; Fig. 6D, E, F).
The ipsilateral changes in cell number seen after amputation can be accounted
for by changes in the number of degenerating cells. Excess cell loss coincided
with excess cell degeneration. Following amputation at stages 52/53, 54, and 58
cell degeneration was higher on the operated as compared to the control side
during the same times when neuronal numbers were decreasing on the operated
Forelimb amputation during development in Xenopus
467
side (Tables 3, 4, 5; Fig. 6B, C, D). When amputations were performed at
stages 61 or 66, excess cell degenerations occurred on the operated side just
prior to excess cell loss, but the number of degenerating cells was too small
to be conclusive (Tables 6, 7; Fig. 6E, F). However, the holes or gaps seen in
ipsilateral spinal cord tissue just after excess cell loss on that side give indirect
evidence that excess cell degenerations were the mode of cell loss (Hughes,
1961).
The relative neuronal excess on the operated side following extirpation at
stages 58, 61, and 66 can also be attributed to differential rates of cell degeneration. In the stage-58 and -61 groups there were many fewer degenerating cells
on the operated side just prior to the period of relative excess on that side
(Tables 5, 6; Fig. 6D, E). In the stage-66 series the numbers of degenerating
cells were again too small to be conclusive.
Loss of the forelimb affected the appearance of individual motor cells as
well as causing changes in cell number. Motor cell differentiation appeared
retarded as compared to the control side after amputation at stages 52/53 and
54. This effect appeared more rapidly after amputation at stage 54 (day 1) than
after amputation at stage 52/53 (day 5). Limb ablation at stages 58, 61, and 66
caused many motor cells on the operated side to become chromatolytic during
the periods of relative cell excess on that side. The fact that chromatolytic
neurons, and also the gaps in spinal cord tissue that were seen after excess cell
loss in the stage-58, -61, and -66 series, were confined to the posterior half of the
brachial ventral horn suggests that these phenomena are related to excess cell
loss since cell loss following amputation at stages 58, 61, and 66 was heaviest in
the posterior sections of the brachial motor horn.
The results demonstrate that the response of the brachial ventral horn to
limb removal varied with the stage at amputation. At stage 52/53 the neurons
seemed to exist independently of the limb since the effects of amputation (cell
loss and retardation of development) did not appear until several days after
amputation at stage 52/53. In contrast, cell loss and delay of differentiation
began almost immediately after forelimb ablation at stage 54. Thus, by stage 54
the brachial motor neurons were under the control of the periphery and depended upon it for survival. At stages 58, 61, and 66 the ventral horn was also
under peripheral control, but limb removal at these stages caused an initial
chromatolysis and suppression of normal cell loss followed by a later decrease in
ventral horn neurons. These results indicate that the cell degenerations that
normally occur in the brachial ventral horn during stages 58-66 are under
peripheral control. So while the limb is necessary for the differentiation and
maintenance of some brachial motor cells, it is necessary for the degeneration of
others.
The results of our experiments agree in general with Prestige's (1967) study of
the lumbar ventral horn. His division of motor neuron development into three
phases is consistent with the results obtained for the brachial ventral horn. If
30-2
468
J. E. FORTUNE AND A. W. BLACKLER
Prestige's phases of neuronal development are applied to the brachial motor
column, all brachial motor neurons are in phase I prior to stage 54. Brachial
neuroblasts enter phase II between stage 54 and 57. By stage 58 brachial motor
cells have become mature phase III neurons. Prestige's hypothesis that the limb
supplies phase III ventral horn neurons with a progressively accumulated
maintenance factor can explain delayed cell loss following forelimb amputation
at stages 58, 61, and 66, but does not explain why normal cell loss was halted
by forelimb removal at these three stages.
The results presented in this paper suggest that the initial migration and
differentiation of brachial ventral horn neurons are not under peripheral control. Between stages 54 and 57 the neurons appear to enter a phase of development in which they are dependent on the limb for further differentiation and
survival. The results thus support the concept that the development of motor
neurons involves both autonomous and dependent differentiation.
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{Received 28 January, revised 26 July 1976)