/ . Embryol. exp. Morph. Vol. 17, 3, pp. 453-71, June 1967
Printed in Great Britain
453
The control of
cell number in the lumbar spinal ganglia during
the development of Xenopus laevis tadpoles
By M. C. PRESTIGE 1
From the Department of Zoology, Bristol University
It is the purpose of this paper to describe the development of the lumbar
dorsal root ganglia after amputation of the leg. This operation can be performed
at a very early stage before any connexions between the limb and the central
nervous system are established. Alternatively, it can be performed at a number of
later stages after the limb has been innervated. The extent of interaction can then
be investigated for each stage by observing the subsequent development of the
ganglia and comparing it with that of normal animals.
Amputation of the limb-bud or the growing leg results in partial removal of
the peripheral field for both sensory and motor neurones; the operation thus
provides a means of investigating the mechanisms that control the processes of
proliferation, maintenance, and degeneration of nerve cells. Detwiler and his
colleagues (Detwiler, 1933) have shown that in Amblystoma loss of cells from the
ganglia (hypoplasia) follows amputation, and that increase in number (hyperplasia) follows grafting of a supernumerary limb. In the chick embryo, the
analysis has been taken further (Hamburger, 1934, 1939; Levi-Montalcini &
Levi, 1942; Bueker, 1943, 1944, 1945 a; Hamburger & Levi-Montalcini, 1949)
to demonstrate that the periphery both 'controls the proliferation and initial
differentiation of undifferentiated cells which have no connexions of their own
with the periphery' and also 'provides the conditions necessary for continued
growth and maintenance of neurons in stages following the first outgrowth of
neurites'. Piatt (1948) has reviewed the evidence on the other groups of vertebrates. Among the anurans, May (1930, 1933) has shown that spinal ganglion
cells in limb regions are dependent on the periphery for normal development in
Discoglossus. In Xenopus, Hughes & Tschumi (1958) have reported the effects
of removal of the early limb-bud by chemical or surgical means. This causes a
slowing of the rate at which new neuroblasts are recruited within the ganglia.
The presence in normal development of degenerating cells (histogenetic
degeneration—Gliicksmann, 1951) has been observed in chick ganglia at trunk
levels (Hamburger & Levi-Montcalini, 1949) and in all ganglia of the Japanese
1
Author's address: Department of Zoology, University of Bristol, Bristol 8, U.K.
454
M. C. PRESTIGE
quail (Palladini, 1961). Their numbers at each stage have been counted in the
trunk and lumbar ganglia of Xenopus (Prestige, 1965); here their number is so
great that for every spinal ganglion cell that matures, some two or three degenerate. We do not know what controls the rate of cell death.
Experimental amputation of the early limb-bud has already been performed
by Hughes & Tschumi (1958) as described above. Here the effect of amputations
later in development on the numbers of ganglion cells and the cell death rate will
be reported, and the effects on cell proliferation will be calculated. In one respect,
the analysis is simplified as the lumbar spinal ganglia of Xenopus do not contain
any large, early differentiating, ventro-lateral neurones, such as are found in the
chick embryo (Levi-Montalcini & Levi, 1942), but instead are all of the same
class (Hughes & Tschumi, 1958).
MATERIAL AND METHODS
Larvae of Xenopus were reared from eggs obtained from adults injected with
gonadotrophins (Nieuwkoop & Faber, 1956). They were kept at temperatures
of between 18 and 23 °C. One leg of tadpoles and juveniles was amputated as
high in the thigh as possible, using scissors or forceps with the animal under
M.S. 222 anaesthesia (1:4000). The other side served as a control. Provided the
water was kept clean, the animals survived well until metamorphic climax.
At this stage tadpoles begin to swim with their legs and cease swimming with
their tails. With only one leg they drown in deep water. If the level is kept so low
that they can expose their mouths above water without swimming, they survive.
The water level can be raised again over the next week or two as they learn to
swim with one leg. This procedure must also be followed after amputating the
legs of juveniles for the same reason. The animals were kept in tap water at all
times, without antibiotics.
Selected specimens were fixed in half strength Bouin; serial paraffin sections
were cut at 10 /i transversely and stained with haematoxylin and eosin. Each
ganglion occupied 30-60 sections and counts were made on every third section.
The ganglia that supply the hind leg in Xenopus are S. 8, 9, 10. Neurones were
identified by the cytoplasm and the presence of the single conspicuous nucleolus.
In each section, only those neurones were counted in which a nucleolus was seen.
This body is small compared with the section thickness and no correction of the
count for duplicate scorings was applied. Degenerating nerve cells were identified
by the presence of a pycnotic nucleus with basophilic cytoplasm, as described by
Hughes (1961) and Prestige (1965). Neurones in chromatolysis were included in
the living group, unless they also had pycnotic nuclei.
The animals were staged according to Nieuwkoop & Faber (1956). The relevant stages are here summarized:
Stage 49 Limb-bud round.
Stage 53 Limb at palette stage. Degeneration is nowfirst seen in the ganglia.
Cell number in spinal ganglia
455
Stage 54 Movement in limb begins.
Stage 58 Fore limbs emerge from pouch.
Stage 61 Tail degeneration begins.
Stage 66 Juvenile.
Limbs were amputated either at stages 53-55, stages 57-59, stage 61, or at
stage 66. The observations in each series are separately described.
In the present work, the sampling procedure adopted was that every third
section throughout the series was counted. Total sample counts of between
2000 and 5000 living cells were recorded; for these, the size of the experimental
changes induced always exceeded the limits expected due to random sampling.
For degenerating cells, counts of between 0 and 250 were recorded; as estimates
of the population mean, the lowest sample counts, if taken from only one individual, may be as much as 100 % in error. However, the error can be decreased
by pooling results from more than one animal, and this has been done where
possible. In addition, rank and sign tests of significance have been used, because
of their great simplicity (Siegel, 1956).
OBSERVATIONS
Series I
Amputation at stages 53-55
Forty-one animals in the early stages of prometamorphosis were used. The
hind limbs at operation measured between 3 and 5 mm long. During the first
week after amputation there is first a depression of the rate of degeneration and
then subsequently a large increase. The latter is much more conspicuous and
results in the loss of half the cells in dorsal root ganglia 8-10. In Fig. 1, the difference between the number of cells on the two sides is plotted against the time
of fixation after amputation. Between 1 and 8 days, each of 22 animals shows a
deficit of cells on the amputated side; the probability that these differences in
number should all be of the same sign is 2~21, which is highly significant.
To calculate the changes in proliferation rate (or more generally, in 'production' rate) and to estimate the significance of the changes in degeneration
rate, the animals have been separated into two groups, as the points of Fig. 1 lie
widely dispersed indicating a variety of rates of cell loss. The division has been
made on the basis of the number of cells in the control ganglia. In the group of
animals in which this count is below 7000, the points indicate one response, and
in the group in which it is above 8000 they show a quantitatively different one;
there is some overlap between 7000 and 8000 (Fig. 1). There is no correlation
between these two responses and the developmental stage of the tadpole at
amputation; they are probably related to the rate of development, which may
vary widely. In both groups, however, the period of excess degeneration on the
operated side is marked by a relative decline in cell number on the same side.
The animals that have less than 7000 cells in the control ganglia show an
456
M. C. PRESTIGE
accumulating cell deficit on the operated side throughout the first week; at the
end they have lost 3000 cells (47 %). Degenerating cells in significant excess of
those present on the control side can be observed (Table 1) on the amputated
side from the second day onwards (t test, P < 0-04). The degeneration rate on
the control side is lower than normal in the period 2-5 days after the operation.
The depression is significant compared with both sides of two normal animals
at the same stage with similar numbers of cells (t test, P < 0-02). Its cause is
3?
0
2
-
»
8
o
o
o
0
o
£> 2000 -
•
C/J
8
°
•
o
0
4000 h
x
W
o
•
•
6000 —
1
10
5
Days
Fig. 1. Early effects of amputation on ipsilateral lumbar dorsal root ganglion cells.
The difference between the two sides is plotted against the time of fixation after
operation. Stages 53-55. All examples, irrespective of rate of development. Open
circles, animals with less than 7000 cells in the contralateral ganglia. Filled circles,
animals with more than 8000 cells in the contralateral ganglia. Half-filled circles,
animals with between 7000 and 8000 cells in the contralateral ganglia.
Table 1. Amputation of hind leg at 3-5 mm (stages 53-55).
Slow developers. Initial period of cell loss
DRG 8 + 9+10
Animal
16/4
24/10
17/4
MIA
16/10
24/10
26/10
Time
after
amputation
Body
length
(mm)
22 h
1d
46 h
53 h
39
—
40
37
—
42
4d
5d
7d
44
Limb
length
(mm)
—
—
—
—
—
Control
f
A
Live
Degen.
60
60
5
6410
6550
6020
6970
6020
6220
4-2
6150
54
33
21
3
35
•\
Amputated
(
Live Degen.
5890
5650
5600
6200
4670
5520
3220
24
15
135
42
75
153
106
Cell number in spinal ganglia
457
unknown. At 24 h, there is no depression on the control side but the two operated
individuals both showed depression of degeneration rate on the amputated
side, prior to the increase which follows at day 2. This result is not significant on
its own, but it will be shown later that this initial decrease of degeneration on the
amputation side is a general phenomenon.
The second group of animals consists of those in which the total number of
cells in the ganglia on the unoperated side is larger than 8000. In these (Table 2),
there is again progressive cell loss of 4000-5000 cells on the side that is amputated
but it is terminated by the fourth day, after which numbers remain steady. The
tadpoles thus again lose about half the available cells (44,44, 56 %). In addition,
degenerating cells in excess are seen on the amputated side starting at about
24 h with a very high peak, but tailing off on the subsequent two days. This
excess is significant (sign test, n = 8, P = 0-008).
Table 2. Amputation of hind leg at 3-5 mm (stages 53-55).
Fast developers. Initial period of cell loss
DRG 8 + 9 + 10
Animal
16/4
16/8 A
19/11
16/4
20/11
25/10
21/1
21/11
15/11
15/10
Body Leg
length length
(mm) (mm)
48
44
—
42
45
—
41
43
39
37
—
3
—
4
—
—
3-5
2
3-5
Control
Stage at Time after
fixation amputation
55-7
54-5
—
56
53-4
57
55-6
53-4
53
54
13 h
18h
Id
29 h
2d
2d
2d
3d
3d
3d
Live
Degen.
10140
10270
9980
8900
10040
9080
9250
9760
9380
8200
12
54
72
12
57
12
54
39
84
9
Amputated
Live Degen.^
9790
10130
7490
8320
5600
7850
7150
5080
5140
5170
0
51
852
48
273
42
534
142
96
66
The number of degenerating cells on the amputated side is lower than on the
control side at 12-18 h (Table 2), that is, before the excess period starts; this
again illustrates the phenomenon seen in the previous group. During the first
week after amputation the rate of degeneration is sometimes depressed, relative
to normal animals, on the control side but this is inconstant and not statistically
significant.
Thus in both groups amputation is followed, in the first place, by a short period
in which the rate of degeneration is depressed ipsilaterally, and then by a decline
in cell numbers ipsilaterally starting 24-48 h after the operation and continuing
for 3-7 days. This is associated with an excess of degenerating cells on the same
side. In the second group, however, these processes appear to take place at about
twice the rate that they do in the first.
The further development of animals operated upon at stages 53-55 will be
29
J'KE M
17
458
M. C. PRESTIGE
considered without subdivision. Due to the wide disparity in rates of development,
the individuals can be arranged either by stage or by time after amputation.
The results have been plotted in Fig. 2 by stage against the outlines of the normal
variation in numbers (Prestige, 1965 and unpublished observations). Each circle
represents the number of cells on the control side. The associated figure is the
number of days after amputation. Fig 2 includes those animals fixed during the
first week, in which the excess degeneration due to amputation had ceased.
Living Degenerating
Contralateral
Ipsilateral
15 000 r
10 000 -
5 000 200
CO
8
100 g
Q
54
56
58
60
62
64
66
Stage
Fig. 2. Amputations at stages 53-55. Further development. Numbers of living and
degenerating cells in DRG 8 + 9 + 10 plotted against stage at fixation, irrespective of
the elapsed time since operation, which is indicated by the attachedfigures.The pairs
of dashed and continuous lines bound the range of observations in control animals,
for numbers of living and degenerating cells respectively.
It can be seen that all the data on cell number on the control side lie within
the range of normal variation; the rate of degeneration is depressed, but not
significantly so (t test against one side of eleven control animals matched for
stage, P > 0-6) All the cell counts on the amputated side lie below the normal
Cell number in spinal ganglia
459
range (horizontal bars, Fig. 2). This is significant (sign test, P < 0-02). During
the first 2 weeks, cell numbers may only have fallen to 6000 but by the third
week there must have been a second loss of cells, because only a final total of
between 2000 and 4000 remains. In the one juvenile animal examined, the
proportion of cells lost was 54 %. At no time is there any evidence of renewed
increase in cell number. Thus we may say that amputation of the leg causes an
irreversible decline in lumbar ganglion cell number. Presumably the remaining
cells are either those that innervate the proximal portion of the thigh and the
pelvic musculature, or are cells which have not yet developed their axons.
Table 3. Amputation of hind leg at 3-5 mm (stages 53-55).
Further development
Animal
Days after
amputation
16/11
4
23/11
5
25/11
7
19/10
7
27/10
8
9/11
13
22/4
15
16/11
20
25/4
18
25/4
18
30/4
23
8/2
44
No. of cells No. of de%of
cells
inDRG generating
cells
8 + 9+10
degenerating
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
Amputated
7120
4190
10620
5980
12270
6840
7950
3520
9230
5200
21
9
129
87
27
12
27
24
78
51
Control
Amputated
Control
Amputated
Control
Amputated
8720
6620
8210
3280
6140
3470
36
60
45
42
33
87
0-41\
0-91
0-55
1-28 'Second loss
0-54
2-54>
Control
Amputated
Control
Amputated
Control
Amputated
7510
3320
13060
2190
9370
3500
6080
2810
12
0
9
3
69
27
0
3
016
0
007
01
0-73
0-77
0 \ meta0 1 / morphosed
Control
Control
Amputated
. 0-28
0-21
1-22
1-44
0-21
018
0-34
0-68
0-84
0-99
In Table 3 the numbers of degenerating cells counted are expressed as a
percentage of the number of living cells. During the first week (4-8 days), and
after the third week (18 days onwards) after amputation, the percentage degeneration rate on the two sides remains the same, notwithstanding the large
differences in cell number. However, in the third week (13, 15, 20 days), that is
29-2
460
M. C. PRESTIGE
during the period when the second loss of ganglion cells must take place, the
percentage degeneration rate on the amputated side rises to at least double that
of the contralateral side. This increase is due to a temporary elevation of the %
degeneration rate on the amputated side (Mann-Whitney rank test, P = 0-042)
rather than a depression contralaterally {t test, P > 0-1).
Since on the amputated side the numbers of degenerating cells are so much
lower than contralaterally (except during the relatively short periods of first and
second loss), the deficit in the number of living cells would be gradually made
good if equal rates of cell production were maintained on the two sides. The
deficit is, however, not made good; therefore the rate of cell production must
also be depressed. When the absolute total does not change, the rate of cell
production must be equal to the rate of cell degeneration both of which are
depressed relative to the control side. These two effects can be spoken of together
as a reduction in the rate of cell turnover. The depression of cell production after
amputation at these stages represents a continuation of the same mechanism
discovered by Hughes & Tschumi (1958) on the limb-bud stages.
Thus at the cellular level, except in the two relatively short periods when cell
numbers are declining, the probability of a single cell dying at any instant is
unaffected by amputation (the % degeneration rates on the two sides are the
same). The effect of amputation is to reduce the size of the population for which
this is true. This reduces the total rate of cell degeneration. At the same time, the
rate of new cell production is diminished as well.
Table 4. Amputation of the hind leg at stages 57-59 {emergence of arms)
Animal
Body
length
(mm)
Leg
length
(mm)
Days after
amputation
Stage
23/5
58
9
2
59
12/10
54
10
4
59
28/10
51
—
5
59
15/8
—
—
5
62
DRG8 + 9 + 10
,
'«.
x
Live Degen.
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
7610
7250
7780
8170
6770
5930
9070
10050
42
45
45
72
24
63
12
12
A
u „•
4. ,
c? ™
Series II
Amputation at stages 57-79
Four animals were operated upon at this stage (Table 4) and were fixed at 2,4,
5 and 5 days. In two, a small excess of cells on the amputated side was counted
(less than 1000 cells) and in two a small deficit (less than 900 cells). In three out
of the four, there was a small excess of cells degenerating on the amputated side.
At this stage, from the first, excess degeneration must be balanced by excess cell
production.
Cell number in spinal ganglia
461
Series III
Amputation at stage 61
Seventeen animals were used in this series. At stage 61, tadpoles are about to
enter metamorphic climax. The tail is still used for propulsion but the hind legs
(length 10-15 mm) assist with vigorous, bilateral kicks. Within the next 6-10
days, metamorphosis will be completed. In this period, the number of cells in
Stage
64
65
61
• Contralateral
1 Ipsilateral
66
12 000
10 000
8
<
T>
8 000,
•Jj 6 000
1
I
I
I
1
1
1
•
I
4 000
1
^
2 000
CD
Contralateral
<U
Q
8
I
&
Ipsilateral
10
20
30'38"
62 " 1 1 9 " 173
Days
Fig. 3. Amputation at stage 61. All examples, plotted against time of fixation after
operation. A. Counts of living cells on control side and on amputated side. The
continuous lines bound the range of observations in control animals. B. Counts of
degenerating cells on control side. C. Counts of degenerating cells on amputated side.
ganglia 8 + 9 + 10 declines from between 7000 and 10000 to between 6000 and
9000, and the number of degenerating cells at any instant falls from nearly
100 to zero.
In Fig. 3A the data on cell numbers have been plotted together with the
boundaries of the normal variation (Prestige, 1965 and unpublished counts)
against the time after amputation. The solid circles mark the number of cells in
the control ganglia and the attached horizontal bars the number on the amputated
462
M. C. PRESTIGE
side. It can be seen that 14 out of 17 of the former are within the normal range.
Since this was constructed from only five individuals, the deviation is not
significant. The number of degenerating cells on the contralateral side falls
within 8 days nearly to its expected zero, where it remains (Fig. 3B, open circles).
Due to the rapidly falling numbers in control animals, it is not possible to test
the significance of this, but apart from a possible depression on day 1, there is
no obvious departure from normality.
During the first 25 days there is no consistent difference in cell numbers
between the sides (Fig. 3 A). After this, the number of cells on the amputated
side declines until, at 2 months, half the cells have been lost. The resulting figure
is then maintained. Thus once again, the proportion of cells that the amputated
limb can no longer support is about half the original (54, 50, 59 %).
On the first 2 days after amputation the rate of degeneration on the operated
side is depressed below that of the opposite side (Fig. 3C). The former rate,
however, is significantly greater than on the contralateral side from 5 days
onwards (sign test for period 5-21 days, seven animals, P = 0016). After this,
a very low level of cell degeneration may continue for as long as 4 months. Thus
during the period 5-25 days there is a mean excess of 10 degenerations on the
operated side at any instant, without any deficit in numbers of viable cells
compared with the unchanged opposite side. Therefore, as after amputation at
stages 57-59, there must be an equivalent excess rate of production of new
Table 5. Initial depression of degeneration rate and cell production
after amputation. All stages
DRG 8 + 9 + 10
Control side
Amputated side
Time after
Animal amputation Number
Degen.
/o
22 h
Id
12 h
13 h
18 h
Id
2d
Mean
60
60
48
12
54
15
66
45
0-94
0-92
0-65
01
0-53
0-2
0-77
0-59
16/4
24/10
25/1
16/4
16/8 A
11/3
9/12
6410
6550
7420
10140
10270
7430
8510
8104
Residual
/o
Number number Degen. Residual
5890
5650
7430
9790
10130
7000
8790
7811
3200
3200
5100
5100
5100
3500
3500
24
15
27
0
51
9
30
22
0-75
0-47
0-53
0
100
0-3
0-86
0-56
neurones. However, it has been shown that on the control side there is only a very
small number of degenerating cells (mean 1-8 per side after 8 days) and the numbers remain unchanged. Therefore the production rate on the control side is also
minimal and the excess production rate observed on the amputated side must be
near the absolute rate.
The depression in this series on the first 2 days may be compared with the
Cell number in spinal ganglia
463
initial depression after amputation at stages 53-55. Taken together, these seven
examples allow the conclusion that immediately after amputation and before the
excess degenerations start, there is a phase when the rate of degeneration on the
amputated side is depressed significantly compared with the contralateral side
(sign test, seven individuals, P = 0-016). If this is unaccompanied by any change
in relative rates of cell production, the numbers of cells will be increased on the
amputated side. However, Table 5 indicates that there is a small mean deficit,
which is, however, not significant (t test, P > 005). Therefore the rate of
production of cells must also be depressed by an equivalent amount to the rate
of degeneration; this once again represents a reduction in turnover. It should
also be pointed out that the early depression of cell production that follows
amputation represents a continuation to stage 61 of the same effect as Hughes &
Tschumi (1958) discovered for limb-bud stages, and as that observed in this work
for the later period after amputation at stages 53-55.
Table 6. Amputation 1-2 months after metamorphosis
DRG8 + 9+10
Animal
Days after
amputation
4/3 A
2
4/3 B
2
22/3
3
23/3
4
3/4
7
7/4
11
15/10
25
1/8
127
Number
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
Control
Amputated
7700
8740
6070
6160
8120
7770
8140
8510
7690
7730
6540
6890
6880
7070
7920
5410
Degen
0
0
0
0
0
0
0
0
0
0
0
18
0
18
0
15
Series IV
Amputation 1-2 months after metamorphosis
In this series, eight young juvenile toads were operated upon 1-2 months
after the tail had been lost. Turnover in the ganglia ceases after metamorphosis;
no degenerating cells are normally visible and cell numbers are constant
(Prestige, 1965). For the first month after amputation (Table 6) there is no
decrease in cell numbers. The apparent increase is not significant (mean 261,
t test, P > 0-4). However, by 4 months, 2500 cells have been lost.
464
M. C. PRESTIGE
No degenerating cells were seen until the eleventh day; after this they continue
to appear for at least 4 months. However, the number of cells does not start to
fall until after day 25, so again, as after amputation at stages 57-59 and at
stage 61, cell production must initially be taking place at the same rate as cell
loss. It is probable that the figure of 5410 at 127 days is not the lowest level
reached, for the rate of degeneration is still substantially elevated.
Early depression of degeneration on the operated side
It is interesting to consider whether the early depression of cell turnover,
which takes place before the period of excess degeneration following amputation, reflects the same mechanism as the later depression of turnover found in
animals operated on at stages 53-55. In the latter, the absolute rate of degeneration varies with the absolute number of ganglion cells present; in the former
instance, the neurones that are destined to die shortly afterwards are also present.
These have to be allowed for. The hypothesis under test is, then, that the absolute
number of cells degenerating in this early period is a function of the residual
number of cells that the limb will be able to support, and not of the number of
cells in the ganglia at that time. These residual totals are, respectively, 3200 for
the animals from Table 1, 5100 for those from Table 2, and 3500 for those amputated at stage 61. In Table 5, these values have been inserted and % degeneration
rates calculated. If the hypothesis is to be upheld, these should be equal on the
two sides. It can be seen that they are not markedly different, supporting the
hypothesis.
DISCUSSION
The unoperated side of the tadpole provides a valuable control for the effects
of amputation. While some effects have been observed there, it is likely that their
cause is also acting similarly on the operated side. The differential effects induced
by the experiment may thus be thought of as those occurring only on the amputated side (Table 7).
Before turnover starts (before stage 53), amputation causes only a decreased
rate of production of cells (Hughes & Tschumi, 1958). When turnover is taking
place (that is, from stages 53-65), amputation decreases both the rate at which
cells are lost by degeneration and also the rate at which new cells are produced.
When these are equal, cell numbers remain steady. Superimposed on this are
periods of excess degeneration which lead to a decline in cell numbers. These
periods may be short and immediate, as after amputation at stages 53-55, or they
may be prolonged and delayed, as after stage 61. After turnover ceases, amputation causes only a decline in cell numbers which is delayed and prolonged. At
stages after 55, excess cell degeneration is temporarily accompanied by an
equivalent excess cell production.
465
Cell number in spinal ganglia
The control that the leg exerts on the cells of the dorsal root ganglia
It has been shown that there are three processes in the ganglia 8 + 9 + 10
which are affected by the removal of the leg.
(1) The production of cells. Amputation at limb-bud stages (Hughes &
Tschumi, 1958), at stages 53-55, and initially at stage 61, causes the rate of production of cells to be lowered. Hughes & Tschumi postulated that some factor
Table 7. The effect of amputation on cells of the dorsal root ganglia
Amputation
at stage
Days
50
(from Hughes &
Tschumi, 1958)
53-55
(57)
61
Juvenile
0
20
30
40
80
Rate of
production
Slowed 6d ->
Depressed Id -*•
Depressed \-2d
Elevated 5-25d
Elevated \\-25d
Rate of
degeneration
Unaffected
Depressed Id -+
with 2 intense
excess periods
in 1st and 3rd
weeks, superimposed
Depressed \-2d
Elevated 5-62d
Elevated \\d-+
(at least
4 months)
Total cell
numbers
Rate of increase
slowed
50% lost in 2
50% lost
between 25
and 62d.
Decline 2Sd. ->•
(still in progress
at 4 months)
Dorsal root
ganglia in
control animals
periods in 1st
and 3rd weeks
TURNOVER
CELL PRODUCTION
CELL DEGENERATION
CELL NUMBERS RISING—FALLING—UNCHANGING
from the limb-bud was normally maintaining the higher rate of production. The
present experiments indicate a similar mechanism continues as far as stage 61,
but not into juvenile life. However, since ganglion cells are present before the
limb-bud can be recognized, the earliest neurones must be produced independently of the limb.
(2) The maintenance of cells. Amputation at all stages after 53 leads to an
irreversible fall in the number of neurones. This may be immediate or delayed
several months. Therefore, in some way the limb is normally preventing these
cells from degenerating.
(3) The death of cells. Amputation, in addition to inducing the extra degenerations which lead to a fall in the number of neurones, causes a depression
in the absolute rate of histogenetic degeneration, to a degree similar to the
466
M. C. PRESTIGE
decrease in cell number. Therefore, in some way the limb normally maintains
the higher rate of cell degeneration.
Some or all of these factors may be identical. The problem is to decide which
are the primary independent processes and which are only secondarily dependent.
The number of cells in the ganglia is necessarily determined by the equation:
Number of neurones = number produced - number which die
There can only be two independent variables; the third must be dependent.
There is also a requirement that the production term is one of the primary
processes, because its actions are earlier than the others. This is satisfied by the
evidence, which in Hughes & Tschumi's experiment shows that the production
process is operating in the absence of the other two (there is no cell loss and no
histogenetic degeneration).
Therefore, of the two remaining, either the process which controls cell
number is dependent on that which determines the rate of degeneration, or vice
versa.
A. If the degeneration rate is directly controlled, then a further hypothesis
is required to explain the decrease in cell number and the excess degenerations
after amputation. It would be possible to ascribe these to a 'traumatic' action on
the nerve: but it is difficult to see how 'trauma' could cause nerve cells to degenerate after a lag of up to 4 months. Conversely, this hypothesis does not
predict an increase in ganglion cell number with a supernumerary graft. This
experiment has not been done on Xenopus, but in other anurans there is evidence
that hyperplasia can be induced: May (1933) briefly reported it in Discoglossus
and Bueker (1945/)) found that in a specimen of Rana with three functional
right legs, there was hyperplasia of over 70 % in ganglia 8 + 9 on the affected
side.
B. If the number of cells in the ganglia is directly controlled, then the rate of
degeneration is determined by the rate at which production causes the number
that can be maintained to be exceeded. Since production itself is directly controlled, amputation will decrease both production and the number that can be
maintained, causing the excess cells to be redundant and ultimately die. This
hypothesis does predict an increase in number of ganglion cells with a supernumerary graft.
Hypothesis A does not explain the facts, while hypothesis B does and renders
hypothesis A unnecessary. It is therefore concluded that after stage 53 some
factor from the limb is directly required to maintain the cells in the dorsal root
ganglia, and that redundant cells die.
The necessary link between changes in degeneration and changes in production can be seen in the early depression of both after amputation at stages 53-55
and also at stage 61, and after excess degeneration has ceased after amputation
at stages 53-55. It can also be seen in the later elevation of both that follows
amputation at stages 57-59, 61, and as juveniles.
Cell number in spinal ganglia
467
The route by which causative factors reach the ganglia has not been specified;
it is ipsilateral only, which argues against a circulating hormone, and in favour
of an intra-axonal transport system.
How does the limb maintain the number of cells within the ganglia?
Information on this subject comes from study of the time after amputation at
which cells die. After amputation at stage 53-55, the period of excess degeneration is short, intense, and immediate. As the tadpole develops further, this period
after amputation becomes lengthened, the peak is flattened out, and the onset is
delayed. However, the final number of cells remaining is nearly the same in each
case.
At stage 53, all the cells in the ganglia are of the same maturity—neuroblasts
with very little cytoplasm. As turnover goes on throughout development, new
cells are brought in and the population becomes asynchronous. After turnover
ceases, no new cells enter, and all cells mature further. There is then a parallel
between the maturity of the cells within the ganglia and the degree to which
degeneration is delayed after amputation. Young neurones die after amputation very quickly and older ones take longer to degenerate. After turnover has
ceased, no cells present are of the youngest ages, and so degeneration is uniformly
delayed. Thus the time taken after amputation for cells to die reflects the differentiation spectrum of the ganglia at the time of the operation, though allowance must be made for the cells that are produced in excess after amputation
at stages 57-59, at stage 61, and in juveniles.
It has already been argued that neurones die because they are no longer
receiving some factor from the leg. Dr A. Hughes has pointed out to me that the
fact that they may take a long time to die suggests that the cells are using a store
of the limb factor or its product.
It is proposed therefore, that the maintaining factor from the limb (or a product of it) is stored within the ganglion cell; it is constantly used and the cell
dies if the store runs out; older cells have bigger stores and so can survive longer
without replenishment. Differentiation can thus be measured by the size of the
store.
The maturation of the ganglia and the effect of amputation is summarized in
Fig. 4.
(1) The initial production of cells is independent of the limb.
(2) The limb controls the rate at which further cells are produced.
(3) The differentiated limb controls the number of cells.
(4) The excess perish (histogenetic degeneration) (Fig. 4A).
(5) Amputation lowers the number of cells that can be maintained.
(6) The excess perish (amputation degeneration) (Fig. 4B).
(7) Amputation also lowers the rate of production of cells so that there is
now a smaller excess (Fig. 4C).
468
M. C. PRESTIGE
This formulation avoids the need to distinguish neurones from neuroblasts by
describing both classes as part of a continuum. Up to stage 53, the ganglia only
contain neuroblasts, that is cells not containing or requiring maintenance
factor; after stage 53, there is progressive differentiation until all cells both concontain and require maintenance factor in different amounts. It is possible that
the stimulus to the change is the production of maintenance factor itself; the
cells are then irreversibly addicted. Other formulations of this would be possible
if it was desirable to make more explicit the distinction between cells that die
immediately after amputation and those that show delayed death. This might
A
Normal
Mumber
i
Maintenance
of
K 1 Degeneration
J
cells
Production
Amputation degeneration
During
After
Degeneration
Number
of
cells
Maintenance
Number
Maintenance
A
| Degeneration
of cells
Production
Production
Fig. 4. Diagram of processes controlling the number of cells in lumbar dorsal root
ganglia. A. Normally, over-production causes the cells surplus to maintenance
capacity to degenerate (histogenetic degeneration). B. Amputation reduces the
maintenance capacity, so the newly redundant cells also degenerate (amputation
degeneration). C. Amputation reduces production by the same factor, so that overproduction is similarly reduced, and thus normal histogenetic degeneration. % rate
is, however, unchanged.
become necessary if it was found that the excess cells produced after amputation
at later stages were degenerating before the cells that had been present in the
ganglia at operation.
The action of the production factor causes the number of cells in the ganglia
to increase with positive feed-back, while the maintenance factor damps this by
ensuring that the number present is not greater than appropriate for the state of
limb development. Thus these two control mechanisms act to match the available
neurones to the demands of the leg.
No explanation is offered of why there is excess cell production at later stages,
Cell number in spinal ganglia
469
nor why second loss takes place after stages 53-55 amputation; these may be
concerned with regeneration of the limb.
There must, in addition to the ipsilateral peripheral control of production and
maintenance, be some unknown bilateral control. This is indicated by the
variation in degeneration rates found between different animals in similar conditions, for example 23/11 and 15/11 in Table 3. Unpublished observations made
in collaboration with Dr. A. Hughes using thyroxine and thyroid-blocking
drugs suggest that thyroxine levels could be responsible for these slow variations.
Lastly, it may be pointed out that at stage 61, although production of new cells
is still going on, only a tiny fraction, if any, of them differentiate further. This
can be deduced from the observation that there is a lag before excess degenerations start after amputation. This is interpreted to mean that the youngest cells
in the ganglia are already slightly differentiated. Thus at this stage the new cells
are almost totally redundant. This is in contrast with stages 53-55, when large
numbers of the recently produced cells mature further and can be observed as
degenerating cells immediately following amputation. Thus the conditions for
successful maturation of young cells become progressively stricter throughout
development.
It is very satisfactory that the elegant model that Hamburger & Levi-Montalcini
(1949) elaborated for the chick is so similar to that proposed in this work for
Xenopus, which has to incorporate histogenetic degeneration. The differences
between the two systems turn out to be quantitative, though the effects of limb
regeneration are not yet understood. Hamburger & Levi-Montalcini inferred
control of proliferation from changes in total mitotic activity, while in Xenopus
it has been inferred by calculation. Both methods are necessarily indirect. Again,
in the chick, differentiation of a cell was equated with argyrophilia, which has
the advantage of enabling a direct count to be made; in Xenopus, it was inferred
from the nature of its response to axotomy, which has the advantage of being
a criterion which can be related to other aspects of the physiology of the cell.
SUMMARY
1. The hind leg of Xenopus laevis has been amputated at the hip at stages 53-55
(first limb movement), at stage 61 (beginning of tail loss), and 1-2 months after
metamorphosis. Counts were made of living and degenerating cells in the lumbar
dorsal root ganglia during subsequent development, and changes in the rate of
cell production were calculated.
2. At stages 53-55, amputation causes the rate of production and degeneration
of cells (turnover) to be depressed ipsilateraly at all times after 1 day, except for
two periods in the first and third weeks, in which the number of cells declines,
relative to the opposite side, with associated extra degenerating cells. The total
loss is approximately 50 % and is not replaced.
3. At stage 61, amputation causes the rate of production and degeneration to
470
M. C. PRESTIGE
be depressed ipsilaterally on days 1 and 2. From days 5 to 25, the rate of production and degeneration is elevated; after day 25, cell number slowly declines to
50 % with associated extra degenerating cells.
4. At juvenile stages, amputation causes production and degeneration to
restart on days 11-25; after this, cell number slowly declines for at least 3 months
with associated extra degenerating cells.
5. It is proposed that the limb both controls the production of cells and also
maintains them. Any surplus cells perish.
6. It is suggested that this control is by two factors, and that differentiated cells
are able to store the maintenance factor, or a product of it, thus putting off cell
death after amputation.
RESUME
La regulation du nombre de cellules dans les ganglions rachidiens
lombaires au cours du developpement de tetards de Xenopus laevis
1. Le membre posterieur de Xenopus laevis a ete ampute a la hanche aux stades
53-55 (premier mouvement du membre), au stade 61 (debut de la regression de
la queue) et 1 a 2 mois apres la metamorphose. On a denombre les cellules
vivantes et en degenerescence dans les ganglions des racines dorsales lombaires
au cours du developpement ulterieur, et on a calcule les modifications du taux
de production des cellules.
2. Aux stades 53-55, l'amputation provoque la baisse ipsilaterale du taux de
production et de degenerescence des cellules (turnover), a toutes les periodes
apres une journee, sauf pour deux periodes au cours de la premiere et de la
troisieme semaine, pendant lesquelles le nombre de cellules diminue, par
rapport au cote oppose, avec en outre des cellules en degenerescence associees.
La perte totale est approximativement de 50 % et n'est pas remplacee.
3. Au stade 61, l'amputation provoque la diminution ipsilaterale du taux de
production et de degenerescence, les deux premiers jours. Du 5e au 25e jour,
le taux de production et de degenerescence est accru; apres le 25e jour, le nombre
de cellules diminue lentement jusqu'a 50 %, avec des cellules associees en
degenerescence.
4. Aux stades jeunes, l'amputation provoque la reprise de la production et de
la degenerescence, du l i e au 25e jour; ensuite, le nombre de cellules diminue
lentement pendant 3 mois au moins, avec les cellules en degenerescence
associees.
5. On propose l'hypothese selon laquelle le membre controle la production
des cellules et en assure egalement le maintien a un niveau donne: tout excedent
de cellules perit.
6. On suppose que cette regulation s'exerce au moyen de deux facteurs, et que
les cellules differenciees peuvent emmagasiner le facteur de maintien, ou Fun
de ses produits, differant ainsi la mort cellulaire apres l'amputation.
Cell number in spinal ganglia
All
It is a pleasure to record my thanks to Dr A. Hughes for his continued advice and encouragement ; to Miss Judith Garraway who looked after the animals, prepared the sections,
and drew the diagrams; and to the Medical Research Council for financial support.
REFERENCES
E. D. (1943). Intracentral and peripheral factors in the differentiation of motor
neurones in transplanted lumbo-sacral spinal cords of chick embryos. J. exp. Zool. 93,
99-129.
BUEKER, E. D. (1944). Differentiation of the lateral motor column in the avian spinal cord.
Science 100, 169.
BUEKER, E. D. (1945 a). The influence of a growing limb on the differentiation of somatic
motor neurons in transplanted avian spinal cord segments. J. comp. Neurol. 82, 335-61.
BUEKER, E. D. (19456). Hyperplastic changes in the nervous system of a frog {Rand) as
associated with multiple functional limbs. Anat. Rec. 93, 323-9.
DETWILER, S. R. (1933). Experimental studies upon the development of the amphibian
nervous system. Biol. Rev. 8, 269-310.
GLUCKSMANN, A. (1951). Cell deaths in normal vertebrate ontogeny. Biol. Rev. 26, 59-86.
HAMBURGER, V. (1934). The effects of wing bud extirpation on the development of the central
nervous system in chick embryos. J. exp. Zool. 68, 449-94.
HAMBURGER, V. (1939). Motor and sensory hyperplasia following limb-bud transplantation
in chick embryos. Physiol. Zool. 12, 268-84.
HAMBURGER, V. & LEVI-MONTALCINI, R. (1949). Proliferation, differentiation and degeneration in the spinal ganglia of the chick embryo under normal and experimental conditions.
J. exp. Zool. Ill, 457-502.
HUGHES, A. (1961). Cell degeneration in the larval ventral horn of Xenopus laevis (Daudin).
J. Embryol. exp. Morph. 9, 269-84.
HUGHES, A. & TSCHUMI, P.-A. (1958). The factors controlling the development of the dorsal
root ganglia and ventral horn in Xenopus laevis (Daud). /. Anat. 92, 498-527.
LEVI-MONTALCINI, R. & LEVI, G. (1942). Les consequences de la destruction d'un territoire
d'innervation peripherique sur le developpement des centres nerveux correspondants dans
l'embryon de Poulet. Arch, de Biol. 53, 537-45.
MAY, R. M. (1930). Repercussions de la greffe de moelle sur le systeme nerveux chez l'embryon
de Tanoure, Discoglossus pictus Otth. Bull. Biol. Fr. Belg. 64, 355-87.
MAY, R. M. (1933). Reactions neurogeniques de la moelle a la greffe en surnombre, ou a
l'ablation d'une ebauche de patte posterieure chez l'embryon de l'anoure, Discoglossus
pictus Otth. Bull. Biol. Fr. Belg. 67, 327-49.
NIEUWKOOP, P. D. & FABER, J. (1956). Normal Table o/Xenopus laevis (Daudin). Amsterdam:
North Holland Publ. Co.
PALLADINI, G. (1961). Neurodegenerazione nella ontogenesi di quaglia (Coturnix coturnix
japonica T. e S.). Riv. Neurobiol. 7, 383-400.
PIATT, J. (1948). Form and causality in neurogenesis. Biol. Rev. 23, 1-45.
PRESTIGE, M. C. (1965). Cell turnover in the spinal ganglia of Xenopus laevis tadpoles.
/. Embryol. exp. Morph. 13, 63-72.
SIEGEL, S. (1956). Nonparametric Statistics for the Behavioral Sciences. New York: McGrawHill Book Company, Inc.
BUEKER,
{Manuscript received 24 October 1966)