/. Embryol. exp. Morph. Vol. 18, 3, pp. 359-87, December 1967
Printed in Great Britain
359
The control of
cell number in the lumbar ventral horns during
the development of Xenopus laevis tadpoles
By M. C. P R E S T I G E 1
The developing limb has been amputated by many workers in several species
and in each case the number of surviving motor neurones on the side of the
operation was less than normal. This may be observed among the mammals
(e.g. Barron, 1945), birds (e.g. Hamburger, 1934), urodeles (e.g. Stultz, 1942),
and anurans (e.g. May, 1930). The loss of motor neurones after amputation in
adults appears to have been first noticed by Vulpian (1868) and Johnson &
Clarke (1868). The early evidence is reviewed by Sherrington (1893) and the
later by Piatt (1948). The control that the developing leg has over proliferation,
migration, maintenance and degeneration of ventral horn cells has been most
completely analysed in the chick, notably by Hamburger (1934, 1939, 1958),
Hamburger & Keefe (1944), Bueker (1943, 1944, 1945a), Barron (1946, 1948),
Mottet (1952) and Mottet & Barron (1954). Less is known about this in Anura.
May (1930, 1933) has recorded that in Discoglossus motor area hyperplasia can
be produced by supernumerary limbs, and that the lateral motor horn (ventral
horn) can be suppressed by replacement of the limb area with skin grafts. Bueker
(19456) found a frog {Rand) with three functional hind legs on one side; this
had a hyperplasia of 22-7 % in the ventral horn. Beaudoin (1955) ablated the
early limb-bud in Rana pipiens and found a terminal hypoplasia of 82 % in
the ventral horn. Counts at intermediate stages were interpreted by him as indicating that the operation had delayed differentiation and the normal loss of cells
on that side. In Bufo vulgaris, Perri (1956a) removed the hind-limb area at tailbud stages, and found that the ventral horn developed normally up to digit
stages, when it regressed. The same happened when the isolated spinal cord
was transplanted (Perri, 19566). Three out of six animals with motile supernumerary limbs had hyperplastic spinal cords on the side of graft; this was not
seen in any of the cases with non-motile grafts (Perri, 1957). Supernumerary
limbs only became motile when innervated by limb plexus fibres, and never
induced formation of new ventral horns in trunk regions. Perri therefore concluded that for the full development of the ventral horn, both regional intrinsic
and also extrinsic limb factors were essential, the former necessarily acting
before the latter.
1
Author's address: The Department of Zoology, University of Bristol, Bristol, U.K.
360
M. C. PRESTIGE
Further evidence has been obtained from the comprehensive series of experiments by Hughes, using batches of Eleutherodactylus embryos to investigate the
conditions for achieving innervation of grafted limbs. Amputation of the limb
at early stages (5-8 days development) causes a rapid degeneration of ventral
horn cells (Hughes, 1962). This is at a time when nerve fibres have already
entered the limb. 'A limb grafted in place of the amputated member has little
effect on halting the loss of cells, and . . . the main effect of the graft is in promoting a partial regeneration of the ventral horn through the outward migration
of new cells from the mantle layer' (Hughes, 1962). Amputation at still later
stages (Hughes, 1964) causes chromatolysis and an excess of cells to develop on
the operated side; this is succeeded by a final cell loss. The latter result was also
obtained by Palladini & Alfei (1966) in Bufo bufo.
In Xenopus, removal of the early limb-bud before fibres enter (Hughes &
Tschumi, 1958) by obstructive isolation, ablation or chemical means, does not
affect the assembly and initial differentiation of motor neuroblasts in animals
fixed before stage 53 (limb at palette stage); but after this stage the ventral horn
was absent. In individuals amputated at these later stages when the developing
neuroblasts require the presence of the limb (stages 55-56), the operation causes
the immediate degeneration of some of the motor cells (Hughes, 1961). Production of an experimental hyperplasia has not been attempted in this species.
Thus in Anura it seems that the limb does not affect the initial development of
the ventral horn, but later becomes essential for its further maintenance. After
fibres have grown into the limb, amputation causes loss of cells; and later still
causes chromatolysis. The leg appears to have some controlling influence over
the further differentiation of new ventral horn cells, and also their ultimate
maintenance.
One factor that has not received much attention is histogenetic degeneration
(Gliicksmann, 1951)—that is, cell deaths occurring normally in the differentiation of functioning organs. These have been observed (Romanes, 1946) and
counted in developing motor cell columns of mouse (Harris, 1965) and the
lumbar ventral horn of Xenopus (Hughes, 1961); they have also been seen in the
motor columns of undisturbed chick embryos (Collin, 1906; Hamburger &
Levi-Montalcini, 1949; Hughes, 1955); and counted on the contralateral side
of chick embryos after amputation of the limb-bud (Hamburger, 1958). Degenerating motor cells in limb regions have also been observed in other Anura,
among them Bufo (Dr A. Hughes, personal communication), Hyla (Hughes,
1963), Eleutherodactylus (Hughes, 1962), and 'few' in Rana (Race & Terry,
1965); also in the lizard, Lacerta (Dr A. Bellairs & Dr A. Hughes, personal
communication). In Xenopus, the species for which we have most information,
Hughes (1961) calculated that the number of degenerating cells was greatly in
excess of that required to account for the decline in cell numbers and therefore
concluded turnover was taking place in the developing ventral horn. The same
may be true of the chick, for Hamburger (1958) observed degenerating cells (on
Cell number in ventral horns
361
the unoperated side in his experimental material) at a time when the number of
cells was not declining; thus new cells must have been entering.
Another factor that has been neglected is the later influence of the periphery
upon the ventral horn. Nearly all the anuran experiments that have been done
were performed on the early limb-bud before the fibres have grown in. Apart
from isolated observations, only in Eleutherodactylus and Xenopus has the leg
been amputated at later stages (Hughes, 1961, 1962, 1964). In the former
species, however, it is very difficult to count degenerating cells, as sites are
numerous and close together. In slowly developing larvae, such as Xenopus,
the sites are almost always separate and distinguishable.
At the present, we do not know what causes ventral horn cells to degenerate
during normal development, nor is it known how this process is controlled. It is
the purpose of this paper to investigate the control of the periphery over the
number of lumbar ventral horn cells in Xenopus by amputating the hind leg
at representative stages throughout development, by observing the consequent
changes in numbers of living and degenerating cells, and by calculating the
changes in rate of 'production' of new cells by immigration. In this way, the
results of acute and chronic decrease of the periphery can be compared at each
stage. This has already been done for the dorsal root ganglia in a previous paper,
to which this is a companion (Prestige, 1967).
MATERIAL AND METHODS
Larvae of Xenopus were reared from eggs obtained from adults injected with
gonadotrophins (Nieuwkoop & Faber, 1956). They were kept at temperatures
between 20 and 23 °C. One leg of tadpoles and juveniles was amputated as high
in the thigh as possible, using scissors or forceps under M.S. 222 anaesthesia
(1:4000). The limb-bud of younger tadpoles was ablated with forceps as close
to the body wall as possible. The other side of the animal served as a control.
Provided the water was kept clean, the tadpoles survived well until metamorphic
climax; at this stage, they normally 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 leg of a juvenile for the same reason. The animals were
kept in tap water at all times, without antibiotics.
The lumbar region of selected specimens was fixed in half-strength Bouin;
the body length and hind-limb length were measured; serial paraffin sections
were cut at 8 fi or 10 fi transversely and stained with haematoxylin and eosin.
Some were silver-impregnated (Palmgren, 1948). The lumbar ventral horn
extended through 130-250 sections. The motor neurones therein were identified
both by position and appearance. While the largest of these are clearly distinct
362
M. C. PRESTIGE
from mantle layer cells, at the other extreme are some neuroblasts which are
similar to them in size and appearance, especially at either end of the column.
Only those cells were reckoned as ventral horn cells in which the nucleus was
considered to be larger and paler than those of the adjacent mantle layer neuroblasts. These criteria are the same as those adopted and illustrated by Hughes
(1961). In each specimen, the neurones of every third section throughout the
series were enumerated; only those cells were counted in which the nucleolus was
present. Ventral horn neurones have only one nucleolus and this body is small
compared with the section thickness. Moreover, as Jones (1937) has pointed out,
nucleoli can be seen on occasion to have been dislodged from the nucleus in the
direction that the microtome knife passes through the tissue, suggesting that
nucleoli are less frequently bisected by sectioning than might be expected on the
basis of their size and numbers, and that instead they may be displaced whole
into one or other of the adjacent sections. Counts of Palmgren and haematoxylin-stained material did not differ. These arguments reduce the magnitude
of a correction factor to an uncertain value, and the counts therefore have not
been corrected.
Degenerating neurones were identified by the presence of a pycnotic nucleus
with basophilic cytoplasm, as described by Hughes (1961). The degenerating
site was counted if the nucleolar mass, or its residue, was contained in the
section; in cases of doubt, reference to the adjacent section could be made. It
has been assumed that changes in the degenerating cell count reflect changes in
the rate of degeneration.
Neurones in chromatolysis were classed as living cells, not as degenerating
cells, though they were counted separately as well. Whereas in the dorsal root
ganglia the nuclei are already eccentric, in the ventral horn the nucleus is
normally centrally placed in the cytoplasm, and its lateral protrusion forms an
excellent criterion for the identification of chromatolytic cells. The other
criterion used was the homogeneous character of the cytoplasm, like 'ground
glass', and the absence of the fine lines of basophilic material that in Xenopus
form the Nissl substance. Larger, wider streaks of basophilia at the cell surface
remain or become intensified, and have to be ignored. The swelling of the cytoplasm per se is not a good criterion as the cells are of different sizes anyway.
Later, during the recovery phase, the characteristic dense ring of basophilia
around the nucleus which may extend towards the poles of the cells is seen;
in Palmgren preparations, this area is not argyrophilic. Thus the number of
cells that react can be counted at two stages of the process: in the chromatolytic phase and in the perinuclear basophilic phase. Such cells are not usually
seen in unoperated tadpoles, nor on the contralateral side in the amputated
ones.
Counts of chromatolytic cells are subject to two errors: first, there are cells
within the ventral horn which are ranked in the total counts with the more mature
Cell number in ventral horns
363
ones, but do not possess sufficient cytoplasm to determine whether or not they
are undergoing chromatolysis; secondly, there are some large neurones which
display some of the characteristics of chromatolysis, but not sufficiently to be
identified with certainty. Identification is easiest in haematoxylin preparations
in which no differentiation with acid alcohol has been attempted. A further
source of variation stems from ignorance of the number of ventral horn cells
in the tadpoles at the time of the operation; only the number at fixation can be
counted.
The counts of living cells in the Tables have been rounded to the nearest 10
and of degenerating cells to the nearest 5.
The sampling procedure adopted was that every third section throughout
the series was counted. Total sample counts of between 100 and 2000 living cells
were recorded: for these, the 95 % confidence limits due to random sampling
are ± 20 % and ± 4-5 % respectively; the size of experimental changes induced
was greater than this. For degenerating cells, sample counts of between 0 and
80 were recorded: as estimates of the population mean, the lowest of these, if
taken from only one individual, may be as much as 100 % in error. However,
the error may be decreased by pooling results from more than one animal, and
this procedure has been followed. In addition to the usual significance tests,
rank and sign tests have been used because of their great power and simplicity
(Siegel, 1956).
In selecting animals for an experiment, it was found that great care in the
matching of the tadpoles made subsequent analysis of the results easier. It was
best to choose examples from the same batch of eggs, reared in the same bath
and with similar sized and shaped legs. Body length is not a good stage criterion.
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—degenerating cells are first seen in ventral horn; stage 54, movement
begins in the limbs; stage 58, forelimbs emerge from pouch; stage 61, tail
degeneration starts; stage 66, juvenile—no more degenerating cells in ventral
horn.
OBSERVATIONS
Limbs were amputated at stages 52-53, 54, 57, 61 or 66. The observations in
each series are separately described. The ventral horns of control animals and of
the unoperated sides in experimental animals contained up to 50 % more cells than
comparable ones at the same stage in Hughes' (1961) study. The data from the
present work are plotted in Fig. 1A (open circles) against the line drawn by
Hughes through his data (continuous line). In Fig. 1B (filled circles), the corresponding data on the number of degenerating cells are similarly plotted. The
peak in number of degenerating cells occurs earlier and to a greater extent than
in Hughes' work.
23
JEEM l8
364
M. C. PRESTIGE
Series 1: amputation at stages 52-53
Fifteen tadpoles at the end of pre-metamorphosis were used. The hind-limb
bud at operation measured between 1 and 2 mm long. In control animals, and
on the control side of experimental ones, there were between 5000 and 6000
cells per side in the ventral horn; the nuclei of these were ovoid and there was
7000
6000
5000
8
4000
00
3
3000
2000
1000
-IHI—
8
200
52
61
Stage
66 2 3 "
Months
Fig. 1. The number of living and degenerating cells in the lumbar ventral horn
during development. Each point is taken from the control side of an animal operated
upon in this work. The continuous lines were drawn by Hughes (1961) through his
counts on normal animals. The interrupted line is a synthetic curve constructed by
summing the numbers of ventral horn cells in phase I, II and III at each stage (see
(Discussion). O, living cells. • , degenerating cells.
little cytoplasm visible. Histogenetic degeneration in the ventral horn had
started, but at a low level. In four out of eight animals that survived long enough
after the operation, a functional leg regenerated, starting during the third week:
in the others, after 4 weeks, a stunted leg regenerated which contained no muscle
except in the most proximal stump portions. The ventral horn of the side
opposite to the amputation site developed normally, both with respect to
numbers of living and degenerating cells and in appearance.
Within 3 days of the operation (Table 1A, Fig. 2) a loss of cells was seen on
the operated side. By the end of the first week over 2000 cells (40 % of those
Cell number in ventral horns
365
Table 1. The effect of amputation of the limb-bud at stages 52-53
on the development of the ventral horn
(C = control; A = amputated.)
Date
Body Limb
length length
fixation (mm) (mm)
of
Days
after
Stage amputation
No. of cells
r
living degen.
Remarks on amount
of regeneration
A. Operation performed 117. x. 66: those in which regeneration was
unsuccessful or not present
—
30
C 5640
0
17.x.
38
1-2
52-53
30
A 5720
—
3
20.x.
15
C 5270
53
36
1-6
A 4670
70
—
24.x.
3-4
7
C 6050
190
46
54
A 3700
55
Ilium not present
55-56
29.x.
5
12
C 4180
185
55
A 1500
100
Pelvic girdle complete but
35
14
C 2250
58-59-60 23
9. xi.
73
A 630
20
femur absent
Ilium + femur present:
16. xi.
C 1730
10
22
30
25
64
A 320
5
only muscles over hipjoint.
—.
Foot regenerated close to
25. xi.
26
39
C 1970
5
66
A 350
0
body: only reduced iliac
muscles present
•—
Complete leg skeleton
20. xii.
25
64
0
66
C 1670
A 110
0
regenerated: some reduced hip-joint muscles
present
Operation i performed 17. x. 66: those inwhich regeneration was successful
Regenerate 2-6 mm:
4. xi.
7
56-7
65
18
C 2620
61
40
A 1280
= stage 54
Regenerate 8 mm:
35
C 2300
10. xi.
58-59-60 24
16
68
A 1690
20
= stage 56-57
20
Regenerate 17 mm:
C 2110
32
17. xi.
64
23
31
A 870
10
= stage 61, thigh flexors
reduced
—
Regenerate 20 mm:
0
C 1760
26. xi.
23
66
40
A 1330
0
= stage 63
15. xi.
39
16. xi.
32
17. xi.
41
B. Preliminary <experimeni: performed 12. xi. 65
—
45
53-54
3
C 5410
A 3380
20
—
2
35
4
C 5790
53
10
A 4940
—
125
2-2
C 5010
54
5
A 2720
55
2
23-2
366
M. C. PRESTIGE
initially present) had degenerated. In the animals in which a functional leg did
not regenerate, this large relative deficit was not maintained after 12 days; the
number of cells in the ventral horn fell smoothly but less steeply on the operated
than on the control side, so that the deficit at metamorphosis was only about
1500 cells (Fig. 2B). This, however, represents a mean cell loss of 86 % in the
6000 \-
I
60
•S
<•
4000
p
A
I
2000
1 .<
-
c
-
j>
Days
I
52
I
1
10
20
30
i I I l I I
I
54
56
58
63
(
'1
40
66
64
Stage
2 *
3
CO
CO
CO
B
1000
2000 L-
S -8
+
0
°O
CO
3
Q>
100
X
W
Fig. 2. Amputation of the leg at stage 53. A, Numbers of living cells in the lumbar
ventral horn on control side (open circles) and on operated side (attached horizontal
bar); those animals in which regeneration was successful are indicated by filled
circles. B, The differences in the number of living cells between the two sides.
C, The differences in the number of degenerating cells between the two sides.
three juveniles. In the chick, Hamburger (1934) found a linear relation between
the amount of muscle lost after amputation of the limb, and the deficit in the
number of motor neurones. If this holds also for Xenopus, then it seems that
the operation removed around 86 % of the limb tissue.
The number of degenerating cells (Fig. 2C) observed on the operated side
was between one-third and two-thirds that of the control side at all times after
Cell number in ventral horns
367
3 days (sign test, six pairs, P = 0-008), but at 3 days there was a large excess
of degenerating cells. It seems that amputation at this stage, as Hughes (1961)
found at the later stages 55-56, causes an early cell loss within the ventral horn
and an excess of degenerating cells; this lasts less than a week, and in the preliminary experiment (Table 1B) must have lasted less than 3 days. Hughes &
Tschumi (1958) showed that removal of the early limb bud did not affect the
differentiation of the ventral horn prior to this stage, but caused its delayed disappearance at about this time. It is possible that in the experiment in which the
excess degeneration had ceased by the third day, the tadpoles were a few days
older, while in the main experiment there was a delay of the same type as Hughes
& Tschumi (1958) observed.
This explanation is supported by examination of the longitudinal pattern of
degeneration and cell loss in the ventral horn. Dr A. Hughes (personal communication) has recently found evidence that the posterior portion of the ventral
horn develops about a stage behind more anterior parts. On the control side,
at stages 52-54 the number of histogenetic degenerating cells is increasing
(Hughes, 1961); in the tadpoles of the present work the anterior two-thirds of
the column always had many more than twice the number in the posterior third.
The extent of cell loss after the operation follows a similar pattern, in that it is
seen to have occurred earlier in the anterior two-thirds than elsewhere. For
example, in animal 15. xi of Table IB, 37 % of the cells have been lost and the
total number of degenerating cells on the operated side (20) was depressed
below the control side (45), as in all other later tadpoles, yet at the posterior
end on the operated side the ventral horn showed no cell deficit and a local
excess of degenerating cells, compared to the unoperated side. It was only this
region on the control side that showed no histogenetic degeneration. Thus although the excess degeneration due to the operation had ceased by the third
day over most of the ventral horn, in the posterior portion it can barely have
started.
These observations indicate that in the ventral horn the capacity to degenerate
after amputation of the limb also develops earlier in its anterior two-thirds, and
at the same time as the capacity for histogenetic degeneration, to a very close
approximation.
Effect of successful regeneration after the operation
The earliest signs of successful regeneration were recognized at 18 days, when
the regenerate was a stage 54 leg, three stages behind the remainder of the tadpole. This represents a difference of about 12 days and was maintained thereafter. The ventral horn on the operated side (Table 1) was apparently almost
totally unaffected by the regenerating leg before this; that is, the numbers of
living and degenerating cells were similar to those expected if there had been no
regeneration. Perri (1956a) and Hughes & Tschumi (1958) showed that the
368
M. C. PRESTIGE
normally developing limb does not affect the ventral horn before stage 53, and
this is therefore also true of the regenerating limb.
After this time the regenerating leg (Fig. 2 A) maintained the number of
ventral horn cells at about the same level, so that the further decline was arrested.
At metamorphosis, the number of ventral horn cells for the regenerate was about
1000, compared with about 300 for the unregenerate leg and about 1700 for a
control leg. Many of these ventral horn cells for the regenerate were as large as
those on the control side. The leg in these circumstances becomes innervated
(judged by sensitivity and motility) with a reduced set of ventral horn cells. The
regenerating limb, however, does not cause the ventral horn cell number to
decrease, as in normal development.
Since the number of ventral horn cells did not decline after 18 days in these
circumstances, and since cell degeneration continued, turnover of the ventral
horn cells must have been taking place while the regenerating leg was being
innervated. Hughes (1961) calculated that a similar process of ventral horn cell
turnover takes place during normal innervation of the leg.
Since the rate of degeneration on the operated side was similar in both regenerating and non-regenerating tadpoles (Table 1), the absence of a decline in
cell numbers after day 18 due to the regenerate must represent an increased
immigration of cells into the ventral horn. Thus a regenerating leg induces
a further influx of neuroblasts from the mantle layer. This is similar to the effect
of a replacing graft of a limb after its amputation at comparable stages in
Eleutherodactylus (Hughes, 1962).
Series II: amputation at stage 54
Tadpoles at these stages have legs between 2-5 and 4 mm long, which perform
'flare' movements (Hughes & Prestige, 1967). They are in the first half of prometamorphosis. Cell degeneration and turnover are at their peak level within
the ventral horn (Hughes, 1961). It is important to emphasize that numbers of
cells in the ventral horn are declining steeply at these stages, and thus that observations on relative differences between the sides can only be made against a
shifting base-line. In the above work, Hughes amputated the legs of tadpoles
high in the thigh at stages 55-56. He found that within 3 days, many cells were
lost from the ipsilateral ventral horn, and this was accompanied by an excess
of degenerating cells. Equilibrium in number of ventral horn cells was restored
some time after the first week, and peristed for a varying time (up to two further
weeks) during which there was on some occasions an actual excess of cells on
the operated side. Ultimately there was a second and final loss of cells, so that
the ventral horn at metamorphosis was reduced to about a third of its normal
size (38 %).
Hughes (1961) found that no regeneration took place at these stages. Thegreater
size of the residual ventral horn, when compared with those from series I,
Cell number in ventral horns
369
reflects the sparing of the iliac musculature at the operation. In series I, it was
possible to remove nearly all the tissue of the pelvic girdle as well.
In the present work, the legs of five tadpoles were amputated at stage 54
(Table 2). The results show that during the first 7 days the ventral horn behaves
as at stages 55-56, though the degree of initial cell loss is much greater. The tadpoles after 3, 5 and 7 days also show the same depression of the rate of degeneration that was seen in series I (Mann-Whitney rank test, P = 005). No chromatolytic cells were seen. The major qualitative difference between the events following amputation at stages 52-53, and those following amputation at stages 55-56,
Table 2. The effect of amputation of the leg below the hip-joint at stage 54
on the development of the ventral horn {operation performed on 18. xi. 65)
(C = control; A = amputated)
Date of
fixation
Body
length
(mm)
Leg
length
(mm)
Stage
Days
after
amputation
19. xi:
—
—
55
1
20. xi.
45
4
55
2
21. xi.
43
3-5
54-55
3
23. xi.
50
5
56
5
25. xi.
50
6
56-57
7
C
A
C
A
C
A
C
A
C
A
No. of cells
r
Living
Degen.
3180
2600
3530
2720
3900
2190
3110
1670
3010
1890
105
235
115
115
115
55
85
55
110
55
is that in the former there is only the early period of relative cell loss, while in
the latter series, a second loss later ensues. After amputation at stages 52-53
there is no evidence of renewed or second loss of cells during later development,
neither as a late increase in the deficit of living cells (Fig. 2B), nor as a late
excess of degenerating cells (Fig. 2C).
Series III: amputation at stage 57
Twelve tadpoles were used. These animals were at the stage just before the
forelimb emerges from the pouch. The hind legs were between 7 and 9 mm long,
and at rest, the thighs were drawn up to protrude laterally; the tadpole swam
by using its tail, while the legs were held extended together against the fin
(Hughes & Prestige, 1967). In the ventral horn, the period of steep decline and
its associated peak in counts of degenerating cells are normally just over: there
were between 2000 and 3000 cells on each side. The animals of Fig. 3 and Table 3
show the following features:
370
M. C. PRESTIGE
(1) On the first 2 days after amputation of the leg an excess in the number of
degenerating cells in the ventral horn on the operated side was found (Fig. 3C).
The magnitude of this excess, at least on the second day, is comparable with
similar early excesses in the number of degenerating cells after amputation at
stages 52-53 (series I), stage 54 (series II) and stages 55-56 (Hughes, 1961). In
contrast to these, however, the resulting cell deficit was small and transitory,
and many of the neurones subsequently become chromatolytic.
57
58
59 61
63
65
66
Stage
3000
o
o
o
8.§
2000
u ft
<a O
1000
z
0
500
15
20
25
30
Days
II
x to
a
500
1000
8-a
50
o
—
o §
50
Fig. 3. Amputation at stage 57. A, Numbers of living cells remaining in ipsilateral
ventral horn. B, The differences in the number of living cells between the two sides.
C, The differences in the number of degenerating cells between the two sides.
(2) The number of living cells on the operated side remained at about the
same level for 2 weeks after the amputation (Fig. 3 A), while the number of
living cells continued to decline on the opposite or control side, as in normal
development (Table 3). This led to a relative excess in the number of living cells
on the operated side over that on the control side (Fig. 3B). This started to develop about the fifth or sixth day, and was at its maximum during the second week.
(3) The numbers of degenerating cells counted on the operated side were
lower than those counted on the control side during the period 6-10 days after
Cell number in ventral horns
371
the operation (Fig. 3C: Mann-Whitney rank test, P = 0014)—that is, when
the relative excess in the number of living cells on the operated side was building
up. This depression in degeneration rate following an excess degeneration rate
after amputation is similar to those described in series I and II. It seems that
Table 3. The effect of amputation of the leg below the hip-joint at stage 57
on the development of the ventral horn
(C = control; A = amputated.)
No. of cells
Date of
fixation
Body
length
(mm)
23/xi
57
24/xi
54
25/xi
57
26/xi
55
27/xi
56
28/xi
60
29/xi
59
30/xi
3/iv
7/iv
62
47
37
12/iv
23
23/iv
—
Leg
length
(mm)
Days
after
Stage amputation
(
Living
Operation performed on 22. xi. 65
1
57
7
C 2700
A 2810
57
2
C 2290
7
A 2370
3
C 2920
7
57
A 2710
11
4
C 2240
59
A 2240
11
5
C 2100
59
A 2290
6
8
57
C 2070
A 2040
11
7
C 1890
59
A 2090
58-59
8
C 2420
10
A 2530
Operation performed on 24. iii. 66
62
24
10
C 2190
A 2710
64
14
C 2120
23
A 2430
65
C 1660
25
19
A 1800
1660
66
30
C
27
A 400
Degen. Chromatolytic
80
95
40
100
25
20
5
10
10
10
20
10
20
0
35
5
25
0
5
5
0
25
0
10
960
840
770
810
1120
1520
1270
900
the failure of the number of living cells to decline on the operated side compared
with the number on the control side can be ascribed to this relative difference
in the numbers of cells degenerating. It does not seem necessary to invoke any
increase in the rate of immigration of cells into the ventral horn in order to
explain the excess.
(4) During the third and fourth weeks the numbers of living cells counted
in the ventral horn on the operated side declined (Fig. 3 A) faster than those of
372
M. C. PRESTIGE
the control side; as a result, the relative excess was converted to a relative
deficit (Fig. 3B). This was accompanied by the presence of cell degeneration on
the operated side throughout this period, while on the control side cell degeneration had ceased (Fig. 3 C), as is usual at the end of metamorphosis. This late
loss of ventral horn cells after amputation was also observed by Hughes (1961)
after amputation at stages 55-56; it was not observed in series I of the present
work after amputation at stages 52-53.
(5) At 30 days, less than 25 % of the ventral horn cells remained. It seems
therefore that the original leg amputation was approximately 75 % complete in
this series (Table 3).
(6) The number of cells in chromatolysis (mean 1024) is somewhat less than
the excess number of cells lost in the final loss period (1500). This latter figure
is the difference between the excess of cells seen in the second week and the
final deficit.
Series IV: amputation at stage 61
A more extensive series was done with 21 tadpoles at this stage: they were
about to enter metamorphic climax, the head had shrunk noticeably, the ankles
were flexed at rest and they swam using repeated bilateral kicks as well as the
tail (Hughes & Prestige, 1967). The legs were 10-15 mm long at operation. At
this stage in the ventral horn there are 1100-1800 cells a side; turnover is
present, but at a much lower level than at stages 54-56 (Hughes, 1961), and the
majority of the cells are large and appear mature. During the next 8 days the
tadpole will change into a juvenile toad.
The animals of Table 4 shows- the following features:
(1) There is no evidence of early cell loss or of an excess of degenerating cells
on the operated side, such as was seen between stages 53 and 56. Many cells
undergo chromatolysis.
(2) The operation depresses the rate of degeneration ipsilaterally after 2 days
(Fig. 4B) for the remainder of the period during which histogenetic degeneration
normally takes place. This is most marked on days 2-4 (Mann-Whitney rank
test, P = 0-036). This depression is similar to that observed in series I—III.
(3) An excess of 150-200 living cells on the operated side builds up at the
end of the first week (Fig. 4 A). This excess appears against a base-line of the
number of cells in the contralateral ventral horn, which is still declining slowly
during this week. The excess appears similar to that observed in series III.
(4) After the first week, the excess in cell numbers is maintained for 2 months;
there are no changes in total cell number and hardly any degenerating cells are
seen on either side (Fig. 4).
(5) A slow decline in the number of ventral horn cells on the operated side
takes place during the third and fourth month. At 62 days there is some evidence
of excess cell degeneration which may be associated with this. The total number
of cells remaining after 6 months is 650 (43 %), most of which are in the anterior
373
Cell number in ventral horns
Table 4. The effect of amputation of the leg below the hip-joint at stage 61 on the
development of the ventral horn: series compiled from operations not all done
simultaneously
(C = control; A = amputated)
No. of cells
Date of
fixation
Body
length
(mm)
Leg
length
(mm)
Stage
11. iii.
—
14
63
1
2. viii. B
43
14
61-62
1
Days
r
after
Living
amputation
e
A
3. viii. A
40
13
61-62
2
25. i.
—
14
61
2
9. xii.
—
15
62
2
4. viii. B
40
15
62
3
23. i.
—
15
—
3
13.x.
36
13
62
3
5.x.
45
15
63
4
15. iii.
—
13
—
5
11. ii.
—
15
64-65
7
18. iii.
—
14
65
8
20. iii.
—
14
65
10
23. iii.
—
13
66
13
20. iii.
—
15
66
15
12. iii.
—
13
66
21
20. iii.
—
21
66
25
13. xi.
—
18
66
38
7. xii.
—
21
66
62
27. i.
—
23
66
119
20. ix.
—
—
66
173
c
c
A
c
A
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
1300
1380
1630
1620
1670
1550
1090
1150
1680
1740
1650
1760
1160
1300
1710
1610
1870
1770
1030
1050
1160
1250
1290
1420
1230
1390
1130
1310
1220
1380
890
1080
1250
1420
1470
1670
1560
1410
1420
800
1520
650
Degen.
Chromatolytic
10
15
15
15
5
10
40
10
5
5
20
10
25
5
20
10
35
10
5
5
5
0
0
0
0
5
5
0
0
5
0
5
0
0
0
5
0
10
0
0
0
0
740
770
820
750
960
1280
374
M. C. PRESTIGE
part of the ventral horn. Delayed loss in this series is thus similar to that observed in series III and in Hughes (1961), though later and more protracted.
(6) The number of cells in chromatolysis (mean 886) is slightly less than the
extra number of cells lost during the final decline (about 1000).
0
5
10
15
Days
20
25.
38 62119 173
200 r
CO
+
•> 2
O Q,
200
400
W
600 L
50
8f
.
0>
+
0
-D
a o/55
50
Fig. 4. Amputation at stage 61. A, The differences in the number of living cells between the two sides. B, The differences in the number of degenerating cells between
the two sides.
The question may be raised whether second loss after amputation at stages
55-56 took place in cells that, though normally present at the time of the
operation, were insensitive to the absence of the limb, but that as development
progressed entered a phase in which they became sensitive, found the limb absent
and so died. This hypothesis predicts that amputation in the present series would
result in immediate loss of the sensitive cells. Such behaviour is not observed,
and so second loss must be due to a delayed reaction to the original operation.
A similar argument and conclusion may be drawn for the final loss of cells
after amputation at stages 57 and 61. In this case the evidence comes from the
results of amputating 1-2 months after metamorphosis (below). Final loss is
therefore due to a delayed reaction in ventral horn cells.
The conclusion from this argument stands in contrast to that obtained from
the delayed degeneration or regression of ventral horn cells after limb-bud
removal at stage 49-50 (Hughes & Tschumi, 1958). In that case, amputation at
stages 53-54, the stages at which delayed regression occurs, causes immediate
loss of cells. Thus the later death of these cells, after amputation at stages 49-50,
is an example of delayed sensitivity to removal of the periphery.
Cell number in ventral horns
375
Series V: amputation early in juvenile life
Thirteen toads were amputated at the hip in the first few months after metamorphosis. The data are shown in Table 5. No early cell loss took place, nor
was there any renewal of cell degeneration. Many cells underwent chromatolysis. Final delayed loss of cells took place mostly between the twelfth and
thirtieth weeks, though some indications of cell degeneration were still present
Table 5. The effect of amputation of the leg below the hip-joint on the ventral
horn of the juvenile: series compiled from operations not all done simultaneously
(C = control; A = amputated)
No. of cells
Date of
fixation
Leg
length
(mm)
3. iii. A
15-18
1
4. iii. A
_
2
23. iii.
17
4
3. iv.
—.
7
7. iv.
.—
11
7. xi.
30
18
15.x.
—
25
5. ix.
26
77
20.x.
33
122
1. viii.
_
127
30. xi.
50
163
27. i.
45
221
21. iii.
60
274
t
Days after
amputation
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
C
A
Living
Degen.
1310
1330
1620
1640
1640
1660
1310
1370
1430
1430
1480
1600
1340
1380
1170
5
0
0
0
0
0
0
0
5
0
0
0
0
0
0
910
1670
1680
1260
1320
2060
1690
2010
1320
1800
1090
Chromatolytic
800
750
5
—
—
0
0
—
—
0
0
0
5
660
750
0
after 9 months. The number of cells remaining was 61 % and 66 % in the last
two animals fixed. The last neurones to recover from chromatolysis and to die
were found in the centre portion of the ventral horn. The number of neurones
that underwent chromatolysis (mean 740) was equal to the number of cells that
376
M. C. PRESTIGE
died in final loss (mean 700); they were present in similar regions of the ventral
horn.
DISCUSSION
It has been recognized for a century that developing neurones are more
susceptible to injury than mature ones. This has been most clearly documented
by Romanes (1946) and LaVelle & LaVelle (1958). These authors described a
critical change in the reactivity of mammalian foetal motor neurones: in the
hamster, for example, after axotomy at birth, the first changes were seen in the
nucleus, and were followed in 4-6 days by cell death, while after the same
operation 7 days post partum, the cells showed only cytoplasmic chromatolysis
and 26 days later one third of the cells were still surviving.
In the anuran Xenopus amputation at stages before 52-53 has no effect on the
ventral horn until stages 52-54 (Perri, 1956a; Hughes & Tschumi, 1958).
Amputation at stages 53-54 provokes a large early loss of cells (series I, II);
at stages 55-56 a smaller early loss and also a later second loss (Hughes, 1961);
at stage 57 a very small early loss and a larger late loss (series III); at stage 61
only a late loss (series IV); and as juveniles only the late loss (series V). These
excess cell losses are accompanied by excess cell degeneration. In the periods
in which excess cell losses are not taking place the rate of histogenetic cell
degeneration on the operated side is significantly depressed below that of the
control side.
During development three phases in the reaction of the ventral horn cells to
amputation can thus be recognized:
(1) Phase I, in which ventral horn cells are unaffected by the removal of the
periphery. These cells are neuroblasts and possess little cytoplasm.
(2) Phase II, in which the ventral horn cell dies within 3-4 days of amputation.
(3) Phase III, in which the ventral horn cell does not die immediately after
amputation but does so only later, usually after an extended period of chromatolysis and recovery. These cells are mature in appearance.
Estimates of the number of cells in each phase at each stage of development
may be made as follows:
Phase I. The ventral horn before stages 52-53 consists entirely of phase I cells,
since it is unaffected by removal of the periphery (Hughes & Tschumi, 1958).
Following removal of the limb-bud at stages 52-53 (series I), any phase II cells
present degenerate, no phase III cells can mature, and therefore the number of
remaining cells at any time on the operated side is equal to the number of phase
I cells present (after making a linear correction for 86 % limb-bud removal).
The graph of this has been plotted in Fig. 5 A as curve I.
Phase II. The cell deficit that develops within 3-4 days of amputation defines
the number of phase II cells within the ventral horn at the operation after
correction for incomplete amputation. This has been applied in Table 6, and
the corrected estimates plotted as solid circles in Fig. 5B. The curve through
311
Cell number in ventral horns
the points is labelled curve II. (It is possible that the number of cells that degenerate after amputation is not equal to the deficit that develops; this would
happen if there was a differential change in immigration rates as well.)
Phase III. The number of cells in phase III is defined as the number of cells
that die in the second or final loss in excess of those normally dying contraaterally; this is equal to the difference between the inter-side differences before
and after final loss, multiplied by the correction factor for incomplete amputation. These have been calculated in Table 7 and plotted as open circles in Fig. 5C,
curve III.
8000
4000
3000
6000 - /
'\
f
\
A
\
'-.
2000
II
B
1000
4000
\
n
\
•
3000 s
v
I + II + IM
2000
2000
*
in
1000 -
1
52
1
1
54
1
1
56
58
62
i
66 JUV.
"
r
y*
l ^j
52
/
*
*
i i i i 1111111
54
56
58
62
C
i
i
66 JUV.
Stage
Fig. 5. The numbers of ventral horn cells in each phase during development. A,
Phase I (continuous line). B, Phase II (filled circles). C, Phase III—estimated by size of
final loss (open circles); by numbers of chromatolytic cells (filled circles). The sum of
the three curves phase I+11+III is plotted in A (interrupted line) and in Fig. 1
(see text).
It might be expected that the number of cells that undergo chromatolysis on
amputation should be related to the number of cells involved in final loss. This
view is supported by the close correspondence in the position of the chromatolytic cells and those finally lost. Both are mainly in the posterior two-thirds.
The numbers of cells either in chromatolysis or in the dense perinuclear
basophilic ring stage of recovery are also plotted as filled circles in Fig. 5C
after correction for incomplete leg removal. The figures for stages 55-56 have
been taken from the same material that Hughes (1961) used. The remaining
data have already been presented in the Tables. Comparison of the two methods
of estimation shows that counts of chromatolytic neurones are unreliable
markers of phase III cells at early stages; that is, when there is little cytoplasm.
378
M. C. PRESTIGE
Table 6
Date of
fixation
Cell deficit
at 3-4 days
Final cell
deficit (%)
Corrected
cell deficit
at 3-4 days
—
Hughes & Tschumi (1958): before stage 52
0
—
0
20. xi.
15. xi.
16. xi.
Series I: stages 52-53
600
)
2030
\ 86
850
J
680
2320
970
21. xi.
Series II: stage 54
1710
(2140)
(80)*
Hughes (1961): stages 55-56
c. 600
62
970
25. xi.
26. xi.
Series 111: stage 57
210
\
0
J 76
280
0
23. i.
13.x.
5.x.
Series IV: stage 61
-140
]
100
[ 57
100
J
-250
180
180
23. iii.
-20
—
Series V: juvenile
37
-60
* The final cell deficit was not determined for the animals of series II.
Table 7
No. of cells ipsilaterally
minus number
contralaterally
i
Stage and source
(a) Before
(b) After
final loss final loss
53 (series I)
No final loss
55-56 (Dr A.
Hughes pers. comm.) 200
57 (series III)
250
61 (series IV)
200
Juveniles
(series V)
0
Estimated
^
(a)-(b)
deficit
(%)
—
Phase III
cells
—
0
0
-810
-1250
- 800
1010
1500
1000
62
76
57
1630
1970
1750
-700
700
37
1900
Cell number in ventral horns
379
This has already been commented on in the section on Materials and Methods.
At later stages, the two methods approximate.
A synthetic total ventral horn cell count curve can then be constructed by
addition of curves I, II and III. This has been plotted in Fig. 1, and 5 A (dashed
line) against the observational data on the ventral horn cell totals on the control
side; considering the paucity of points and the assumption of the linear correction factor, the fit is satisfactory, despite the peak at stage 53. The following
points can be noted:
(1) Phase I cells decrease in number from being the sole constituents of the
ventral horn at stage 52 to almost zero by stage 61.
(2) Phase II cells are only present in observable numbers between stages 52
and 58: they account for half the total numbers around stage 54.
(3) Phase III cells increase in number from zero at stage 53 until at stage 57
they are almost the only type of cell in the ventral horn.
(4) All these maturation processes occur most rapidly at stages 53-56—that
is, when the total ventral horn cell number is decreasing most rapidly and there
is a peak in the number of histogenetically degenerating cells (Hughes, 1961;
Fig. 1).
(5) Small numbers (less than 200) of phase 1, II and III cells could be present
at times other than those of Fig. 5, but not resolved by the analysis.
(6) The counts are made on the state of the ventral horn. They do not
describe the flux of neurones through the phases. To do this would require
knowledge of the pattern of immigration of cells into phase I, and whether this
was also controlled by the periphery, as suggested by the successfully regenerating animals of series I.
How does a ventral horn cell know that the leg has been amputated!
Why does it subsequently die ?
The effects of amputation are ipsilateral only, which excludes any hypothesis
of a systemically borne substance which may cause motor neurones to
degenerate. A direct 'trauma' substance is ruled out for three reasons: first,
because cell death may be postponed for many months after the operation.
Secondly, in the CSL series of Hughes & Tschumi (1958), in which innervation
of the growing leg was hindered by a slit before fibres had grown out, and in
which no subsequent trauma or amputation took place, the only ventral horn
cells which survived were those few in which successful contact with the periphery was established by means of a spinal nerve; the remainder, though they
all completed their initial differentiation, degenerated or regressed. This shows
that it is the absence or interruption of the spinal nerve that is signalled and
leads to cell death (ventral roots and limb nerves are present at all times when
amputation causes degeneration in the ventral horn—unpublished observations
on Palmgren preparations). Thirdly, the cells that die as a result of amputating
the leg below the hip-joint are related to the amputation stump not so much by
24
JEEM l8
380
M. C. PRESTIGE
proximity as by the pattern of spinal nerves; they lie in the posterior two-thirds
of the ventral horn, which supplies spinal nerves 9 and 10. These two spinal
nerves make up the sciatic nerve in the normal plexus with only a small contribution from other sources. Cells of spinal nerve 8, which supply the iliac
musculature by the crural nerve, are largely unaffected.
A number of effects follow the cutting of a peripheral nerve, which involve the
passage of information from the site of the lesion to the cell body. We are here
concerned only with the information that leads to the ultimate death of the cell.
The evidence that it is not due to the mechanical effects of changing the resistance to axoplasmic flow by opening or squashing the axon comes from Weiss &
Hiscoe (1948), who showed that the pressure changes within the fibre which
follow squashing are only local, and so no mechanical information can be passed
back to the cell body; this will be true also of cutting the axon. The injury
discharge that is set up on cutting the axon probably lasts only for a few seconds,
and so this form of information transfer cannot be responsible, nor can it be due
to the injury current, for this would at most only spread electrotonically a few
hundred micra. The information must therefore be sensed either by reduction of
the impulse traffic, or by the absence of a chemical normally transported centripetally, or by central accumulation of a metabolite due to a failure to remove it
centrifugally. It is difficult to see how the last alternative can be correct, for
cutting the axon does not halt growth and is unlikely to stop the removal of the
metabolite. Moreover, axoplasmic damming (Weiss & Hiscoe, 1948) is only
temporary and passes off long before the cell dies in mature cases. Centrifugal
metabolites of this kind would be 'lethal'; centripetal ones would be 'trophic'.
It is possible that trophic information is carried by the impulse traffic; this
would then have to be in sensory nerve fibres, as the direction of travel is wrong
for motor fibres. However, Taylor's (1944) experiment in Rana, in which he
found that motor nerves were not lost, and became functional in the absence of
sensory nerves, suggests that the maintenance information is carried centripetally along motor nerve fibres. The concept of a trophic chemical, transported
intra-axonally in motor nerve fibres, is therefore preferred. Interruption of the
nerve at amputation leads to cell death, due to absence of this 'maintenance
factor'.
The relationship between the maturity of a cell and its ability
to postpone death after cutting of its axon
Phase II cells (by definition) are not able to postpone death after amputation
of the leg. Phase III cells (again by definition) can. The degree to which this is
so depends on the maturity of the cell at the time of the operation. When this
is performed at stages 55-56, final loss takes place after about 3 weeks (Hughes,
1961); after amputation at stage 57, during the fourth week (Table 2); after
amputation at stage 61, during the third and fourth month (Table 4); and after
amputation as juveniles, not until the fourth to seventh months (Table 5).
Cell number in ventral horns
381
Young neurones therefore die after amputation very quickly, while older ones
take longer. Thus the times at which cells die after amputation plot out a maturity
spectrum for the cells in the ventral horn at the time of the operation.
It has already been argued that neurones die because they are no longer
getting something 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 up 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 ventral
horn cell; it is constantly used and the cell dies if the store runs out; and more
mature cells have bigger stores and so can survive longer without replenishment.
Maturity can thus be measured in terms of store size. This mechanism has adaptive significance, for it allows nerve fibres to be broken during natural life
without loss of the parent cell. This also provides a reason, though not an
explanation, why the store is only large enough to allow protein synthesis and
axon growth to proceed far enough to re-establish connexions.
The maintenance factor proposed comes into a general category of cellulipetal neurotrophic substances. It is known that, following section of a peripheral muscle nerve in adults, reinnervation of the muscle can to some extent
reverse the effects of the lesion on the injured nerve cell, and that this must
involve passage of information from the axon tip to the perikaryon (Weiss,
Edds & Cavanaugh, 1945; Aitken, Sharman & Young, 1947; and many others).
Similarly, Evans & Yizoso (1951) showed that the characteristic maturation of
motor nerve fibres in post-natal life is completely dependent on the establishment of peripheral connexions. Recently, Kerkut, Shapira & Walker (1967)
have shown that in an isolated CNS—nerve trunk—muscle preparation,
[14C]labelled material placed in the muscle compartment is carried antidromically along the motor nerves to the CNS.
The maintenance factor hypothesis leaves unanswered any questions as to
the origin of the substance, and as to the nature or location of the store. It may
be asked whether the same mechanism can be extended from these results on
developing neurones to those of axotomy in adults. It may be that some of the
great variety in the timing and severity of reactions to axonal lesions in differing
cell groups comes from the particular quantitative combination of the size of
the store and the change in the supply of maintenance factor. In this connexion,
it is interesting to recall that adult dorsal root ganglion cells survive following
section of the appropriate dorsal root for 2-3 years (Hinsey, Krupp & Lhamon,
1937), whereas after section of the spinal nerve there is substantial cell loss
(Ranson, 1906). For these cells, it apears that it is only the absence of peripheral
connexions that leads to their death, rather than axonal trauma.
24-2
382
M. C. PRESTIGE
The presence of degenerating cells in the ventral horn during normal
development {histogenetic degeneration)
The most plausible explanation is that, at least from stages 53-56, they
represent phase II cells which have failed to develop due to a lack of a maintenance factor from the leg. The evidence supporting this view is:
(1) There is a close correspondence between the shapes of the graphs of the
number of phase II cells (Fig. 5B, curve II) and of the observed numbers of
degenerating cells (Fig. IB; Hughes, 1961).
(2) In series I it was pointed out that this approximation of the capacity to
degenerate after amputation and the capacity for histogenetic degeneration
held even within different zones of the same ventral horn. This suggests
that the two types of cell death occur for the same reason. It has already been
argued that cell death after amputation is due to lack of a maintenance
factor.
(3) It is improbable that they are phase I cells, because the amount of cytoplasm present around the pycnotic nuclei is greater than that associated with
ventral horn cells at stage 52, when all the cells are in phase I. Also the rate of
degeneration can be altered by amputation, while phase I cells are independent
of the leg.
(4) Following incomplete leg amputation, such as that performed in these
experiments, the early loss of cells only reduces the number of phase II cells;
the numbers of phase I and phase III cells remain unaltered by definition.
When the number of phase II cells is thus reduced, the number of cells degenerating is similarly reduced: this was observed in series I-IV between the
excess periods of cell loss.
(5) If for any reason the supply of maintenance factor from the limbs was
liable to fluctuate, phase II cells (when present) would be more likely to die
than phase III cells, being intrinsically more sensitive to such a deficiency.
An alternative hypothesis is that the leg instructs the ventral horn cells to die
by an 'execution warrant' substance travelling centripetally. This would explain
the decrease in number of degenerating cells in the intermediate period after
partial amputation. But it does not explain why any excess cell loss occurs in
the first place. Neither can it account for the cell loss in the CSL series of Hughes
& Tschumi (1958) in which there was no contact between the leg and the ventral
horn. This hypothesis is therefore untenable without the additional hypothesis
of a maintenance factor to explain the effects of amputation, whereas the latter
makes the former unnecessary.
For the later stages of development there are two exceptions to the concept of
histogenetically degenerating cells all being in phase II. First, after stage 57,
no phase II cells are present in the ventral horn (Fig. 5B), yet histogenetic
degeneration continues at a low rate until stage 65. This situation is due to a
limitation in the definition of phase II (the observed cell loss): cells may be
Cell number in ventral horns
383
prevented from degenerating that otherwise would have, or there may be
associated changes in the immigration of cells into the ventral horn. Either of
these events if present, would cause false estimates to be made of the real
number of cells that die after amputation. These would be most acute when the
observed numbers are low, as after stage 57.
The second objection is that the concept does not explain the relative build-up
of cells ipsilaterally after amputation at stages 57 and 61. In each case this is
accompanied by a depression in the ipsilateral rate of degeneration. If this
latter is due only to the decrease in the number of phase II cells following
partial amputation, the continuing decline of numbers on the control side (also
by loss of phase II cells) cannot do more than restore the balance finally. For
the relative pile-up to take place, there must be either prevention of the degeneration of some cells that otherwise would have died, or there must be changes in
the immigration rate. While it is not yet possible to estimate the relative importance of these, there is some evidence that phase III cells do degenerate during
the latter half of normal development. Some of the cells seen with pycnotic
nuclei at these times have as large a quantity of cytoplasm as any others. It may
be that the chromatolytic reaction holds up cell death that otherwise might have
occurred. It is concluded that the cells which die in the ventral horn during
normal development are phase II cells, and that phase III cells may also die in
the later stages.
The decline in cell number during development
The high sensitivity of phase II cells to loss of maintenance factor and the
steadily increasing independence of the phase III cell as it matures means that
in competition between cells of different ages, the older cell has the advantage.
The design of the system is such that achievement by a neurone of even a slightly
larger peripheral connexion than its fellows confers a virtual guarantee that its
own supply of maintenance factor will not dry up. Furthermore, it could cause
the disparity between itself and its fellows to grow greater. This means that the
system produces a small number of neurones, each with a large, stable peripheral
field; the remaining neurones, those which have failed to achieve peripheral
connexions or those whose peripheral field was not large enough, become
redundant and die. In this respect, neurones behave like industrial concerns in
a laissez faire economy.
This arrangement ensures that neurones have an efficient, though not necessarily specific, connexion with the periphery. The establishment of specific, coordinated contacts must require a subsidiary mechanism.
The development of the neuromotor system
In the period between stages 53 and 57, some 2^—3 weeks, and especially the
week of stages 54-55:
(1) Cell number in the ventral horn falls to half its starting figure, accom-
384
M. C. PRESTIGE
panied by the appearance of large numbers of pycnotic nuclei (Hughes,
1961).
(2) At stage 53 ventral horn neurones are small and neuroblastic; at stage 57
they are large cells with dendrites and Nissl substance, cholinesterase-positive
(Hughes, 1961) and acid phosphatase-positive (Palkama & Prestige, 1964).
(3) The leg muscles differentiate (Nieuwkoop & Faber, 1956).
(4) At stage 53 ventral horn cells are still all in phase I; by stage 57 practically all are in phase III.
(5) The neuronal circuitry for the 'flare' and 'stepping' movements of the
leg is established (Hughes & Prestige, 1967).
(6) The spinal cord becomes extremely sensitive to strychnine, indicating the
genesis of post-synaptic inhibition (Hughes & Prestige, 1967).
SUMMARY
1. The development of the lumbar ventral horn cells in Xenopus tadpoles
after amputation of the hind leg at the hip has been studied by counting the
numbers of living, degenerating and chromatolytic cells present at intervals after
the operation.
2. During development, each cell passes through three phases, defined by its
reaction after amputation: the operation either has no effect on the cell (phase I)
or it causes it to degenerate within 3 days (phase II), or it may cause it to chromatolyse first and only degenerate weeks or months later (phase III). Cell loss,
relative to the opposite side, takes place in steps—either early or late or both—
and is accompanied by an excess of degenerating cells. At other times, cell
degeneration, if normally present, is reduced in rate.
3. Removal of the limb-bud has no effect until the palette stage. At the palette
stage, amputation causes a large early but no late loss; at the stepping stage
(hind-leg about 4-5 mm), a small early and a small late loss; at the beginning
of metamorphic climax, a negligible early and a large late loss; at tail loss, no
early and a large late loss; and as juveniles, no early and a large late loss.
4. The numbers of cells in phases I, II and III throughout development have
been independently estimated. The algebraic sum of these three is the same as the
number of cells on the control side for each stage.
5. The period for which cells in phase III can survive after amputation increases with maturity.
6. It is argued that cells in phases II and III die after amputation because they
are no longer getting from the leg an essential 'maintenance factor' which is
normally carried in the motor axons. It is suggested that phase III cells are able
to store this substance, and thus put off death temporarily; and that more
mature cells have larger stores and so can survive longer without replenishment.
Early maturing cells thus have a selective advantage over their fellows.
Cell number in ventral horns
385
RESUME
Regulation du nombre de cellules dans les comes medullaires lombaires
ventrales, au cours du developpement de tetards de Xenopus laevis.
1. On a etudie le developpement des cellules de la corne ventrale lombaire de
tetards de Xenopus apres amputation du membre posterieur, en comptant le
nombre de cellules vivantes, en degenerescence et chromatolytiques presentes a
divers intervalles de temps apres l'operation.
2. Au cours du developpement, chaque cellule passe par trois phases, definies
par sa reaction apres l'amputation: l'operation, ou bien n'a aucun effet sur la
cellule (phase I), ou la fait degenerer dans les 3 jours (phase II), ou peut d'abord
entrainer sa chromatolyse et ensuite sa degenerescence, des semaines ou des
mois plus tard (phase III). La perte en cellules, par rapport au cote oppose, a
lieu par etapes, soit precocement, soit tardivement, ou les deux, et s'accompagne
d'un exces de cellules en degenerescence. A d'autres moments, la degenerescence
cellulaire, si elle est normalement presente, voit son taux reduit.
3. L'ablation du bourgeon de membre n'a pas d'effet jusqu'au stade palette.
Au stade palette, l'amputation provoque une forte perte precoce mais non
tardive; au stade du membre posterieur de 4 a 5 mm, une faible perte precoce
et une faible perte tardive; au debut du climax metamorphotique, une perte
precoce negligeable et une forte perte tardive; a la regression de la queue, pas
de perte precoce et une forte perte tardive, de meme sur des jeunes metamorphoses.
4. On a evalue de maniere independante le nombre de cellules des phases I,
II et III tout au long du developpement. La somme algebrique de ces trois
valeurs est la meme que la nombre de cellules du cote temoin pour chaque
stade.
5. La periode pendant laquelle des cellules en phase III peuvent survivre
apres l'amputation augmente avec la maturite.
6. On suppose que les cellules en phase II et III meurent apres l'amputation
parce qu'elles ne recoivent plus de la patte un 'facteur de maintien' essentiel qui
se trouve normalement transports dans les axones moteurs. On suggere que les
cellules en phase III sont aptes a stocker cette substance et echappent ainsi
temporairement a la mort; et que les cellules plus mures ont des reserves plus
importantes et peuvent ainsi survivre plus longtemps sans 'refaire le plein'. Les
cellules a maturation precoce ont ainsi un avantage selectif sur leurs soeurs.
I am very grateful to Dr A. Hughes for encouragement and advice throughout this work;
to Miss Judith Garraway who ably assisted at all times; and to the Medical Research Council
for support.
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{Manuscript received 24 May 1967, revised 20 July 1967)