J. Embryol. exp. Morph. Vol. 28, 1, pp. 1-11, 1972
Printed in Great Britain
Neurotrophic dependence of
macromolecular synthesis in the early limb
regenerate of the newt, Triturus
By MARCUS SINGER 1 AND J.DOUGLAS CASTON 1
From the Department of Anatomy, Case Western Reserve University,
Cleveland
SUMMARY
The well-documented nerve dependence of limb regeneration in the newt was analysed by
study of accumulation of newly synthesized macromolecules following denervation. The specific activity of RNA and DNA in the denervated early regenerate bud was determined following intraperitoneal injection of [3H]-uridine and [3H]-thymidine. Results showed an outburst
in the incorporation into RNA and DNA which reached a peak 3 h after denervation for the
former and 7 h for the latter. There was then a decline in incorporation to a plateau about
50-60% of the control non-denervated side within 48 h. Combining these results with our
previous demonstration of a similar outburst in the accumulation of newly synthesized protein with a peak at 4 h, the sequence of the outbursts was in order RNA, protein and DNA.
The results are interpreted to mean that the nerve influences either macromolecular synthesis
or macromolecular processing and turnover, and therefore accumulation in the regenerate.
INTRODUCTION
Regeneration of the salamander limb requires the presence of an adequate
nerve supply at the amputation wound (review, Singer, 1952). If the stump is
denervated at the time of amputation or during early limb growth, regeneration
is interrupted and only resumes after nerve fibres have regrown to the amputation site. The nature and the precise effect of the neuronal contribution to regeneration is not known. The agent of the nerve is commonly considered to be
chemical, and its effect is to cause accumulation of mesenchymatous cells and
their subsequent multiplication to form the blastema of regeneration (review,
Thornton, 1970).
There are recent attempts to define, by biochemical means, neurotrophic activity
during regeneration. Dresden (1969) reported that the synthesis of protein, DNA
and RNA during a late stage of regeneration declined after nerve transection.
The greatest rate of change occurred within the first two days and was followed
by a levelling off of macromolecular synthesis which reached a plateau at about
1
Authors' address: Department of Anatomy, School of Medicine and Developmental
Biology Center, Case Western Reserve University, Cleveland, Ohio 44106, U.S.A.
I
EM B 28
2
M. SINGER AND J. D. CASTON
60 % of normal. Studies of protein synthesis in the early regenerate, a stage
more sensitive to nerve transection than the later stage (Singer & Craven,
1948), showed a more rapid decrease reaching a value of about 50-60 % of the
control within about a day after denervation (Lebowitz & Singer, 1970). However, in these studies the decline was preceded by an initial outburst in protein
synthesis which reached a peak at about 3-5 h after denervation. The studies
also showed that crude nerve homogenates when infused into the 48 h denervated regenerate stimulated the recovery of about 50 % or more of the lost
protein synthesis. The evidence was interpreted to mean that the neurotrophic
agent is indeed chemical in nature and that protein synthesis in the denervated
regenerate could serve as an assay of the neuronal effect. The meaning of the
initial outburst in protein synthesis after denervation is not known. The present
paper reports our continuing analysis of the phenomenon and deals with the
influence of nerve transection on overall incorporation of labeled substrates
into RNA and DNA in the blastema stage of regeneration with an emphasis on
the early hours after denervation.
MATERIAL AND METHODS
The forelimb of adult Triturus viridescens, collected in Massachusetts, was
amputated bilaterally in the lower third of the upper arm. The left stump was
denervated 10-13 days after amputation; the right served as the non-denervated
control. A sham operation was performed on the right side in some instances
but not in others; since there was no difference in results, no further mention
will be made of the sham comparison. At 10-13 days postamputation, the regenerate is in the early stage (see stages of Singer, 1952) and consists of a small
mound of blastema covered by a thickened epithelium. Denervation at this stage
stops further regeneration and the blastema withers and is resorbed. Since
significant variation exists among animals in the speed of regrowth, animals in
the same stage of limb regeneration were selected for denervation. Little variation
is exhibited between the two forelimbs of the same animal. Animals were kept at
25 °C throughout the experimental period. To avoid the possible interference of an
anesthetic with neurotrophic activity, the animals were inactivated by wrapping
them in moist cotton with only the operation site exposed. The contact and
pressure stimuli of the wrapping apparently served to minimize response to
painful stimuli. Denervation was performed in the brachial plexus; it involved
transection of spinal nerve 3 and the combined nerve trunk of 4 and 5. The
sympathetic postganglionic fibers, which in the newt follow the arteries into the
limb, were not interrupted; previous studies showed that these fibers by themselves are quantitatively inadequate to sustain regeneration (see review, Singer,
1952).
Except in one experiment, the animals were injected intraperitoneally 3 h
before harvesting. In the one exception to the 3 h 'pulse' time, harvesting of the
Neurotrophic activity in limb regeneration
3
regenerates for RNA determination occurred 2 h after denervation and isotope
injection. For RNA determinations 90/*Ci of [5-3H]-uridine (25-4 Ci/mmole)
in 0-1 ml aqueous solution was injected into each animal. In one of the 5 h
denervation series [2-14C]-uridine (90 JLLC\/animal; specific activity 59-8 mCi/
mmole) was employed; since no difference in results was observed, no further
mention will be made of it. For DNA studies [5-3H]-thymidine (90/iCi/animal;
specific activity 20 Ci/mmole) was used.
At the selected postdenervation time, the regenerates were removed without
anesthesia using an iris scissors and avoiding adult stump tissues as much as
possible. The left (denervated) and right (innervated) regenerates were separately pooled and homogenized in 0-2 ml 5 % cold trichloracetic acid (TCA)
with a small ground glass homogenizer at 0-2 °C. The homogenate was transferred to another tube, the homogenizer rinsed three times with 0 1 ml 5 %
TCA, and the washing combined to yield 0-5 ml final volume of homogenate.
After removing aliquots for determination of total radioactivity, RNA, DNA
and protein were separated from each other by the method of Schmidt & Thannhauser (1945) and measured by the colorimetric methods of Mejbaum (1939),
Burton (1956) and Lowrey, Rosenbrough, Farr & Randall (1951), respectively.
Radioactivity was measured on aliquots of these samples by use of a threechannel liquid scintillation spectrometer equipped with external standardization. The counting fluid was dioxane:anisole:dimethoxyethane (750:125:125)
and contained 7 g PPO (2,5-diphenyl-oxazole) and 0-5 g POPOP (1,4-bis[2-(4-methyl-5-phenyloxazolyl)]-benzene) per litre. The efficiency of counting
was 30 % for 3H and 87 % for 14C.
In selected cases, the recovery and distribution of labeled substrate from the
acid soluble fraction of denervated and innervated blastemas of the several time
periods was checked by first separating the bases, nucleosides and nucleoside
phosphates on thin-layer chromatography sheets (Randerath & Randerath,
1967) and then measuring the distribution of radioactivity with a liquid scintillation spectrometer.
Since the amount of each nucleic acid in a single early regenerate is too small
for reliable determination (approximately 5/tg DNA and 13 peg RNA), the
regenerates from a number of animals were pooled for each postdenervation
time. The regenerates of five animals were found sufficient for DNA determinations, and seven to nine for RNA (see Table 1 and Table 1 (cont.)). The use of
these quantities of blastemas permitted colorimetric measurements by spectral
analysis (Schneider, 1957) on at least three different-sized aliquots of each
sample and always gave readings of more than 0-15 absorbency unit. Hence, a
high degree of confidence can be placed on each individual measurement, the
variation being less than ± 5 %. The average wet weight of an early regenerate, based on 10 readings, was 1-15 mg, S.D. ±0-32.
After correcting for quench, the results were normalized to equivalent volumes
and then expressed as specific activity in counts per minutes (cpm) per micro-
M. SINGER AND J. D. CASTON
160
140
o RNA
DNA
— Protein
120
100
o =?-
80
0. o
60
40\\>
0
0
10
15
20
25
Hours after denervation
45
50
Fig. 1. Specific activity of RNA, DNA and protein of the denervated forelimb
regenerate expressed as percentage of the opposite non-denervated control limb.
Note the sequence of the outburst in macromolecular syntheses in the early hours
after denervation and then the decline to a plateau at about 2 days.
gram (^g) of the labeled macromolecule (see Table 1 and Table 1 (cont.)). The
specific activity for the left denervated regenerate was then expressed as a
fraction of that for the right control side and plotted as a percentage value in
Fig. 1 relative to the non-denervated control which was set at 100 %.
RESULTS
The effect of denervation on the accumulation of newly synthesized macromolecules in the early limb regenerate is shown in Fig. 1 and the essential data
of the experiments are recorded in Table 1 and Table 1 (cont.).
Incorporation of [uC]-leucine into protein of the denervated regenerate
In a previous study (Lebowitz & Singer, 1970) we presented the data for
postdenervation protein synthesis using [14C]-leucine as a marker. The curve
is reproduced in Fig. 1 for comparison with those for RNA and DNA. It
shows a postdenervation rise in the accumulation of newly synthesized protein
culminating in a peak at about 4 h then a continuous decline to a plateau at
about 50 h reflecting about a 45 % loss in protein synthesis. The plateau persisted with a small decrement to 100 h when the experiment was terminated.
We then showed that it is possible to recover partially the loss in ability of the
denervated plastema at the 48 h postdenervation time to accumulate labeled
protein by infusing crude homogenates of intact nerves directly into the nerveless regenerate.
Neurotrophic activity in limb regeneration
5
In the present study we affirmed selected points on the protein synthesis
curve, pooling two to four regenerates. We obtained averages of 0-59 ± 0-05 for
seven readings at 51 h; 0-68 ± 006 for eight readings at 26 h; and 0-85 for two
runs at 12 h.
Incorporation of [sH]-uridine into RNA of the denervated regenerate
As already noted, the amount of RNA in an individual blastema of the early
regenerate stage was too low for reliable determinations. Dresden (1969) employed the 'palette', an advanced stage of development, which weighs about
5 times that of the early regenerate bud; yet, the RNA content of this later stage
was also too small and had to be estimated by normalization with the protein
content. In our studies it was necessary to pool 7-9 regenerating blastemas of
each time period to obtain a reliable measurement of RNA. When this was done,
the determinations as recorded in Table 1 were relatively close for each postdenervation time. The averages are plotted in Fig. 1. The graph depicts an outburst in the accumulation of newly synthesized RNA within the early hours after
denervation. The increase in specific activity of the RNA reached a peak at 3 h,
then fell rapidly to a value of the control at 5 h. The rate of change fell off during
subsequent hours to a plateau of less than about 60 % of the control value.
Initial studies of 72 and 96 h after denervation suggest a further decline to a value
of about 30 % of the control. The peak value for specific activity of RNA was
almost 40 % above that for the control side. The value at 2 h was based upon a
labeling period of 2 rather than 3 h. If the value was normalized to the 3 h
labeling time, it would not alter the curve significantly.
Incorporation of [3H]-thymidine into DNA of the denervated regenerate
The specific activity of DNA likewise showed a similar increase after denervation (Fig. 1 and Table 1 (cont.)). However, the peak value which was at least
1-5 times that of the control was not reached until about 7 h after denervation,
although the onset in the outburst was much earlier. Moreover, the decline
was less precipitous than that for RNA and the increase extended over a longer
period of time. A plateau was also reached similar to that for protein and RNA.
Initial observations at 72 and 96 h suggest that this level of accumulation
persisted at least through 4 days after denervation.
Availability of labelled substrates
During the course of these experiments we have measured the distribution
of labeled nucleoside and nucleoside triphosphate in the denervated and nondenervated blastemas at several time periods following transection of the nerves.
From the results summarized in Table 2, it is apparent that both types of blastema contained similar levels of labeled substrates. Also, it appears that the conversion of the nucleoside to the nucleoside triphosphate reached a steady state
during the first hour after the labeled nucleoside was injected into the animal.
M. SINGER AND J. D. CASTON
Table 1. RNA synthesis in denervated and control regenerates
/tg RNA in
regenerate pool
Hours after
denervation Denerv.
Cpm//tg RNA in
regenerate pool
A
\
A
r
Innerv.
Denerv.
Innerv.^
2
3*
3
3
135
147
89
69
159
138
108
88
286
327
531
930
278
282
354
637
4
4
4
171
72
62
201
74
51
452
773
1264
418
700
1244
5
131
50
122
42
506
643
518
653
12
12
85
91
63
104
752
416
1051
561
24*
27
63
49
59
103
749
468
1107
807
50
119
109
484
845
5t
Cpmjfig
RNA
denerv./
innerv.
103
1-16
1-49
1-46
Av. 1-37
108
1-10
102
Av. 1-06
0-97
0-98
Av. 0-98
0-72
0-74
Av. 0-73
0-68
0-58
Av. 0-63
0-57
Cpm RNA//«g
protein
denerv./innerv.
1-33
106
118
1-40
1-21
119
1-13
105
1-12
1-20
104
1-12
0-82
0-71
0-77
0-71
0-63
0-67
0-53
* Seven animals pooled, f Nine animals pooled. All other runs had 8 pooled animals.
Labeling time was 3 h preceding harvest except for the earliest determination which was
2h.
These results indicate that the differential labeling pattern of macromolecules in
the denervated and non-denervated regenerates was probably due to factors
other than differential availability of radioactive substrates.
The mitotic rate in denervated regenerates
The relation of the outburst of DNA synthesis to the results of previous studies
on mitotic activity in the denervated regenerate (Singer & Craven, 1948) should
be remarked upon. In those studies mitotic counts showed an outburst of mitosis
within 24 h after denervation followed by a precipitous drop to a low level.
Counts were not made before the 24 h period; it may be that the peak occurred
sooner. The outburst was particularly evident in the early regenerate and less so
in later stages. The present biochemical results are in accord with these cytological observations. DNA synthesis reached a peak at about 7 h and the mitotic
outburst occurred sometimes afterward, not later than 24 h postdenervation.
The difference in timing conforms to our present understanding of the relation
between DNA synthesis and mitosis, the peak outburst falling within the S
phase of the cycle.
Neurotrophic
activity in limb
regeneration
Table 1 (cont.)
DNA synthesis in denervated and control regenerates
Cpm//*g D N A in
regenerate pool
/*g D N A in
regenerate pool
Hours after <
denervation '.Denerv.
A
Innerv. \
f
Denerv.
Innerv.
3520
1996
2825
3
3
3
29
25
39
29
20
37
3738
2429
3067
5
5
5
7
27
31
8
23
32
5492
2932
4483
7
7
7
40
22
29
34
20
40
2423
1837
2727
9
9
21
25
20
26
2676
2043
12
13
12
28
33
29
22
35
33
2555
6654
2730
24
24
34
43
32
42
1169
1849
51
51
37
34
44
41
1474
1305
Labeling time was uniformly 3 h preceding
sach analysis.
Cpm//*g
DNA
denerv./
Innerv.
Cpm DNA/^g
protein
Denerv./innerv.
106
1-22
109
Av. 1-12
4075
1-34
1-27
2300
3570
1-26
Av. 1-29
1436
1 69
1178
1-56
1-40
1948
Av. 1-55
1-29
2064
1952
1-05
Av. 1-17
2258
1-13
7083
0-94
2714
100
Av. 102
0-71
1138
2572
0-72
Av. 0-72
0-54
2760
0-51
2558
Av. 0-53
harvesting. Five regenerates were
109
1-47
106
1-21
109
1-56
1-12
1-26
1-49
1-29
1-24
1-34
1-39
1-03
1-21
096
0-85
104
0-95
0-74
0-75
0-75
0-61
0-54
0-58
pooled for
Normalization of RNA and DNA synthesis with the protein content
For comparison with the results of Dresden (1969) in which the RNA and
DNA counts were normalized with the protein content of the sample, protejn
determinations were also made on the homogenate pools used in our experiments. Normalization of the nucleic acid counts with the protein content yielded
values which are recorded in the last column of Table 1 and Table 1 {cont).
Although somewhat erratic, the results are similar to those for the specific
activity of the nucleic acids. Both methods of expressing the results showed an
outburst in accumulation of newly synthesized nucleic acids of the same character
and timing followed by the same sort of decline to a similar plateau.
8
M. SINGER AND J. D. CASTON
Table 2. Recovery of labelled nucleosides and nucleoside triphosphates from
denervated and non-denervated blastemas at different time periods after nerve
transection*
Cpm per /ig blastemal protein
Uridine triphosphate
or
Thymidine triphosphate
Uridine or Thymidine
l~Tf~\iit"o a ft At"
nuuii diici
A
A
r
denervation
Denervate
Non-denervate
Denervate
Non-denervate
I. Uridine injected
1
1
2
2
3
3
5
5
24
24
1255
2569
902
902
993
628
1881
970
674
699
1255
2522
919
890
1327
698
1937
1197
683
666
140
32-5
14-7
13-7
350
22-8
56-2
31-3
21-6
20-1
15-2
30-6
15-9
13-5
43-9
26-8
55-8
37-1
20-4
20-6
II. Thymidine injected
16-5
1017
1010
1
15-4
15-2
1073
1081
1
18-7
11-6
470
476
12-7
3
32-1
1156
938
3
201
30-5
923
804
7
28-9
14-0
587
491
7
14-1
190
597
424
24
14-9
23-3
753
830
24
27-4
* All blastemas were labeled continuously for 3 h excepting those at 1 and 2 h after
denervations which were labeled for• 1 and 2 h respectively.
DISCUSSION
A previous work from this laboratory (Lebowitz & Singer, 1970) showed that
denervation of the early regenerate bud results in an initial outburst in accumulation of newly synthesized protein. This outburst was followed by a decline to a
plateau about 50 % of the control at 48 h. The present work demonstrates a
similar response to denervation in the specific activity of RNA and DNA.
The results thus support the view that a level of control of RNA and DNA
metabolism (and/or accumulation) in some cellular components of the early
regenerate is also nerve dependent.
The decline in accumulation of newly synthesized macromolecules follows
logically from well-established information on the influence of the nerve on
I
Neurotrophic activity in limb regeneration
9
limb regeneration, namely that growth of the young regenerate ceases after
denervation. What is less understandable is the initial outburst in synthesis
and/or accumulation of newly synthesized macromolecules in the early postdenervation hours. Dresden (1969) reported a decline but not an outburst in
protein, RNA and DNA synthesis. However, his results cannot be strictly compared to ours because his earliest postdenervation reading was 7 h, whereas
the outbursts seen in our experiment peaked at 3 and 7 h for RNA and DNA
respectively; and by 7 h protein synthesis had returned to a normal level and
RNA synthesis was already greatly depressed. Moreover, he used a later
regenerate, whereas we employed the early bud which is affected more profoundly by denervation (Singer, 1952); also, his analytical procedures were
quite different from ours. An outburst in RNA and protein synthesis in the
lateral geniculate nucleus of the monkey following transection of the optic
nerve was reported by Kupfer & Downer (1967). It persisted for 2 days and was
then followed by a prolonged decline to a subnormal level, in the case of RNA
to about 30 %.
In a previous paper (Lebowitz & Singer, 1970) we likened the outburst in
accumulation of protein to the supersensitivity of denervated effectors to stimulating agents, a phenomenon embodied in W. B. Cannon's Law of Denervation
(1939) which states: 'When in a series of efferent neurons a unit is destroyed, an
increased irritability to chemical agents develops in the isolated structure or
structures, the effects being maximal in the part directly denervated' (see also
Cannon & Rosenblueth, 1949). However, the law defines physiological phenomena and not biochemical changes although Cannon & Rosenblueth speculated
upon the chemical basis for the increased sensitivity. Furthermore, the outburst
in accumulation of macromolecules develops within hours of denervation
whereas the physiological changes are much slower in onset and are pronounced
only days later. The difference in time course may mean that it is not the initial
outburst but rather the decline in accumulation of macromolecules that is the
chemical basis for the subsequent altered sensitivity if, indeed, these biochemical
changes are directly related to altered sensitivity. However, it may be that
biochemical changes other than those measured here cause altered sensitivity
and that they may occur within the first few hours of denervation.
The mechanism whereby nerve interruption alters the synthesis and accumulation of macromolecules is not elucidated in the present experiments. Perhaps
cutting the nerve releases a nervous restraint on macromolecular synthesis which
in a short time is reasserted by controls within the synthetic mechanisms themselves. Assuming that the neurotrophic agent is chemical in nature, an assumption for which experimental evidence is already presented (Lebowitz & Singer,
1970), it may be that the outburst occurs after exhaustion of the neurotrophic
agent from the cut nerve and reflects an 'overshooting' of the synthetic
mechanisms before a new equilibrium is established. Alternatively, one may
imagine that the initial amplification reflects increased release of the chemical
10
M. SINGER AND J. D. CASTON
agent due to the transection causing a corresponding augmentation in macromolecular synthesis, and that the later decline to a plateau reflects exhaustion
of the contribution. If the observed outbursts are due to exhaustion or to
hurried emptying of the trophic substance, it is possible to calculate the approximate speed of movement of the neurotrophic agent in the axon based on the
observed time of the outburst. The length of the distal segment of the transected
nerve from the brachial plexus to the regenerate was about 15 mm and the outburst in RNA synthesis began to rise at about 2 h. Therefore, the velocity in the
transected part is in the order of 180 mm per day. Such a velocity places the
trophic agent among the faster components of axoplasmic flow (compare
Lasek, 1970).
We have avoided the use of the term 'synthesis' in reporting the effect of
denervation on the macromolecular content of the regenerate because our experiments do not distinguish between synthesis and accumulation. It may be that
the primary effect is on control systems which regulate turnover of macromolecules and only indirectly macromolecular synthesis. The neurotrophic influence
may be likened in this way to the reported effect of phytohaemagglutinin on the
lymphocyte. In the 'resting' lymphocyte rRNA wastage appears to be very high;
but upon addition of the growth stimulant, the waste is diminished dramatically
(Cooper, 1968, 1969). Our data also do not reveal the primary target of the
nerve effect. It may be RNA or its polymerase since the RNA response to
denervation precedes that of protein and of DNA.
The authors are grateful for the assistance of Mrs Kai-Yu Clara Lin and Charles S. Maier.
This work was supported by grants from the National Multiple Sclerosis Society, the American Cancer Society and the National Institutes of Health.
i
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{Manuscript received 22 November 1971, revised 2 March 1972)