J. Embryol. exp. Morph. Vol. 28, 1, pp. 1-11, 1972

Printed in Great Britain

Neurotrophic dependence ofmacromolecular synthesis in the early limb

regenerate of the newt, Triturus

By MARCUS SINGER1 AND J.DOUGLAS CASTON1

From the Department of Anatomy, Case Western Reserve University,Cleveland

SUMMARY

The well-documented nerve dependence of limb regeneration in the newt was analysed bystudy of accumulation of newly synthesized macromolecules following denervation. The speci-fic activity of RNA and DNA in the denervated early regenerate bud was determined follow-ing intraperitoneal injection of [3H]-uridine and [3H]-thymidine. Results showed an outburstin the incorporation into RNA and DNA which reached a peak 3 h after denervation for theformer and 7 h for the latter. There was then a decline in incorporation to a plateau about50-60% of the control non-denervated side within 48 h. Combining these results with ourprevious demonstration of a similar outburst in the accumulation of newly synthesized pro-tein 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 synthesisor macromolecular processing and turnover, and therefore accumulation in the regenerate.

INTRODUCTION

Regeneration of the salamander limb requires the presence of an adequatenerve supply at the amputation wound (review, Singer, 1952). If the stump isdenervated at the time of amputation or during early limb growth, regenerationis interrupted and only resumes after nerve fibres have regrown to the amputa-tion site. The nature and the precise effect of the neuronal contribution to re-generation is not known. The agent of the nerve is commonly considered to bechemical, and its effect is to cause accumulation of mesenchymatous cells andtheir subsequent multiplication to form the blastema of regeneration (review,Thornton, 1970).

There are recent attempts to define, by biochemical means, neurotrophic activityduring regeneration. Dresden (1969) reported that the synthesis of protein, DNAand RNA during a late stage of regeneration declined after nerve transection.The greatest rate of change occurred within the first two days and was followedby a levelling off of macromolecular synthesis which reached a plateau at about

1 Authors' address: Department of Anatomy, School of Medicine and DevelopmentalBiology 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 stagemore sensitive to nerve transection than the later stage (Singer & Craven,1948), showed a more rapid decrease reaching a value of about 50-60 % of thecontrol within about a day after denervation (Lebowitz & Singer, 1970). How-ever, in these studies the decline was preceded by an initial outburst in proteinsynthesis which reached a peak at about 3-5 h after denervation. The studiesalso showed that crude nerve homogenates when infused into the 48 h dener-vated regenerate stimulated the recovery of about 50 % or more of the lostprotein synthesis. The evidence was interpreted to mean that the neurotrophicagent is indeed chemical in nature and that protein synthesis in the denervatedregenerate could serve as an assay of the neuronal effect. The meaning of theinitial outburst in protein synthesis after denervation is not known. The presentpaper reports our continuing analysis of the phenomenon and deals with theinfluence of nerve transection on overall incorporation of labeled substratesinto RNA and DNA in the blastema stage of regeneration with an emphasis onthe early hours after denervation.

MATERIAL AND METHODS

The forelimb of adult Triturus viridescens, collected in Massachusetts, wasamputated bilaterally in the lower third of the upper arm. The left stump wasdenervated 10-13 days after amputation; the right served as the non-denervatedcontrol. A sham operation was performed on the right side in some instancesbut not in others; since there was no difference in results, no further mentionwill be made of the sham comparison. At 10-13 days postamputation, the re-generate is in the early stage (see stages of Singer, 1952) and consists of a smallmound of blastema covered by a thickened epithelium. Denervation at this stagestops further regeneration and the blastema withers and is resorbed. Sincesignificant variation exists among animals in the speed of regrowth, animals inthe same stage of limb regeneration were selected for denervation. Little variationis exhibited between the two forelimbs of the same animal. Animals were kept at25 °C throughout the experimental period. To avoid the possible interference of ananesthetic with neurotrophic activity, the animals were inactivated by wrappingthem in moist cotton with only the operation site exposed. The contact andpressure stimuli of the wrapping apparently served to minimize response topainful stimuli. Denervation was performed in the brachial plexus; it involvedtransection of spinal nerve 3 and the combined nerve trunk of 4 and 5. Thesympathetic postganglionic fibers, which in the newt follow the arteries into thelimb, were not interrupted; previous studies showed that these fibers by them-selves are quantitatively inadequate to sustain regeneration (see review, Singer,1952).

Except in one experiment, the animals were injected intraperitoneally 3 hbefore 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 isotopeinjection. 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 hdenervation series [2-14C]-uridine (90 JLLC\/animal; specific activity 59-8 mCi/mmole) was employed; since no difference in results was observed, no furthermention 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 withoutanesthesia using an iris scissors and avoiding adult stump tissues as much aspossible. The left (denervated) and right (innervated) regenerates were separ-ately 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 trans-ferred to another tube, the homogenizer rinsed three times with 01 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, DNAand protein were separated from each other by the method of Schmidt & Thann-hauser (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 three-channel liquid scintillation spectrometer equipped with external standardiza-tion. 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 countingwas 30 % for 3H and 87 % for 14C.

In selected cases, the recovery and distribution of labeled substrate from theacid soluble fraction of denervated and innervated blastemas of the several timeperiods was checked by first separating the bases, nucleosides and nucleosidephosphates on thin-layer chromatography sheets (Randerath & Randerath,1967) and then measuring the distribution of radioactivity with a liquid scintil-lation spectrometer.

Since the amount of each nucleic acid in a single early regenerate is too smallfor reliable determination (approximately 5/tg DNA and 13 peg RNA), theregenerates from a number of animals were pooled for each postdenervationtime. The regenerates of five animals were found sufficient for DNA determina-tions, and seven to nine for RNA (see Table 1 and Table 1 (cont.)). The use ofthese quantities of blastemas permitted colorimetric measurements by spectralanalysis (Schneider, 1957) on at least three different-sized aliquots of eachsample and always gave readings of more than 0-15 absorbency unit. Hence, ahigh degree of confidence can be placed on each individual measurement, thevariation being less than ± 5 %. The average wet weight of an early regener-ate, based on 10 readings, was 1-15 mg, S.D. ±0-32.

After correcting for quench, the results were normalized to equivalent volumesand then expressed as specific activity in counts per minutes (cpm) per micro-

M. SINGER AND J. D. CASTON

o =?-

0. o

160

140

120

100

80

60

40\\>

0

o RNADNA

— Protein

0 10 15 20 25Hours after denervation

45 50

Fig. 1. Specific activity of RNA, DNA and protein of the denervated forelimbregenerate expressed as percentage of the opposite non-denervated control limb.Note the sequence of the outburst in macromolecular syntheses in the early hoursafter 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.)). Thespecific activity for the left denervated regenerate was then expressed as afraction of that for the right control side and plotted as a percentage value inFig. 1 relative to the non-denervated control which was set at 100 %.

RESULTS

The effect of denervation on the accumulation of newly synthesized macro-molecules in the early limb regenerate is shown in Fig. 1 and the essential dataof 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 forpostdenervation protein synthesis using [14C]-leucine as a marker. The curveis reproduced in Fig. 1 for comparison with those for RNA and DNA. Itshows a postdenervation rise in the accumulation of newly synthesized proteinculminating in a peak at about 4 h then a continuous decline to a plateau atabout 50 h reflecting about a 45 % loss in protein synthesis. The plateau per-sisted 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 thedenervated plastema at the 48 h postdenervation time to accumulate labeledprotein by infusing crude homogenates of intact nerves directly into the nerve-less regenerate.

Neurotrophic activity in limb regeneration 5In 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 forseven readings at 51 h; 0-68 ± 006 for eight readings at 26 h; and 0-85 for tworuns 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 earlyregenerate stage was too low for reliable determinations. Dresden (1969) em-ployed the 'palette', an advanced stage of development, which weighs about5 times that of the early regenerate bud; yet, the RNA content of this later stagewas also too small and had to be estimated by normalization with the proteincontent. In our studies it was necessary to pool 7-9 regenerating blastemas ofeach 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 post-denervation time. The averages are plotted in Fig. 1. The graph depicts an out-burst in the accumulation of newly synthesized RNA within the early hours afterdenervation. 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 duringsubsequent 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 valueof about 30 % of the control. The peak value for specific activity of RNA wasalmost 40 % above that for the control side. The value at 2 h was based upon alabeling period of 2 rather than 3 h. If the value was normalized to the 3 hlabeling 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 denerva-tion (Fig. 1 and Table 1 (cont.)). However, the peak value which was at least1-5 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 declinewas less precipitous than that for RNA and the increase extended over a longerperiod 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 accumulationpersisted at least through 4 days after denervation.

Availability of labelled substrates

During the course of these experiments we have measured the distributionof labeled nucleoside and nucleoside triphosphate in the denervated and non-denervated blastemas at several time periods following transection of the nerves.From the results summarized in Table 2, it is apparent that both types of blas-tema contained similar levels of labeled substrates. Also, it appears that the con-version of the nucleoside to the nucleoside triphosphate reached a steady stateduring 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

Hours afterdenervation

23*33

444

5t5

1212

24*27

50

/tg RNA inregenerate pool

Denerv.

1351478969

1717262

13150

8591

6349

119

A\

Innerv.

15913810888

2017451

12242

63104

59103

109

Cpm//tg RNA inregenerate pool

ArDenerv.

286327531930

452773

1264

506643

752416

749468

484

^Innerv.

278282354637

418700

1244

518653

1051561

1107807

845

CpmjfigRNA

denerv./innerv.

1031-161-491-46

Av. 1-37

1081-10102

Av. 1-06

0-970-98

Av. 0-98

0-720-74

Av. 0-73

0-680-58

Av. 0-63

0-57

Cpm RNA//«gprotein

denerv./innerv.

1-331061181-401-21

1191-131051-12

1-201041-12

0-820-710-77

0-710-630-67

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 inthe denervated and non-denervated regenerates was probably due to factorsother 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 studieson mitotic activity in the denervated regenerate (Singer & Craven, 1948) shouldbe remarked upon. In those studies mitotic counts showed an outburst of mitosiswithin 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 occurredsooner. The outburst was particularly evident in the early regenerate and less soin later stages. The present biochemical results are in accord with these cyto-logical observations. DNA synthesis reached a peak at about 7 h and the mitoticoutburst occurred sometimes afterward, not later than 24 h postdenervation.The difference in timing conforms to our present understanding of the relationbetween DNA synthesis and mitosis, the peak outburst falling within the Sphase of the cycle.

Neurotrophic activity in limb regeneration

Table 1 (cont.)

DNA synthesis in denervated and control regenerates

Hours after <denervation '.

333

555

777

99

121312

2424

5151

/*g DNA inregenerate pool

Denerv.

292539

72731

402229

2125

283329

3443

3734

A \Innerv.

292037

82332

342040

2026

223533

3242

4441

Labeling time was uniformly 3 hsach analysis.

Cpm//*g DNA inregenerate pool

f

Denerv.

373824293067

549229324483

242318372727

26762043

255566542730

11691849

14741305

Innerv.

352019962825

407523003570

143611781948

20641952

225870832714

11382572

27602558

preceding harvesting. Five

Cpm//*gDNA

denerv./Innerv.

1061-22109

Av. 1-12

1-341-271-26

Av. 1-29

1 691-561-40

Av. 1-55

1-291-05

Av. 1-17

1-130-94100

Av. 102

0-710-72

Av. 0-72

0-540-51

Av. 0-53

regenerates

Cpm DNA/^gprotein

Denerv./innerv.

1091-471061-21

1091-561-121-26

1-491-291-241-34

1-391-031-21

0960-851040-95

0-740-750-75

0-610-540-58

were pooled for

Normalization of RNA and DNA synthesis with the protein content

For comparison with the results of Dresden (1969) in which the RNA andDNA counts were normalized with the protein content of the sample, protejndeterminations were also made on the homogenate pools used in our experi-ments. Normalization of the nucleic acid counts with the protein content yieldedvalues 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 specificactivity of the nucleic acids. Both methods of expressing the results showed anoutburst in accumulation of newly synthesized nucleic acids of the same characterand 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 fromdenervated and non-denervated blastemas at different time periods after nervetransection*

l~Tf~\iit"o a ft At"nuuii diici

denervation

I. Uridine injected

11

2233

55

2424

II. Thymidine injected

11

33

77

2424

Cpm per /ig blastemal protein

Uridine or Thymidine

Denervate

12552569

902902993628

1881970

674699

10171073

4701156

923587

597753

A

Non-denervate

12552522

919890

1327698

19371197

683666

10101081

476938

804491

424830

* All blastemas were labeled continuously for 3 hdenervations which were labeled for

Uridine triphosphateor

Thymidine triphosphateAr

Denervate

14032-5

14-713-735022-8

56-231-3

21-620-1

16-515-2

11-632-1

30-514-0

19023-3

excepting those at• 1 and 2 h respectively.

Non-denervate

15-230-6

15-913-543-926-8

55-837-1

20-420-6

15-418-7

12-7201

28-914-1

14-927-4

1 and 2 h after

DISCUSSION

A previous work from this laboratory (Lebowitz & Singer, 1970) showed thatdenervation of the early regenerate bud results in an initial outburst in accumula-tion of newly synthesized protein. This outburst was followed by a decline to aplateau about 50 % of the control at 48 h. The present work demonstrates asimilar 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 DNAmetabolism (and/or accumulation) in some cellular components of the earlyregenerate is also nerve dependent.

The decline in accumulation of newly synthesized macromolecules followslogically from well-established information on the influence of the nerve on

INeurotrophic activity in limb regeneration 9

limb regeneration, namely that growth of the young regenerate ceases afterdenervation. What is less understandable is the initial outburst in synthesisand/or accumulation of newly synthesized macromolecules in the early post-denervation hours. Dresden (1969) reported a decline but not an outburst inprotein, RNA and DNA synthesis. However, his results cannot be strictly com-pared to ours because his earliest postdenervation reading was 7 h, whereasthe outbursts seen in our experiment peaked at 3 and 7 h for RNA and DNArespectively; and by 7 h protein synthesis had returned to a normal level andRNA synthesis was already greatly depressed. Moreover, he used a laterregenerate, whereas we employed the early bud which is affected more pro-foundly by denervation (Singer, 1952); also, his analytical procedures werequite different from ours. An outburst in RNA and protein synthesis in thelateral geniculate nucleus of the monkey following transection of the opticnerve was reported by Kupfer & Downer (1967). It persisted for 2 days and wasthen followed by a prolonged decline to a subnormal level, in the case of RNAto about 30 %.

In a previous paper (Lebowitz & Singer, 1970) we likened the outburst inaccumulation of protein to the supersensitivity of denervated effectors to stimu-lating 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, anincreased irritability to chemical agents develops in the isolated structure orstructures, the effects being maximal in the part directly denervated' (see alsoCannon & Rosenblueth, 1949). However, the law defines physiological pheno-mena and not biochemical changes although Cannon & Rosenblueth speculatedupon the chemical basis for the increased sensitivity. Furthermore, the outburstin accumulation of macromolecules develops within hours of denervationwhereas the physiological changes are much slower in onset and are pronouncedonly days later. The difference in time course may mean that it is not the initialoutburst but rather the decline in accumulation of macromolecules that is thechemical basis for the subsequent altered sensitivity if, indeed, these biochemicalchanges are directly related to altered sensitivity. However, it may be thatbiochemical changes other than those measured here cause altered sensitivityand that they may occur within the first few hours of denervation.

The mechanism whereby nerve interruption alters the synthesis and accumula-tion of macromolecules is not elucidated in the present experiments. Perhapscutting the nerve releases a nervous restraint on macromolecular synthesis whichin a short time is reasserted by controls within the synthetic mechanisms them-selves. Assuming that the neurotrophic agent is chemical in nature, an assump-tion for which experimental evidence is already presented (Lebowitz & Singer,1970), it may be that the outburst occurs after exhaustion of the neurotrophicagent from the cut nerve and reflects an 'overshooting' of the syntheticmechanisms before a new equilibrium is established. Alternatively, one mayimagine 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 macro-molecular synthesis, and that the later decline to a plateau reflects exhaustionof the contribution. If the observed outbursts are due to exhaustion or tohurried emptying of the trophic substance, it is possible to calculate the approxi-mate speed of movement of the neurotrophic agent in the axon based on theobserved time of the outburst. The length of the distal segment of the transectednerve from the brachial plexus to the regenerate was about 15 mm and the out-burst in RNA synthesis began to rise at about 2 h. Therefore, the velocity in thetransected part is in the order of 180 mm per day. Such a velocity places thetrophic agent among the faster components of axoplasmic flow (compareLasek, 1970).

We have avoided the use of the term 'synthesis' in reporting the effect ofdenervation on the macromolecular content of the regenerate because our experi-ments do not distinguish between synthesis and accumulation. It may be thatthe primary effect is on control systems which regulate turnover of macromole-cules and only indirectly macromolecular synthesis. The neurotrophic influencemay be likened in this way to the reported effect of phytohaemagglutinin on thelymphocyte. 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 thenerve effect. It may be RNA or its polymerase since the RNA response todenervation 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 Ameri-can Cancer Society and the National Institutes of Health.

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{Manuscript received 22 November 1971, revised 2 March 1972)