Download Amphibian Limb Regeneration and its Relation to Nerves

A M . ZOOLOGIST, 10:113-118 (1970).
Amphibian Limb Regeneration and its Relation to Nerves
CHARLES S. THORNTON
Department of Zoology, Michigan State University,
East Lansing, Michigan 48823
SYNOPSIS. Much circumstantial evidence points to a neurotrophic influence in amphibian limb regeneration. Although fine-structural observations of nerves in regenerating
limbs have indicated the possibility that neurosecretory vesicles accumulate distally in
these axons, there is no clear-cut demonstration available that these organelles are
neurotropic. Evidence is accumulating that the neural influence in newt limb
regeneration is transneuronal. There is also evidence that trophic substances other
than those found in the nerve itself may be involved in the supporting limb
regeneration. The characterization of the neurotrophic substance is considered a central
task for students of regeneration in the future.
THE ROLE OF THE NERVE
True limb regeneration, as illustrated in
the salamander, is dependent on the accumulation of a mass of mesenchymatous
cells at the tip of the stump beneath the
wound epithelium. These cells, the bluslemal cells, are derived from a limited dissociation ("dedifferentiation") of injured
stump tissues, and the uniqueness of the
phenomenon in the salamander's limb is
that these cells do aggregate and proliferate to form a single rudiment, or blastema,
from which the missing parts of the limb
are reconstituted. In the mammal, or even
in the adult frog, limb-stump tissues also
undergo a limited "dedifferentiation," but
relatively few such mesenchymatous cells
are produced and they are utilized immediately for repair of the injured tissue that
gave rise to them. They do not aggregate
to form an apical blastema. Repair of tissues in the adult newt limb can apparently
proceed in the absence of nerves, but the
accumulation of a blastema can not. The
importance of the nerves for newt limb
regeneration was first recorded by Todd
(1823) and much later by Schottc (1926)
and others who concluded that the sympathetic innervation of the limb exerted an
important control on regeneration. Singer
(1952, for review) in an admirable series
Supported by grants-in-aid from the National
Science Foundation (GB-2618; GB-7748) and the
National Institutes of Health (NB-04128).
of studies, determined that the qualitative
nature of the nerve was of little importance for newt limb regeneration, but that
the number of nerves at the amputation
surface was of critical significance. Indeed,
he discovered that if the ratio of nerve
fibers to amputation surface area fell below approximately 9 per (100 /x)2 regeneration failed. Furthermore, the neural influence is local. Thus Kamrin and Singer
(1959) transplanted sensory ganglia of
newts into young blastemata of denervated
newt limb stumps and obtained continued
regeneration despite the fact that no central nervous connection was available to
the amputated limb.
The neural influence is more critical for
early stages of limb regeneration than for
later ones. In Ambysloma larvae, denervalion of regenerating limbs inhibited continued regeneration if the neural deprivation
occtirred before 9 days post-amputation
(Schottc and Butler, 1944), or before 17
days in the adult newt (Singer and
Craven, 1948). These critical phases of regeneration correspond with the initiation
of the period of great blastemal growth. It
is not surprising, therefore, that Singer and
Craven (1948) found that denervation of
the regenerating newt limb inhibited mitotic proliferation of the blastemal cells.
Recently, Dresden (1969) has analyzed
biochemically the effects of denervation of
the paddle-stage blastema in the newt. He
finds that denervation decreases markedly
113
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CHARLES S. THORNTON
the synthesis of RNA, DNA, and protein. ter of the axons in these fibers is signifiSynthesis of RNA decreases within 7 hours cantly greater, so that the total amount of
after denervation and synthesis of DNA neuroplasm at the amputation surface in
and protein is affected only after 24 hours. Xenopus limbs is equivalent to that of the
Since these effects are obtained also in de- newt. As the authors point out, these renervated, but not in innervated, blastema- sults are interpretable in terms of an axonta cultured for 20 hours in vitro, Dresden fiow mechanism of TS transport. Since the
suggests that they indicate a direct control original demonstration of axon flow by
by the nerve on synthesis of DNA, RNA, Weiss and Hiscoe (1948), there have been
and protein in the blastema.
many confirmations and in a variety of
Xhe mechanism of the "neurotrophic" animals (Weiss, 1969). Indeed, in the cat,
effect in newt limb regeneration has been for example, there has been described both
extensively investigated, particularly by a fast and a slow rate of axoplasmic flow
Singer and his associates (Singer, 1960, (Ochs, Sabri, and Johnson, 1969), alfor review). An early theory that sympa- though the mechanisms responsible for
thetic nerves were chiefly responsible for these rates of transport are unknown.
the neurotrophic effect led Schotte (1926)
Morphological evidence of a possible
to apply various drugs associated with met- neurosecretory material in nerve fibers of
abolism of sympathetic nerves, but without regenerating amphibian limbs is suggestive
success. Taban (1955) also failed to in- but not fully convincing. Inoue (1960)
duce regeneration in denervated limbs by describes vesicles of 300A to 600A in axons
injecting acetylcholine and other neurody- of the regenerating newt limb but is
namic substances. Singer (1960), ap- doubtful that these are neurosecretory
proaching the problem from the other di- granules. Hay (1960) also describes vesirection, infused into regenerating limbs a cles of 300A to 1000A in diameter which
variety of substances known to block the accumulate in the end bulbs of nerve fibers
acetylcholine mechanism—atropine, pro- penetrating the apical epidermal cap of
caine hydrochloride, tetraethylammonium regenerating larval limbs of Ambystoma.
hydroxide—and stopped further regener- She speculates, within the limits of her
ation. The toxicity of the concentrations data, that "the morphology of these nerves
used, however, caused him to doubt the invites interpretation in terms of a trophspecificity of their action on regeneration. ic neurosecretory material which is manuThe nature of the trophic substance (TS) factured in the perikaryon, travels down
still remains unknown. Whatever its the nerve fiber in the endoplasmic reticunature, Singer (1965) proposes that TS is lum and, when released, stimulates epiderproduced in great abundance in the neu- mal hyperplasia" (page 314). Van Arsdall
ron primarily to maintain its great mass of and Lentz (1968) also have described vesiactive neuroplasm, but that significant cles, (1000-2500A in diameter) filled with a
amounts spill over onto other tissues which moderately dense material, which are
then come to depend on the nerve for found in nerve fibers of regenerating limbs
their own regenerative activity.
of newts. These same nerve fibers conThe transport of TS to the limb tissues tained material which stained with aldeis by way of sensory as well as motor nerve hyde fuchsin, a classical stain for neurosefibers. More important than the quality of cretory granules. Staining of the nerve
the nerve is the size of the axon. Singer, fibers in the blastema was observed from
Rzehak, and Maier (1967), for example, 14 to 28 days postamputation, a stage of
have shown that although the number of regeneration, however, (Singer and Cravnerve fibers present in regenerating limbs en, 1948), when the blastema is losing its
of Xenopus is below the threshold level dependence on nerves.
characteristic of the newt limb, the diameIn my laboratory, some particularly in-
REGENERATION1 OF LIMBS
O 10
15
20
25
30
35
DAYS AFTER AMPUTATION
FIG. 1. Comparison of mean lengths o£ limb regenerates distal to the level of amputation for five
groups of Ambystoma mexicanum larvae (n=55).
These groups consisted of amputation of: A. one
forelimb; B. one forelimb and one hindlimb; C. one
hindlimb; D. both hindlimbs; E. both forelimbs. Measurements of length were made with an
optical micrometer (1 micrometer unit = 0.15mm).
In animals with both forelimbs and those with
both hindlimbs amputated, just the forelimb regenerate was measured. At 35 days after amputation a one-way analysis of variance and new multiple range test showed that there was a significant
difference (P<0.05) between the upper three
groups on the graph (denoted by solid lines) and
the lower two groups (denoted by dashed lines).
teresting experiments by Charles Tweedle
(I969a,b) further illuminate the mechanisms of interaction between nerves and regeneration of limbs. His work began with
an investigation of how amputation of one
limb might affect the rate and morphogenesis of regeneration of a second
limb in the adult newt. Surprisingly, it was
found that amputation of two limbs
caused a significantly slower rate of regeneration than is found after amputation of
one limb, but only if the two limbs removed were contralateral (Fig. 1). It
would seem, therefore, that the amount of
tissue removed did not significantly affect
the rate of regeneration but that from
where it was removed did. In seeking an
explanation, Tweedle recalled the early
experiments of Detwiler (1936, for review) in which extirpation of the contralateral limb discs in salamander embryos caused a greater hypoplasia of the
115
associated sensory ganglia than did removal of a single limb disc. He, therefore,
investigated the effects on nerve cell bodies
in the brachial sensory ganglia (as well as
in spinal motor horns) of amputating one
and two forelimbs in adult newts and in
axolotl larvae. Nuclei of these neurons
showed typical chromatolytic changes after
amputation of one forelimb. Of particular
interest, however, was the fact that neurons in the opposite brachial ganglia (and
motor horns) also showed chromatolytic
effects, although not as severe. Chromatolysis in neurons of brachial ganglia (and
motor horns) was more intense and lasted
longer when both forelimbs were amputated. It is known that synthesis of RNA
increases in chromatolytic neurons (Cole,
1968). Tweedle, therefore, injected Hsuridine intraperitoneally into adult newts
with (a) no limbs amputated; or (b)
with one forelimb amputated; or (c) with
two forelimbs amputated. Newts with
one forelimb amputated exhibited statistically more uptake of H3-uridine in motor
horn and sensory ganglionic neurons of
both sides of the spinal cord than did
unamputated controls; newts with both
forelimbs amputated incorporated significantly more label still, and for a longer
period of time. These results pointed to a
transneuronal effect whereby a greater degree of chromatolysis accompanied amputation of both forelimbs. This increased
nerve reaction is thought to lessen the normal trophic ability of the nerve and thus
bring about a slower rate of regeneration
in the limbs. Evidence for a transneuronal
effect was further strengthened when
Tweedle was able to demonstrate, by the
method of Fink and Heimer (1967), that
degeneration of nerve fibers could be seen
in the motor horns of both sides of the
brachial spinal cord for 7 days after the
amputation of one forelimb. Furthermore,
amputation of both aneurogenic forelimbs
in Ambystoma larvae resulted in rates of
regeneration statistically indistinguishable
from those found in amputated, single
aneurogenic forelimbs. In these cases a
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CHARLES S. THORNTON
iransneuronal effect is eliminated since the
spinal cord was removed in the tailbud
embryonic stage.
Suggestive as the data may be, morphological and experimental studies have nevertheless failed to demonstrate incontrovertibly a mechanism for a neurotrophic
control of limb regeneration. 1 find it surprising, therefore, that more attention has
not been given to Overton's (1950; 1955)
interesting discovery that a protein found
in spinal cord stimulates dramatic growth
of the tail fin epidermis in Ambystomn
larvae. This system surely needs further
analysis and may provide insights into the
neurotrophic mechanism of regeneration
which have eluded us so far.
THE ROLE OF NON-NEURAL LIMB TISSUES
Under the impact of new evidence that
nerves are not always needed for regeneration, the original neurotrophic theory of
regeneration has recently undergone considerable refinement (Singer, 1965). Thus,
Yntema (\9b9a,b; 1962), Thornton and
Steen (1962), Steen and Thornton (1963),
and Thornton and Tassava (1969) have
described regeneration, under a variety of
conditions, of aneurogenic limbs produced
by excising the neural tube of tailbud embryos of Ambystoma macula turn. To account for this apparently decisive negation
of the neurotrophic theory of regeneration, Singer (1965) proposed the possibility that the trophic substance (TS) was
not necessarily limited to the neuron but
that in embryos other cell types could also
manufacture it. During ontogeny, he suggested, the neuron synthesizes much more
TS than other cells and the excess, bathing
the limb tissues, quenches the production
of TS in them, so that they come to depend on this neural supply for their own
growth. One can, therefore, visualize a
type of feedback of end-product in which
the abundant neural TS gradually inhibits synthesis of TS in non-neural limb
tissues by repressing specific biochemical
mechanisms. If such a repression is involved, then one might expect that pro-
longed denervation of the limb might result in a return of TS-synthesis in nonneural tissues no longer under neural inhibition. This has not been found in denervated adult newt limbs (see Powell, 1969).
Perhaps the long-continued functioning
of limb nerves during ontogeny and later
development and growth produces such a
strong inhibition that interventions in addition to simple nerve withdrawal are
needed to reactivate synthesis of TS in the
non-neural tissues. The experiments of
Singer and Mutterperl (1963) point to this
possibility. They found that limb segments grafted auloplastically to the back
of adult newts would regenerate with subthreshold numbers of nerve fibers. It
was suggested that the trauma of transplantation either reduced the tissue threshold to neural TS or that the tissues were
induced, by the traumatization, to manufacture some TS themselves. Therefore,
the question arises: Will a shorter term of
innervation allow limbs, subsequently denervated, to recuperate the ability to regenerate after simple amputation? The aneurogenic limb system provides an excellent
means of examining this possibility. During ontogeny, nerves are absent in the
limb, yet nerves can be introduced naturally by orthotopic transplantation of the
aneurogenic limb to normal larvae, when
brachial nerves may then invade the graft.
This new innervation can be withdrawn at
will and the effect of this on regeneration
observed (Thornton, 1968, 1969). Aneurogenic, 4-digit forelimbs can be transplanted in place of forelimbs of normal
larvae quite successfully (Thornton and
Tassava, 1969). After healing is complete,
amputated graft-forelimbs regenerate normally, whether allowed to become innervated or not. Indeed the newly introduced
brachial nerves seem not to influence the
rate of regeneration in the formerly aneurogenic forelimbs. Of particular interest,
however, is the fact that the grafted,
formerly aneurogenic limbs, become dependent on their new nerves for regeneration. Thus, when brachial nerves are al-
117
REGENERATION OF LIMBS
lowed to glow naturally into the aneurogenie limb graft, the limb tissues become
fully innervated by 10 clays after transplantation. If, from 10-13 days, the limb grafts
are denervated by sectioning their new
nerves, and simultaneously amputating
through the upper arm, the following results are obtained: 14 of the 19 limbs denervated on day 10 regenerated (74%); four
of 9 limbs denervated on day 11 regenerated (44%); two of 16 limbs denervated on
day 12 regenerated (12%); none of 12
limbs denervated on day 13 regenerated
(0%). Thus, from the tenth to the thirteenth day post-transplantation, changes
were taking place in the limbs which rendered them progressively dependent on
nerves for successful regeneration. Perhaps
this is a period during which neural TS is
actively inhibiting synthesis of non-neural
tissue-TS.
Now comes a question of crucial importance to the neurotrophic theory: Having
become nerve-dependent, can these transplanted limbs recover their former ability
to regenerate without nerves? Indeed,
about half of them can. Aneurogenic
limbs, transplanted orthotopically to normal host larvae and allowed to become
"nerve-dependent", underwent section of
their host-derived brachial nerves on the
nineteenth day post-transplantation and
were maintained in a denervated condition
for 40 days by subsequent nerve sections,
repeated at 5-day intervals. Histological
examinations of sample limbs checked the
adequacy of denervation throughout the
period of the experiment. On day 30 the
limbs were amputated. By day 40, 16 of the
33 limbs (49%) had clearly defined regenerates, even though nerve counts of 14 of
these regenerates proved them to be aneurogenic or very sparsely innervated. Nerve
dependence, therefore, was reversed in
these cases by the simple expedient of
maintaining limbs, previously innervated
for a relatively short time, in a nerveless
condition for 40 days. It is concluded that
these results are in accord with, but do not
necessarily prove, the theory that the
ingrowth of nerves quenches synthesis of
TS in other limb tissues and that simple
elimination of the neural TS from the
limb can bring about recovery of TS
synthesis in non-neural limb tissues.
SUMMARY AND CONCLUSIONS
There is much circumstantial evidence
that the amphibian limb regenerates under
the influence of a trophic substance which
in the typical course of ontogeny is mediated by peripheral nerves. Regeneration is
apparently dependent on a threshold supply of TS. Fine-structural studies of regenerating limbs indicate that vesicles filled
with an electron-dense material progressively accumulate distally in regenerating
nerve fibers, but there is no proof yet that
these vesicles contain a neurotrophic substance. Axoplasmic flow has been demonstrated in nerve fibers, and suggestions as
to how this mechanism may be involved in
transport of TS to the regenerate have
been made. However, no critical evidence
is yet at hand to establish this mechanism
as an important one for regeneration. Isolation and identification of TS have not
been obtained despite intensive efforts. Evidence that, under certain condtions, tissues of the limb other than nerves can
manufacture TS is accumulating and may
indicate that TS is not necessarily a single
substance. Efforts now must be concentrated on defining the biological activity of TS
and on determining its mode of synthesis
and its chemical composition. These will
not be easy tasks but they do provide significant challenges for future investigators.
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