J. Embryol. exp. Morph. Vol. 69, pp. J83-192, 1982
Printed in Great Britain © Company of Biologists Limited 1982
183
Neurotrophic control of events in injured
forelimbs of larval urodeles
By ANTHONY L. MESCHER1
From the Department of Biological Sciences,
The George Washington University,
Washington, D.C.
SUMMARY
Denervated forelimbs and contralateral innervated forelimbs of Ambystoma larvae were
injured internally distal to the elbow by compression with watchmaker's forceps. Innervated
controls completely repaired the crush injury within one week; denervated limbs failed
to repair the injury and exhibited varying degrees of limb regression. Histological examination revealed that the process of tissue dedifferentiation initiated by injury was more
extensive in denervated, regressing limbs than in controls. In innervated limbs, both the
DNA labelling index and the mitotic index peaked approximately 4-6 days after the injury
and returned to baseline levels by 10 days. In denervated limbs, the DNA labelling index
also increased and remained at an elevated level for at least 2 weeks after the injury, but
significant mitotic activity was not observed. The data indicate that intact nerves are riot
needed for cellular dedifferentiation, cell cycle re-entry, and DNA synthesis in injured
limbs, but are required for the cells to proliferate and repair the injury. These results are
discussed together with those of similar experiments on the role of nerves during the initiation
of epimorphic regeneration in amputated limbs.
INTRODUCTION
The process of limb regeneration in amphibians depends directly on at least
three factors: a stimulus produced by the tissue injury which initiates the
'dedifferentiative' cellular events leading to regeneration; an agent released
from nerves which promotes mitotic activity in the cells affected by the injury;
and an inductive influence from an apical wound epithelium which causes the
underlying cells to remain in the proliferative state and give rise to a blastema
out of which the regenerate develops (Thornton, 1968; Tassava & Mescher,
1975; Wallace, 1981). The importance of nerves in promoting cell division in
the injured limb tissues is demonstrated more dramatically in larval urodeles
than in adults. The latter fail to regenerate denervated and amputated limbs,
but do heal the stump with a fibrocellular scar and cartilage over the bone
end (Schotte, 1926). If larval limbs are denervated before or shortly after
amputation, however, the consequent absence of normal mitotic activity in
1
Author's present address: Medical Sciences Program/Anatomy Section, Indiana
University School of Medicine, Bloomington, Indiana 47401, U.S.A.
184
A. L. MESCHER
the stump tissues results not only in the failure of the limb to establish
a blastema and regenerate, but also in the abrogation of the tissue repair
process (Butler & Schotte, 1941). Barring re-innervation, dedifferentiation
continues unchecked and the resultant cells are eliminated, causing the structural
regression of the limb as far as the shoulder (Schotte & Butler, 1941; Schotte &
Karczmar, 1944; Karczmar, 1946). Thornton (Thornton, 1953; Thornton &
Kraemer, 1951) showed that amputation trauma was not necessary to cause
regression of denervated larval limbs, but that simple compression of the
limb with watchmaker's forceps so as to injure the muscle and cartilage without
breaking the skin was sufficient to induce the histological changes leading
to limb regression. Thus in larval limbs the neural influence is required even
for the relatively simple process of tissue repair.
Recent histological and autoradiographic studies have attempted to elucidate
the role of nerves in amphibian limb regeneration by examining the effects of
denervation on cell cycle parameters during tissue dedifferentiation or in the
blastema. It has been learned that the neurotrophic influence is not needed for
the initial events of dedifferentiation which follow amputation, including reentry of muscle and connective tissue cells into the proliferative cycle and
replication of their DNA (Tassava, Bennett & Zitnik, 1974; Mescher & Tassava,
1975; Tassava & Mescher, 1976; Maden, 1978). Although the percentage of
cells incorporating tritiated thymidine in denervated limbs increases in parallel
with that of control limbs, the reports by Tassava and co-workers indicate
that the denervated cells do not divide, i.e. the percentage of cells in mitosis
does not increase significantly in the denervated limb stumps. This finding led
to the suggestion that the neurotrophic effect on the dedifferentiating cells is
exerted during the G2 phase of the cell cycle (Tassava & Mescher, 1975;
Tassava & McCullough, 1978). Maden's (1978) cell cycle studies were similar
to those of Tassava etal. (1974) but yielded results which suggested other, less
specific, controls of cell cycle events by the nerves in the amputated limb. The
methods of cell cycle analysis have been applied here to injured larval limbs
with and without nerves, like those originally studied by Thornton (1953), in
an attempt to clarify the neurotrophic control of cellular events following injury
to the urodele limb.
MATERIALS AND METHODS
Ambystoma maculatum embryos, collected locally, were allowed to hatch in
the laboratory and the larvae were raised in separate dishes to a length of
25-30 mm with daily feedings of freshly hatched brine shrimp {Anemia salina).
Fifty animals were anaesthetized lightly with MS 222 (Sigma) and the left
forelimbs were denervated by transecting the third, fourth, and fifth brachial
nerves distal to the brachial plexus. At the same time both forelimbs were
injured according to the method of Thornton & Kraemer (1951) by compressing
0-5 mm of the limb between the elbow and wrist with watchmaker's forceps,
Neurotrophic control of limb repair in urodele larvae
185
taking care not to break the skin. Left forelimbs were re-denervated every six
days. Every other day for 2 weeks following the initial operations, three larvae
were selected at random and each was injected intraperitoneally with 5 [iQ\
[3H]thymidine (ICN Pharmaceuticals, specific activity 73-5 Ci/mmole). After
3 h of incorporation, each larva was fixed in Carnoy's fluid. Three uninjured,
undenervated animals were labelled similarly to establish 'day 0' levels of cell
labelling. Experimental larvae not used for DNA labelling were maintained
for six weeks to observe the morphological changes accompanying regression
of the denervated limbs.
Forelimbs of the labelled animals were embedded in paraffin and sectioned
longitudinally at 10 /im. Sections from each limb were processed either for
autoradiography or by the Feulgen method as described previously (Mescher
& Tassava, 1975). Mesodermal tissues in the area between the elbow and wrist,
i.e. the radius, ulna, adjacent muscles and loose connective tissue, were examined
quantitatively with the aid of a grid in the microscope's eyepiece. For each
limb a DNA labelling index (percentage of cells labelled with at least a dozen
silver grains) was determined from the haematoxylin- and eosin-stained autoradiographs and a mitotic index (percentage of cells with mitotic figures) w&s
determined from both the Feulgen-stained sections and the autoradiographs.
Approximately 2500 cells were counted per limb, using non-adjacent sections
to give representative areas throughout the entire tissue sample. Statistical
significance of the data was determined by means of a t test.
RESULTS
Morphology and histology
The innervated limbs appeared to recover quickly from the injury, and by
one week after the operations no visible indication of the tissue crushing could
be observed. However, none of the denervated limbs ever recovered completely,
and the deleterious effects of the injury gradually became more pronounced
in the continued absence of nerves. Within three days of the operations, four
denervated limbs detached at the point of compression and the stumps regres$ed
to various levels in the upper arm region over the next five weeks. All of the
denervated, injured limbs showed morphologically some signs of the regression
described for such limbs by Thornton & Kraemer (1951). Reduction in the
overall size of the still-compressed forearm and shrinkage of the digits were
apparent within 10-20 days of the injury and in many cases gradual disappearance of the forearm, digits and elbow had occurred by the end of the
6-week observation period.
The histological changes accompanying the onset of regression in denervated,
injured limbs have been detailed by Thornton (1953) and resemble the dedifferentiative events that occur in denervated, amputated larval limbs (Butler
& Schotte, 1941). Only the first two weeks after denervation and crushing
186
A. L. MESCHER
Fig. 1. Autoradiographs of sections through the forearm region of the denervated
(a) and innervated (b) limbs of an Ambystoma larva 12 days after crush injury. The
approximate level of the crush is indicated by the large arrows. Note the overall
reduction in size and amount of soft tissue, as well as the higher percentage of
nuclei labelled with [3H]thymidine (small arrows), in the denervated limb. Both
photographs were taken at 100 x magnification.
30 n
20-
10-
4
6
8
10
12
14
Days after injury
Fig. 2. DNA labelling indices of innervated ( # ) and denervated (O) larval limbs
during the two weeks following crush injury. Each point indicates the mean of
three limbs. Standard errors are given in Table 1.
Neurotrophic control of limb repair in urodele larvae
187
Table 1. DNA labelling andmitotic indices in injured denervated and
innervatedforelimbs o/Ambystoma larvae
Labelling index*
Mitotic index*
Days after
injury
Denervated
Innervated
Denervated
Innervated
2
4
6
8
10
12
14
10-73 ±1-53
16-63 ±3-89
10-40 ±3-59
12-40 ±3-52
13 93 + 2-99
ll-23±3-18
13-34 + 602
14-50 ±1-62
28 -40 ±1-22
18-30±2-28
12-33 ±3-94
7-43 ±1-96
5-60 ±1-70
6-83 ±209
0
0066 ±0067
0086 ±0087
0
0053 ±0053
0
0
0-373 ±0-233
0-683 ±0-366
0-623 ±0047
0-400 ±0-208
0083 ±0083
0116±0117
0097 ±0097
A
Control undamaged limbs (day 0) had a labelling index of 4-96 ±0-47 and a
index of 0083±0068.
* Mean ± standard error of three limbs.
have been studied histologically here. Sections of the forelimbs from one
animal 12 days after injury are shown in Fig. 1. In the denervated limbs, the
cartilage of the radius and ulna at the site of injury became progressively
vacuolated and devoid of cells, while adjacent muscle detached and dedifferentiated. Wrist elements and the epiphyseal cartilage of the radius and ulna
were more resistant to vacuolation and remained intact with little histologic&l
change during the 14 days of observation. Tissues of the innervated limbs,
although injured in a similar manner, showed much less extensive dediffereritiation. Cells lost from the crushed cartilage were quickly replaced and muscle
remained present. By 10-14 days after the injury, innervated limbs were
completely repaired and the presence of new, less deeply stained cartilage at
the level of limb compression was the only histological evidence that an injury
had occurred.
DNA labelling and mitotic indices
Autoradiography revealed significant increases in the number of cells undergoing DNA synthesis in the'mesodermal tissues of the larval forearms in the
first few days following the injury. This was true both for the innervated limbs,
involved in the process of tissue repair, and the denervated limbs, which
underwent the phenomenon of regression rather than repair. Moreover, while
the DNA labelling index of the innervated forearms peaked on day 4 and
returned to the normal level by day 10, that of the denervated limbs remained
at a significantly (P < 0025) elevated level throughout the two-week period
of measurement (Fig. 2). The mean labelling index and standard error for
each day are given in Table i. Figure 3 shows the high percentage of labelled
cells still found in the denervated, regressing limbs 14 days after the injury.
188
A. L. MESCHER
Fig. 3. Autoradiograph of a section through the injured region of a denervated
forelimb 14 days after crush injury. Note the large number of nuclei labelled
with [3H]thymidine (arrows). 160 x magnification.
100-8-
0-6-
0-4-
0-2-
4
6
8
10
12
14
Days after injury
Fig. 4. Mitotic indices of innervated ( # ) and denervated (O) larval limbs during
two weeks following crush injury. Each point indicates the mean of three limbs.
Standard errors are given in Table 1.
Thus cellular dedifferentiation in the denervated, injured larval limbs resembles
that of amputated limbs, with or without nerves, in being characterized by
cell cycle re-entry or acceleration and increased levels of DNA replication.
Mitotic activity in the mesodermal cells of the innervated forearms was
significantly increased for eight days after the injury, but by ten days had
returned to the level shown by uninjured larval limbs (Fig. 4 and Table 1).
In marked contrast to the innervated limbs, however, the denervated limbs
were found to contain very few or no mitotic cells despite searches through
Neurotrophic control of limb repair in urodele larvae
189
every section at 400 x magnification. The mitotic index of the denervated forearms remained at or below that of control, uninjured limbs. It appears therefore that the failure of these limbs to heal the injury and the loss of tissue
which results in limb regression are due to failure of the dedifferentiated cells
to divide normally in the absence of nerves.
DISCUSSION
The results presented here confirm the original observations of Thornton
(Thornton, 1953; Thornton & Kraemer, 1951) with denervated, injured forelimbs
of Ambystoma larvae and extend those findings in terms of the cell cycle. The
tissue dedifferentiation which characterizes the onset of limb regression in
these limbs has been found to resemble that which follows amputation of
denervated larval limbs, not only histologically (Thornton, 1953), but also in
the increased percentage of mesodermal cells replicating DNA. Like amputation
(Tassava et al. 1974; Tassava & Mescher, 1976; Maden, 1978), the crush
injury causes renewed or accelerated cell cycling and an increased DNA labelling index in the absence of intact nerves. The dedifferentiation/cell cycling
stimulus elicited by injury apparently involves only the internal limb tissues,
since open skin wounds affecting only the superficial tissues do not promote
dedifferentiation and regression of denervated limbs (Thornton & Kraemer,
1951). Renewed cell cycling in response to tissue injury probably involves
agents released during inflammation (see, e.g., Greenburg & Hunt, 1978).
However, from an analysis of the regression process in denervated, amputated
larval Ambystoma forelimbs, Karczmar (1946) suggested that nerves, degenerating as a result of the proximal transection, release a factor promoting the
cellular events leading to regression. This was based on his finding that denervation of the limbs 4 days after amputation greatly accelerated dedifferentiation
and regression, but that this process was retarded if denervation was performed
10 days prior to amputation. Thus one can speculate that nerves may participate
in modulating the tissue dedifferentiation following limb amputation either
by axoplasmic trophic factor(s) (Maden, 1978) or by factors released from
degenerating myelin (Abercrombie & Johnson, 1946; Gospodarowicz &
Mescher, 1980).
While any sort of neural involvement in the onset of dedifferentiation is
uncertain, there is clear evidence for a neurotrophic influence being required
to effect repair of injured larval limbs (Thornton, 1953) and, in the case of
amputated urodele limbs, to act together with the wound epithelium to bring
about formation of a regeneration blastema (Singer, 1952, 1978). In the events
leading to blastema formation, the principal role of the nerves seems to be
to promote mitotic activity in the dedifferentiating cells (Singer, 1952; Thornton,
1968). Tassava & Mescher (1975), discussing the factors which initiate limb
regeneration, suggested that nerves act during the G2 phase of the cell Cycle
7
EMB 69
190
A. L. MESCHER
to allow mitosis. This hypothesis was based primarily on the observation that
the early events of dedifferentiation and the initial increase in DNA labelling
index were found to occur normally, but without subsequent cell division in
denervated, amputated limbs of both larval axolotls (Tassava et al. 1974) and
adult newts (Mescher & Tassava, 1975). The rinding reported here, that during
the initiation of regression in denervated, injured larval limbs there is an
increase in the percentage of cells replicating DNA but no concomitant increase
in the percentage of cells in mitosis, emphasizes once again that intact nerves
are not needed for cell cycle re-entry and the S phase, but are necessary for
the mitotic activity which leads to repair of the injury. The present results
also support the explanation suggested by Tassava & Mescher (1975) for
regression of denervated, injured larval limbs: that the dedifferentiated, nondividing cells of denervated limbs are not viable and are removed from the
limb. According to that hypothesis, gradual resorption of the limb would
occur because the injury effect persists, causing more and more cells to
dedifferentiate and replicate DNA, and without neural support the cells do
not complete a normal cell cycle, become non-viable and are eliminated. This
view predicts continued incorporation of [3H]thymidine by cells of denervated,
injured limbs as reported here (Fig. 2). The elevated DNA labelling index and
barely detectable level of cell division are consistent with the possibility of
a G2 block to the dedifferentiating cells' proliferative cycle in the absence of
nerves. However, these data do not rule out other possibilities, such as a
general neurotrophic effect on the rate of cell cycling so that in denervated
limbs levels of mitosis leading to regeneration or repair would be precluded
(see Mescher & Tassava, 1975; Tassava & McCullough, 1978; Globus, 1978).
Maden (1978) has repeated (with a variety of procedural differences) the
experiment of Tassava etal. (1974) and, unlike those authors, found a significant
number of mitotic cells in denervated limb stumps of larval axolotls. The
reason for the difference in results is not clear, but the possibility of incomplete
elimination of all nerve fibres by the denervation procedure of crushing the
brachial plexus (Maden, 1978) should be considered. The presence of nerve
branches outside the primitive, anastomosing plexus of amphibian limbs and
the generalized distribution of nerves in this region of premetamorphic urodele
limbs in particular are discussed in reviews by Detwiler (1933), Strauss (1946)
and Hughes (1968). Despite this, Maden did not verify the absence of nerves
in the limbs by nerve staining. His suggestion that in denervated limbs fewer
cells than normal enter the cell cycle and divide appears to be contradicted by
his own data, which show identical labelling indices in both innervated and
denervated limbs until the onset of increased mitosis four days after amputation.
In a related study Maden (1979) used Feulgen microdensitometry to show that
cells of 'cone stage' blastemas accumulate in Gx after denervation, i.e. have
a 2C DNA content, and cited this finding as evidence against the hypothesis
of Tassava & Mescher (1975). However, the result he obtained could have
Neurotrophic control of limb repair in urodele larvae
191
been predicted, since it is well known that larval Ambystoma blastemas denervated at such a late stage of growth almost always continue to develop
(Schotte & Butler, 1944; Butler & Schotte, 1949; Singer, 1952). Denervated
cone-stage blastemas of newts show continued mitotic activity for at least two
weeks (Singer & Craven, 1948) and normal tissue differentiation (Powell, 1969).
The cells of these nerve-independent blastemas would be expected to accumulate
in GX/GQ with a 2C DNA content as they begin to differentiate. The finding
of a 'Gj block' (Maden, 1979) in such blastemas is, therefore, not surprising
and is irrelevant to neurotrophic control of the cell cycle during earlier
preblastemic or nerve-dependent blastema phases of regeneration when nerves
are of major importance.
Thus the question of whether nerves control a specific phase of the proliferative
cycle of mesodermal cells during the initiation of amphibian limb regeneration
or repair remains open. The nerve-dependent repair of injured limbs in
Ambystoma larvae is a system well suited for studies designed to answer that
question, since it is not complicated by the influence of a wound epithelium
converting the process of tissue repair into one of epimorphic regeneration
(Tassava & Mescher, 1975; Mescher, 1976). Finally, as the discussion above
emphasizes, it is important for future work on the neurotrophic control of
cell cycle events during limb regeneration to be based on what is already
known about the role of nerves in this process.
This work was supported by NIH grant GM 27735 to the author and a NIH Biomedical
Research Support Grant to the George Washington University.
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{Received 26 August 1981, revised 23 November 1981)