/. Embryol. exp. Morph. Vol. 48, pp. 169-175, 1978
Printed in Great Britain © Company of Biologists Limited 1978
169
Neurotrophic control of the cell cycle
during amphibian limb regeneration
By M. MADEN 1
From the National Institute for Medical Research, London
SUMMARY
It is shown here that amputated and denervated limbs of larval axolotls dedifferentiate
and a proportion of the cells released undergo DNA synthesis and mitosis. When the limb
is denervated prior to amputation fewer cells go through the cell cycle, implying the existence
of a pool of trophic factor in the limb. Recent work has demonstrated that denervated
blastemal cells accumulate in the d phase of the cycle. These results strongly argue against
the theory that the trophic factor controls the G2 phase. Rather, it is proposed that this
factor regulates either the total number of cells cycling or the rate at which they cycle by
varying the length of the Gx phase.
INTRODUCTION
The role of the nervous system in amphibian limb regeneration has been the
subject of considerable research since the observation of Todd (1823) that a
denervated newt limb would not regenerate. It is now thought that the nerves
supply a trophic factor which must be in sufficient quantity to permit regeneration (review Singer, 1974). Recently Singer and his colleagues have concentrated
their attention on the biochemical activity of this trophic factor and have
isolated a protein from newt brains which can largely restore the level of DNA
and protein synthesis in the denervated blastema (Singer, Maier & McNutt,
1976; Jabaily & Singer, 1977).
The activity of the trophic factor has also been explored at the cellular level
by observing the early events following amputation of the denervated limb.
Dedifferentiation occurs as normal and it has been reported that the cells thus
liberated incorporate labelled thymidine (enter S phase) but do not divide
(Tassava, Bennett & Zitnik, 1974; Mescher & Tassava, 1975; Tassava &
Mescher, 1976). This observation led to the hypothesis that the trophic factor
controls the progress of blastemal cells through the G2 phase of the cell cycle
(Tassava & Mescher, 1975). However, it has recently been demonstrated by
microdensitometry(Maden, 1979) that denervated blastemal cells accumulate in
the G1 phase either due to a Gx block or a greatly protracted cell cycle time.
These results are clearly contradictory to the hypothesis of a G2 neurotrophic
1
Author's address: Division of Developmental Biology, National Institute for Medical
Research, The Ridgeway, Mill Hill, London NW7 1AA, U.K.
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M. MADEN
control and in order to resolve this dilemma the experiments of Tassava et al.
(1974) on larval axolotls have been repeated and extended. Contrary to these
authors, dividing cells were observed in denervated limbs. Furthermore, extending the interval between denervation and amputation provides evidence for
the existence of a pool of trophic factor in the limb.
MATERIALS AND METHODS
These experiments were performed on larval axolotls Ambystoma mexicanum,
50-60 mm long, kept in an incubator at a constant water temperature of 20 °C.
The animals were divided into two groups, Series I was used to duplicate the
experimental conditions of Tassava et al. (1974) and Series II to investigate the
effects of denervation prior to amputation.
In Series I both forelimbs were denervated by crushing the brachial plexus
at the time of amputation through the distal humerus. The hind limbs served
as innervated controls after amputation through the distal femur. In Series II
the forelimbs were similarly denervated 6 days prior to amputation, the hindlimbs again serving as controls. All forelimbs were redenervated every 3 days
to prevent reinnervation. On days 1-7 post-amputation one animal from each
series was injected with 5 /tCi [3H]-6-thymidine (20-6 jtiCi/jtig). After a 4 h
incorporation period all limbs were fixed in Bouins. In addition, several unamputated animals were similarly injected with tritiated thymidine to determine
the normal rate of incorporation in larval axolotls.
The samples were sectioned at 10/tm, prepared for autoradiography with
Ilford K2 emulsion and left for 5 weeks. The slides were then developed and
stained with haematoxylin and eosin. For each sample the percentage of
labelled mesodermal cells at the distal tip of the limb was estimated on eight
different sections. At the same time the number of mitoses in these eight sections
was recorded and since the total number of cells counted was known, a value
for the mitotic index (per thousand cells) could be calculated.
RESULTS
Concerning the initial events following amputation - that is, epidermal wound
healing and dedifferentiation - there were no observable differences between
the experimental and control series, confirming that they are independent of
any nervous control.
Labelling indices
The mesodermal limb tissues of unamputated animals had labelling indices
of up to 5 % (shaded area in Fig. 1). Since these were small larvae, still growing
rapidly, such a level is not unexpected.
The data from the two amputated control series were virtually identical and
so in Fig. 1 these results have been pooled (dotted line). Here it can be seen
Neurotrophic control of cell cycle during limb regeneration
60 i
111
I,
45-
I 303
15-
1
2
3
4
5
6
7
Days
Fig. 1. The increase in percentage labelled cells at the distal tip of amputated limbs
in control amputated limbs (
), limbs which were denervated at the time of
amputation (
) and limbs which had been denervated 6 days prior to amputation
(
) The shaded area represents the level of labelling in normal, unamputated
limbs. Each experimental point is composed of 16 estimates (total of about 4000
cells) from two samples. Control points are from pooled data (32 estimates from
four samples), ± standard errors.
that in otherwise normal amputated limbs the cells became labelled significantly
above normal on day 3. Thereafter the percentage rose rapidly throughout the
sampling period to 60 % by day 6. The drop on days 5 and 7, recorded in both
control series, perhaps reflects some degree of synchrony in the population(s)
of cells liberated by dedifferentiation. Synchrony has also been observed in the
mitotic index of the epidermis following amputation (Maden & Wallace, 1976).
In those limbs which had been denervated at the same time as amputation
(Series I), some cells entered the cell cycle (dashed line in Fig. 1). By day 4 as
many as 30 % of the mesodermal cells were labelled after the 4 h incorporation
period but over the next 3 days the percentage declined to near normal levels.
This confirms the conclusion of Tassava et al. (1974) and Tassava & Mescher
(1976) that DNA synthesis commences in some cells of denervated axolotl
limbs. However, despite the identical labelling regime and other experimental
conditions (except the temperature was slightly lower here) the above authors
only reported a maximum of 10% labelling in denervated limbs. This discrepancy was similarly reflected in the control series: they obtained a mere 20 %
172
M. MADEN
labelling by day 7 (Tassava et al. 1974) and 40-50% by day 9 (Tassava &
Mescher, 1976) whereas here in Fig. 1 60% of the cells were labelled by day 6.
The reason for these differences remains an enigma - it would be expected in
older larvae which regenerate more slowly but similar-sized larvae were intentionally used here.
Limbs which had been denervated 6 days before amputation (Series II) had
fewer cells labelled (solid line in Fig. 1) than in Series 1. The maximum obtained
here was only 20 % and the two denervation curves are significantly different
from day 3 to 6. Thus it is clear that prior denervation decreases the number of
cells that become labelled after amputation. Of these labelled cells scored in
Series II up to a half were perichondrial cells and fibroblasts surrounding the
humerus and it is conceivable that these might be involved in a non-neurotrophic
wound-healing response.
Mitotic indices
Very few mitoses were seen in the mesodermal tissues of unamputated limbs
and the mitotic index varied between 0 and 0-5 (shaded area in Fig. 2).
The rise of mitotic index in control amputated limbs (dotted line in Fig. 2)
was delayed by 24 h relative to the rise in cell labelling. Mitoses were significantly above normal levels on day 4 and rapidly increased from day 5 onwards.
It is interesting to note that three mitotic figures seen in control limbs were
labelled. Since the incorporation period was only 4 h and G 2 has been estimated
to be 6-6-5 h (Maden, 1976) this suggests seme heterogeneity in cycle
times.
The most important observation here, however, was that significant numbers
of mitoses were recorded in both experimental series after day 3 (dashed line
and solid line in Fig. 2). As in the labelling studies, fewer mitoses were seen in
the prior denervation series.
DISCUSSION
These results clearly demonstrate that denervating the limb prior to amputation decreases the labelling index of the dedifferentiated cells at the amputation
plane. This phenomenon has not previously been considered in detail, although
a brief mention was made concerning similar levels of RNA synthesis in prior
denervated and normally denervated limbs sampled only on day 6 after amputation (Kelly & Tassava, 1973). The simplest explanation of these results is that
there is a pool of trophic factor in the limb probably provided by the degenerating
nerve fibres between the brachial plexus and the amputation plane. There is
evidence that these isolated axons can furnish a limited supply of trophic factor
since neuromuscular junctions degenerate more rapidly and other denervation
changes appear earlier when a nerve is cut close to the muscle than if it is cut
far away (Harris & Thesleff, 1972). Similarly both the lateral line and taste buds
degenerate in a proximal-distal direction when denervated (Geraudie & Singer,
1977).
Neurotrophic control of cell cycle during limb regeneration
12
173
n
6-
Days
Fig. 2. The increase in mitotic index of the same limbs as in Fig. 1
, controls;
, denervated at the time of amputation;
, denervated 6 days prior to
amputation. The shaded area represents the mitotic index of normal, unamputated
limbs. ± standard errors.
The second important finding reported here is that significant numbers of
mitoses were found in denervated limbs, with less in the prior denervated series.
This was not observed by Tassava et ah (1974): on their graph of mitotic index
the curve for denervated limbs remains firmly at 0. Yet Mescher & Tassava
(1975) using newts and Kelly & Tassava (1973) and Tassava & Mescher (1976)
on similarly sized larval axolotls all recorded mitoses in denervated limbs.
These values apparently were not significantly above normal but those reported
in Fig. 2 were. It is to be emphasized that denervated limbs were redenervated
every 3 days, so reinnervation cannot be advanced as an explanation for this
discrepancy. It has recently been demonstrated that the cells of denervated
blastemas accumulate in the Gx phase of the cell cycle (Maden, 1979). This,
together with the above results, strongly argues against the existence of a G2
neurotrophic block (Tassava & Mescher, 1975). Rather, I suggest that in
denervated limbs fewer cells than normal enter S, go through G2, divide and then
accumulate in the next Gx due to depletion of the trophic factor. Prior denervation would reduce the size of the pool of trophic factor, thus even fewer cells
would progress through the cell cycle. This view holds that the trophic factor
controls the number of cells cycling by the operation of a Gx block.
12
KMB 48
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M. MADEN
An alternative view is that the trophic factor is a rate-limiting molecule
(Globus & Vethamany-Globus, 1977). This could be combined with the above
data to suggest that the trophic factor controls the speed of progression through
Gv A highly protracted Gx after denervation would give the same microdensitometry data (Maden, 1979) as a complete block and would provide a simple
explanation for the observations of occasional mitoses many weeks after
denervation (Singer & Craven, 1948). Unfortunately the data reported here are
not sufficient to distinguish between these two alternative views. In principle,
when the same number of cells cycle at a slower rate the labelling and mitotic
index curves should peak later than when fewer cells enter the cycle at the same
rate as normal. However, preliminary evidence has been obtained from continuous labelling studies which argues in favour of the rate-limiting suggestion
(Maden, unpublished). Thus apart from furthering our understanding of limb
regeneration and the function of trophic molecules, this in vivo system provides
exciting possibilities for fundamental research into the nature of cell cycle
controls.
I thank J. Cooke and H. Wallace for discussions and helpful comments during the
preparation of the manuscript.
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{Received 8 May 1978, revised 3 July 1978)