/ . Embryol. exp. Morph. Vol. 56, pp. 309-317, 1980
Printed in Great Britain © Company of Biologists Limited 1980
309
Regeneration of reversed aneurogenic arms
of the axolotl
By H. WALLACE 1
From the Department of Genetics, University of Birmingham
SUMMARY
Aneurogenic arms of young axolotls were implanted into the flank as heterotopic autografts with reversed proximo-distal orientation. The formerly proximal ends of such arms
regressed to a variable extent, and then either regenerated or could do so following a second
amputation. The regenerate always contained a complete sequence of skeletal elements
between the adjacent stump skeleton and terminal digits, being a mirror image of the
implanted arms with identical transverse axes but an opposed proximo-distal axis. Many
reversed arms also regenerated fingers from the implanted hand. Identical results were
obtained from reversed arms of control larvae, confirming previous studies on reversed
well-innervated arms. Nerves are not required for the establishment of a new proximo-distal
axis, therefore, and probably have no influence on the determination of any limb axis.
Morphogenesis of the regenerate is clearly related to the position along the proximo-distal
axis where the blastema originates. Although this axis is reasonably envisaged in terms of
a gradient, its polarity is ignored during regeneration.
INTRODUCTION
The technique of removing most of the central nervous system from an
amphibian embryo maintained in parabiosis has been exploited for several
studies of limb development and regeneration. Sparsely innervated and aneurogenic arms produced in this way develop normally at first and can regenerate
(Yntema, 1959), although the arms of older larvae and adult urodeles apparently
require a considerable innervation in order to regenerate (Singer, 1952). This
contradiction has not been fully resolved but comparisons between aneurogenic
and normally innervated larval arms should reveal what aspects of regeneration
are influenced by nerves. Local irradiation, for instance, inhibits regeneration
in both cases, implying a direct action of X rays which is not mediated by
nerves (Wallace & Maden, 1976). I have applied the same technique to enquire
whether or not nerves influence the proximo-distal (PD) polarity of regenerating
arms.
Milojevic & Grbic (1925) first tested the permanence of the limb's PD axis
by implanting the distal ends of limb segments into the backs of adult newts.
The free end them sometimes regenerated an almost complete limb with a
1
Author's address: Department of Genetics, University of Birmingham, P.O. Box 363,
Birmingham B15 2TT, U.K.
310
H. WALLACE
normal PD polarity, opposed to that of the implanted stump. Subsequent
experiments have confirmed this result while attempting to reduce wastage
and resorption of the reversed limb by improving its blood supply and innervation (Efimov, 1933; Monroy, 1942). Butler (1955) adapted the operation
for larval urodeles by allowing an implanted wrist to heal into the flank for
several days before amputating the dually attached arm close to the shoulder.
Even then, most of the upper arm was resorbed before healing or regeneration
occurred near to the elbow (Deck, 1955). Deck & Riley (1958) also noticed
considerable regression and a common failure of regeneration when repeating
this operation on the hind limbs of larval urodeles. Dent (1954) increased the
frequency of regeneration after the same operation on adult newts by deflecting
brachial nerves to the implanted wrist and delaying the amputation for 2 weeks.
Oberheim & Luther (1958) ingeniously grafted the arm of one salamander
larva through the tail muscle of another, diverted sciatic nerves into the host's
wound region and kept the two larvae attached for three days under a light
narcosis. Amputating the grafted arm through the humerus and wrist then
sometimes caused 'bipolar regeneration', at both ends simultaneously.
The extent of the regenerated structures encountered in these experiments
varied according to the level of amputation and amount of regression. From
a base which conformed to the adjacent region of the limb stump, each regenerate
contained the usual sequence of structures which normally lie distal to that
region. This feature is commonly ascribed to the law of distal transformation
governing normal limb development and regeneration (Stocum, 1978). All the
regenerates apparently preserved the anterio-posterior and dorso-ventral axes
of the limb from which they arose. Consequently, the formerly proximal end
of a right-arm stump regenerated a left hand. Even though the PD polarity
of such a regenerate is opposed to that of the implanted stump, it is perfectly
normal in respect to the rest of the body and to the local innervation which
has grown into the implant. The direction of nerve growth could dictate distal
transformation, as several investigators have suggested (e.g. Needham, 1952).
If that were so, a reversed aneurogenic arm might be incapable of regenerating
distal structures or even show some form of proximal transformation. Precedents
for both of these expectations have been described for reversed limb buds
(Swett, 1927, 1928; Graper, 1922), and it is of general interest to discover if
the analogy between developing and regenerating limbs can be extended to
this situation.
MATERIALS AND METHODS
Reversed aneurogenic arms were produced by combining the techniques of
Yntema and Butler outlined in the introduction. That entailed subjecting
axolotls (Ambystoma mexicanum) to four successive operations, each of which
involved some wastage or mortality. Pairs of embryos were joined in parabiosis
before the tailbud stage. The hindbrain and trunk spinal cord were extirpated
Reversed arms
311
from the right embryo of each pair on the following day. Further details of
these operations are provided by Wallace & Maden (1976). Nerve fibres tend
to spread from the nurse embryo to the defective one, so only the right arm
of the latter could be treated as sparsely innervated or sometimes completely
free from nerves. All such right arms are termed aneurogenic in this report.
Reversal was performed on well grown arms of 25-30 mm specimens. The
digits were removed from aneurogenic arms (or the right arms of control
larvae) to create a wounded hand which was implanted into the peritoneal
cavity. The specimens were kept virtually immobile in dilute MS 222 overnight, as movement of either the arm or body tends to dislodge the implanted
hand. The right arms of a second control series were denervated immediately
before implanting the hand. Implanted arms were amputated close to the
shoulder one week later, and displaced repeatedly to ensure a complete
separation.
Regular inspection during the next 4 weeks provided records of regeneration
from the shoulder, from the free end of the arm and from the implanted hand.
Reversed arms which showed no regenerate on the free end after five weeks
were reamputated and observed for a further period. The orientation of each
regenerate was assessed before fixation and preparation of whole mounts
stained with methylene blue. Aneurogenic arms were finally sectioned longitudinally and impregnated with silver according to Blest (1976) to display
nerve fibres.
A few additional reversals were performed on non-parabiotic aneurogenic
larvae which are completely free of nerves (Popiela, 1976), using Oberheim
and Luther's technique outlined in the introduction. One such arm was
processed for electron microscopy (cf. Egar, Yntema & Singer, 1973; Popiela,
1976).
RESULTS
Less than half of the parabiotic twins (Fig. 1) survived to feed and grow
normally. Subsequent operational failures, fungal infection, and a novel recessive
gene which impedes limb development all reduced the number of useful twins
to about 20 % of the starting material. Aneurogenic arms grew more slowly
than those of nurse or control larvae but reached a relatively slender 3- to
4-digit stage. Implanting the hand into the flank only interrupted the brachial
blood flow for a few days. The subsequent shoulder amputation eliminated
the normal blood supply to the arm but circulation resumed in the implanted
wrist two days later. This amputation provoked regeneration from the shoulder,
resulting in new 3-digit right hands in as many weeks. The implants regressed
initially to lose at least the free end of the upper arm. Some were completely
resorbed, most healed near the elbow as a permanent stump, and the rest
regenerated as left hands from about the same region (Fig. 2). Differences in
healing of the implanted hand affected those responses to a considerable extent,
312
H. WALLACE
Table 1. Classification of reversed arms 25 days after amputation
Type of arm
Innervated
Denervated
Aneurogenic
Total amputated
34
32
26
2
5
4
Completely resorbed
12 (44%)
Free end regenerated
7(23%)
10* (42%)
18 (67%)
Implanted wrist regenerated
26(87%)
14(58%)
20%
30%
24%
Expected coincidencef
7 (26%)
Bipolar regeneration
7(23%)
5(21%)
* Only one of these cases had no detectable nerves, the other nine were sparsely innervated,
t Explained in the text. In the first column, for instance, 23% x 87% = 20%.
indeed the most poorly implanted arms became detached during later handling.
Table 1 shows the results from the remaining reversed arms. A few were weakly
connected by a narrow neck of tissue and were slowly resorbed without gaining
a blood supply. The majority were only partially implanted, allowing several
digits to regenerate from an exposed portion of the wrist. Less than half of
these partially implanted arms regenerated at the free ends. Deeply implanted
arms which healed smoothly to the flank were generally more successful at
regenerating from the free end.
Considering first the reversed aneurogenic arms, those which gained a blood
supply were usually able to regenerate at one end or the other, or at both
ends (Table 1). All these regenerates displayed a normal proximo-distal succession
of skeletal elements, with the most proximal region corresponding to that of
the adjacent stump (Fig. 3 c). Most of the control innervated arms were only
partially implanted and relatively few regenerated from the free end, possibly
because flank nerves had not penetrated that far. The second control series of
denervated arms was designed to enhance regeneration in the manner pioneered
FIGURES
1-4
Fig. 1. Parabiotic twins: the host or nurse larva is 12 mm long; the defective larva
larva lacks a hindbrain and most of the trunk spinal cord.
Fig. 2. Ventral view of denervated control specimen, 28 days after amputation close
to the shoulder (S). Scale bar of 1 mm. A normal right arm has regenerated from
the shoulder. The partially implanted original hand has regenerated three digits
and is attached to the flank by its posterior side. The free end of the implanted
arm has regenerated a left hand (arrow).
Fig. 3. Largest and smallest reversed arm regenerates from each series: (a) innervated control; (b) denervated control; (c) aneurogenic. All are dorsal views with
the implanted hand at the bottom of the figure and at the same magnification
(1 mm scale bar).
Fig. 4. Reversed arms which regenerated at the free end following a second
amputation. The arrangement and magnification are identical to Figure 3. The
largest arms in each series contain regenerated distal humerus.
Reversed arms
313
t
4(a)
314
H. WALLACE
by Dent (1954). These arms could not be actively flexed and so might become
implanted more securely. The implanted hand might also attract flank nerves
earlier because its own innervation has been destroyed. Table 1 offers mild
support for these predictions, in that denervated control arms were less liable
to regenerate from the implantation site but regenerated at their free ends
more frequently than innervated control arms. In fact, the denervated arms
resemble aneurogenic arms more closely in this respect. The expected frequency
of bipolar regenerates can be obtained by treating regeneration at the free end
and implanted wrist as independent events and calculating their coincidence.
The observed frequency of bipolar regenerates is remarkably close to this
expectation in each series (Table 1). More importantly, the two control series
included cases of regeneration at either or both ends of the reversed arm. Such
regenerates were identical to those obtained from reversed aneurogenic arms
in position, structure and orientation (Fig. 3).
The static reversed arms, which had merely healed after the first amputation,
were reamputated close to the elbow after 5 weeks. Except for one aneurogenic
arm, each regenerated a complete wrist and hand from the new wound surface.
All these regenerates, like those resulting from the first amputation, clearly
preserved the antero-posterior and dorso-ventral axes of the limb stump but
showed distal transformation (Fig. 4). The stained skeletal cartilages seen in
cleared whole mounts leave no doubt that the free end of the implanted arm
regenerated a mirror image of itself. Many of these regenerates contained a
surprisingly complete skeleton, often including part of the humerus, but others
had regressed further and regenerated correspondingly fewer skeletal parts
(Figs. 3, 4). Since regeneration invariably involved distal transformation, these
results confirm previous observations and extend them to aneurogenic aims,
suggesting that the amount of innervation has no appreciable influence on the
structure and orientation of a regenerate.
As expected from previous studies (cf. Egar et at. 1973), most of the reversed
aneurogenic arms were sparsely innervated by one or two small bundles of
axons which were easily detected in longitudinal sections. Only one regenerate
lacked any visible nerve fibres, an isolated case which clearly required more
rigorous confirmation. The reamputated aneurogenic arms failed, to provide
this confirmation, for they had all become innervated in the nine weeks since
implantation. Aneurogenic axolotls do not survive long enough to support
this experiment unless they are maintained in parabiosis, when they are inevitably invaded by nerves from the nurse larva. Arms of advanced single
aneurogenic larvae can be implanted into younger ones, however, and then
have time to regenerate. Two advanced regenerates obtained in this way showed
the typical features of distal transformation. Only one of them was successfully
processed for electron microscopy, but it was certainly devoid of nerves.
Re versed arms
315
DISCUSSION
The regenerated structures noted in this study correspond perfectly to the
categories described by Butler (1955) and Deck (1955) for reversed arms of
A. maculatum larvae, and provide a means of reconciling their results to those
obtained from embryos of the same species. When Swett (1927, 1928) reversed
limb buds after Harrison stage 37, the implanted tip often managed to grow
out from the flank but the formerly proximal end invariably healed over without
regenerating. That was also the commonest result in the present control series.
Butler (1955) only described his successful operations on relatively advanced
larvae, presumably meaning well-embedded arms which did not grow any
fingers at the implantation site. He noted that the free ends of such reversed
arms could grow a new forearm and hand but usually did so only after a second
amputation, as the present results confirm. Reversed arms commonly regenerate
digits at the implantation site and perhaps do so whenever the formerly distal
end is partly exposed. The formerly proximal free end of the arm sometimes
regenerates promptly and almost always does so following a later amputation.
There is clearly no distal dominance of regenerative power, for both ends of
the arm can regenerate simultaneously (cf. Monroy, 1942; Oberheim & Luther,
1958). In an analogous experiment performed by Eiland (1975), regeneration
occurred at a better innervated site in preference to a more distal one. Better
innervation of the implanted hand of a reversed arm could also explain why
regeneration occurs more frequently there than at the free end, especially as
the reduced innervation of aneurogenic arms tend to reduce this disparity
(Table 1).
Since the control reversed arms, like other heterotopically transplanted arms,
only gain a subnormal innervation (Deck, 1955; Singer & Mutterperl, 1963), it
is tempting to suppose those which did not regenerate promptly had not yet
acquired an adequate nerve supply. That is, the innervation of control arms
fell within the threshold level of the quantitative neurotrophic theory (Singer,
1952). The identical behaviour of the aneurogenic arms casts doubt on this
explanation for they would have to respect a lower threshold level, being
more sparsely innervated both before and after implantation. Perhaps a limb
can adapt to any amount of innervation, but fails to regenerate only when its
nerve supply has been reduced recently. That seems preferable to the threshold
concept as a means of explaining both the present results and experiments
designed to test the neurotrophic theory (Thornton & Thornton, 1970).
All the regenerates considered here faithfully perpetuated the antero-posterior
and dorso-ventral axes of the limb from which they arose, and all showed
distal transformation. The former two axes evidently remain fixed after their
determination during early development, as Harrison (1921) and others have
demonstrated. Contrary to Swett's (1927) contention, however, the proximodistal axis is reversible by regeneration. Since this also applies to aneurogenic
316
H. WALLACE
arms, distal transformation is not determined by nerves which do not seem
to have any polarising influence on the regenerate. It is difficult to avoid the
conclusion that distal transformation is an autonomous property of the regeneration blastema and one shared by the developing limb bud (Graper, 1922).
Admittedly, the base of the blastema redifFerentiates in conformity with the
region of the stump from which it originated. That conformity is still commonly
ascribed to a stump influence or field affect, but it can equally well be interpreted as a predetermined property of the local cells which form the blastema
(Wallace & Watson, 1979). If the original blastemal cells are predetermined
in this way, their determination is most easily envisaged in terms of a PD
gradient along the intact limb (ignoring the transverse axes). The direction of
that gradient, however, is evidently unimportant and probably meaningless
once regeneration begins. Perhaps that is implicit in the numerical gradient
proposed by Maden (1977) and elaborated by Stocum (1978).
In conclusion, blastemal morphogenesis corresponds closely to that of a
limb bud in being an autonomous but internally regulative process. Such a view
is reinforced by finding that the establishment of limb axes, which anticipates
innervation during development, also occurs without reference to nerves during
regeneration.
REFERENCES
A. D. (1976). A new method for the reduced silver impregnation of arthropod
central nervous systems. Proc. R. Soc. Lond. B 193, 191-197.
BUTLER, E. G. (1955). Regeneration of the urodele forelimb after reversal of its proximodistal axis. /. Morph. 96, 265-281.
DECK, J. D. (1955). The innervation of urodele limbs of reversed proximo-distal polarity.
/. Morph. 96, 301-331.
DECK, J. D. & RILEY, H. L. (1958). Regenerates on hindlimbs with reversed proximo-distal
polarity in larval and metamorphosing urodeles. /. exp. Zool. 138, 493-504.
DENT, J. N. (1954). A study of regenerates emanating from limb transplants with reversed
proximo-distal polarity in the adult newt. Anat. Rec. 118, 841-856.
EFIMOV, M. (1933). Uber den Mechanismus des Regenerationsprozesses. Biol. Zbl. 2,
214-220.
EGAR, M., YNTEMA, C. L. & SINGER, M. (1973). The nerve fiber content of Amblystoma
aneurogenic limbs. /. exp. Zool. 186, 91-95.
EILAND, L. C. (1975). The relation of nerves to multiple regeneration in a single newt limb.
/. exp. Zool. 194, 359-371.
GRAPER, L. (1922). Extremitatentransplantationen an Anuren. Wilhelm Roux Arch.
EntwMech. Org. 51, 587-609.
HARRISON, R. G. (1921). On the relations of symmetry in transplanted limbs. /. exp. Zool.
22, 1-136.
MADEN, M. (1977). The regeneration of positional information on the amphibian limb.
/. theoret. Biol. 69, 735-753.
MILOJEVIC, B. D. & GRBIC, N. (1925). La regeneration et l'inversion de la polarite des
extremites chez les tritons adultes, a la suite d'une transplantation heterotope. C. r.
Seanc. Soc. Biol. 93, 649-651.
MONROY, A. 0942). La rigenerazione bipolare in segmenti di arti isolati di Triton crist.
Archo ital. Anat. Embriol. 48, 123-142.
NEEDHAM, A. E. (1952). Regeneration and Wound Healing. London: Methuen.
BLEST,
Reversed arms
317
K. W. & LUTHER, W. (1958). Versuche iiber di Extremitaten-regenerationen von
salamanderlarven bei umgekerter polaritat des Amputationsstumpfes. Wilhelm Roux Arch.
EntwMech. Org. 150, 373-382.
POPIELA, H. (1976). In vivo limb tissue development in the absence of nerves: a quantitative
study. Expl Neurol. 53, 214-226.
SINGER, M. (1952). The influence of the nerve in regeneration of the amphibian extremity.
Q. Rev. Biol. 27, 169-200.
SINGER, M. & MUTTERPERL, E. (1963). Nerve fiber requirements for regeneration in forelimb
transplants of the newt Triturus. Devi Biol. 7, 180-191.
STOCUM, D. L. (1978). Organization of the morphogenetic field in regenerating amphibian
limbs. Am. Zool. 18, 883-896.
SWETT, F. H. (1927). Differentiation of the amphibian limb. /. exp. Zool. 47, 385-435.
SWETT, F. H. (1928). Further experiments on the mediolateral axis of the forelimb of
Amblystoma punctatum (Linn). /. exp. Zool. 50, 51-70.
THORNTON, C. S. & THORNTON, M. T. (1970). Recuperation of regeneration in denervated
limbs of Amybstoma larvae. /. exp. Zool. 173, 293-302.
WALLACE, H. & MADEN, M. (1976). Irradiation inhibits the regeneration of aneurogenic
limbs. /. exp. Zool. 195, 353-358.
WALLACE, H. & WATSON, A. (1979). Duplicated axolotl regenerates. /. Embryol. exp. Morph.
49, 243-258.
YNTEMA, C. L. (1959). Regeneration in sparsely innervated and aneurogenic forelimbs of
Amblystoma larvae. /. exp. Zool. 140, 101-123.
OBERHEIM,
{Received 20 November 1979, revised 5 December 1979)
EMB 56